The Journal of Microbiology

, Volume 46, Issue 5, pp 482–490 | Cite as

Isolation and molecular characterization of Xylella fastidiosa from coffee plants in Costa Rica

  • Mauricio Montero-Astúa
  • Carlos Chacón-Díaz
  • Estela Aguilar
  • Carlos Mario Rodríguez
  • Laura Garita
  • William Villalobos
  • Lisela Moreira
  • John S. HartungEmail author
  • Carmen Rivera


Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica’s Central Valley. The symptoms are referred to by coffee producers in Costa Rica as “crespera” disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 μm in width and 1.5 to 3.0 μm in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-l-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.


citrus variegated chlorosis coffee leaf scorch crespera Pierce’s disease 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aguilar, E., W. Villalobos, L. Garita, and C. Rivera. 2005a. Confirmation of the presence of Xylella fastidiosa in plants of grapevine in Costa Rica. Phytopathology 96, S162.Google Scholar
  2. Aguilar, E., W. Villalobos, L. Moreira, C.M. Rodríguez, E.W. Kitajima, and C. Rivera. 2005b. First report of Xylella fastidiosa infecting citrus in Costa Rica. Plant Dis. 89, 687.CrossRefGoogle Scholar
  3. Araújo, W.L., J. Marcon, W. Maccheroni, Jr., J.D. VanElsas, J.W.L. Van Vuurde, and J.L. Azevedo. 2002. Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl. Environ. Microbiol. 68, 4906–4914.PubMedCrossRefGoogle Scholar
  4. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.H. Smith, and K. Struhl. 1992. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, New York, N.Y., USA.Google Scholar
  5. Beretta, M.J.G., R. Harakava, C.M. Chagas, K.S. Derrick, G.A. Barthe, T.L. Ceccardi, R.F. Lee, O. Paradela, M. Sugimori, and I.A. Ribeiro. 1996. First report of Xylella fastidiosa in coffee. Plant Dis. 80, 821.Google Scholar
  6. Berisha, B., Y.D. Chen, G.Y. Zhang, B.Y. Xu, and T.A. Chen. 1998. Isolation of Pierce’s disease bacteria from grapevines in Europe. Eur. J. Plant Pathol. 104, 427–433.CrossRefGoogle Scholar
  7. Campanharo, J.C., M.V.F. Lemos, and E.G. De Lemos. 2003. Growth optimization procedures for the phytopathogen Xylella fastidiosa. Curr. Microbiol. 46, 99–102.PubMedCrossRefGoogle Scholar
  8. Chen, J., R. Groves, E.L. Civerolo, M. Viveros, M. Freeman, and Y. Zheng. 2005. Two Xylella fastidiosa genotypes associated with almond leaf scorch disease at the same location in California. Phytopathology 95, 708–714.PubMedCrossRefGoogle Scholar
  9. Chen, J.C., J.S. Hartung, D.L. Hopkins, and A.K. Vidaver. 2002. An evolutionary perspective of Pierce’s disease of grapevine, citrus variegated chlorosis, and mulberry leafscorch diseases. Curr. Microbiol. 45, 423–428.PubMedCrossRefGoogle Scholar
  10. Davis, M.J., W.J. French, and N.W. Schaad. 1981. Axenic culture of the bacteria associated with phony disease of peach and plum leaf scald. Curr. Microbiol. 6, 309–314.CrossRefGoogle Scholar
  11. De Lima, J.E.O., V.S. Miranda, J.S. Hartung, R.H. Brlansky, A. Coutinho, S.R. Roberto, and E.F. Carlos. 1998. Coffee leaf scorch bacterium: axenic culture, pathogenicity, and comparison with Xylella fastidiosa of Citrus. Plant Dis. 82, 94–97.CrossRefGoogle Scholar
  12. Godoy, C., J. Garita-Cambronero, C. Rivera, and W. Villalobos. 2006. Two new species of Kapateira Young from Costa Rica (Auchenorrhyncha: Cicadellidae: Cicadellinae). Zootaxa 1282, 29–38.Google Scholar
  13. Godoy, C. and W. Villalobos. 2006. Two new species of Graphocephala Van Duzee from Costa Rica (Cicadellidae: Cicadellinae). Zootaxa 1298, 61–68.Google Scholar
  14. Goheen, A.C., B.C. Raju, S.K. Lowe, and G. Nyland. 1979. Pierce’s disease of grapevines in Central America. Plant Dis. 63, 788–792.Google Scholar
  15. Güldür, M.E., B.K. Çaglar, M.A. Castellano, L. Ülnü, S. Güran, M.A. Yilmaz, and G.P. Martelli. 2005. First report of almond leaf scorch in Turkey. J. Plant Pathol. 87, 246.Google Scholar
  16. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98.Google Scholar
  17. He, C.X., W.B. Li, A.J. Ayres, J.S. Hartung, V.S. Miranda, and D.C. Teixera. 2000. Distribution of Xylella fastidiosa in citrus rootstocks and transmission of citrus variegated chlorosis between sweet orange plants through natural root grafts. Plant Dis. 84, 622–626.CrossRefGoogle Scholar
  18. Hendson, M., A.H. Purcell, D. Chen, C. Smart, M. Guilhabert, and B. Kirkpatrick. 2001. Genetic diversity of Pierce’s disease strains and other pathotypes of Xylella fastidiosa. Appl. Environ. Microbiol. 67, 895–903.PubMedCrossRefGoogle Scholar
  19. Hernandez-Martinez, R., H.S. Costa, C.K. Dumenyo, and D.A. Cooksey. 2006. Differentiation of strains of Xylella fastidiosa infecting grape, almonds and oleander using a multiprimer PCR assay. Plant Dis. 90, 1382–1388.CrossRefGoogle Scholar
  20. Hopkins, D.L. and A.H. Purcell. 2002. Xylella fastidiosa: Cause of Pierce’s disease of grapevine and other emergent diseases. Plant Dis. 86, 1056–1066.CrossRefGoogle Scholar
  21. Kostka, S.J., J.L. Sherald, S.S. Hearon, and J.F. Rissler. 1981. Cultivation of the elm leaf scorch-associated bacterium. Phytopathology 71, 768.Google Scholar
  22. Kostka, S.J., T.A. Tattar, J.L. Sherald, and S.S. Hurtt. 1986. Mulberry leaf scorch, new disease caused by a fastidious, xylem-inhabiting bacterium. Plant Dis. 70, 690–693.CrossRefGoogle Scholar
  23. Lacava, P.T., W.L. Araujo, and W.A.J.L. Maccheroni, Jr. 2001. RAPD profile and antibiotic susceptibility of Xylella fastidiosa, causal agent of citrus variegated chlorosis. Lett. Appl. Microbiol. 33, 302–306.PubMedCrossRefGoogle Scholar
  24. Lacava, P.T., W.L. Araújo, J. Marcon, W. Maccheroni, Jr., and J.L. Azevedo. 2004. Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacterium Xylella fastidiosa, causal agent of citrus variegated chlorosis. Lett. Appl. Microbiol. 39, 55–59.PubMedCrossRefGoogle Scholar
  25. Lee, I.M., Davis, R.E., and J. Fletcher. 2001. Cell-wall free bacteria. In N.W. Schaad, J.B. Jones, and W. Chun (eds.). Laboratory Guide for the Identification of Plant Pathogenic Bacteria, p. 289–290. American Phytopathological Society. St. Paul, Minnesota, USA.Google Scholar
  26. Leu, L.S. and C.C. Su. 1993. Isolation, cultivation and pathogenicity of Xylella fastidiosa, the causal bacterium of pear leaf scorch disease in Taiwan. Plant Dis. 77, 642–646.Google Scholar
  27. Li, W.B., W.D. Pria, Jr., D.C. Teixera, V.S. Miranda, A.J. Ayres, C.F. Franco, M.G. Costa, C.X. He, P.I. Costa, and J.S. Hartung. 2001. Coffee leaf scorch caused by a strain of Xylella fastidiosa from citrus. Plant Dis. 85, 501–505.CrossRefGoogle Scholar
  28. Mehta, A., R.P. Leite, Jr., and Y.B. Rosato. 2001. Assessment of the genetic diversity of Xylella fastidiosa isolated from citrus in Brazil by PCR-RFLP of the 16S rDNA and the 16S–23S intergenic spacer and rep-PCR fingerprinting. Antonie Van Leeuwenhoek 79, 53–59.PubMedCrossRefGoogle Scholar
  29. Mehta, A. and Y.B. Rosato. 2001. Phylogenetic relationships of Xylella fastidiosa strains from different hosts, based on 16S and 23S intergenic spacer sequences. Int. J. Syst. Micrbiol. 51, 311–318.Google Scholar
  30. Minsavage, G.V., C.M. Thompson, D.L. Hopkins, R.M.V.B.C. Leite, and R.E. Stall. 1994. Development of a polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology 84, 456–461.CrossRefGoogle Scholar
  31. Mollenhauer, H.H. and D.L. Hopkins. 1974. Ultrastructural study of Pierce’s disease bacterium in grape xylem tissue. J. Bacteriol. 119, 612–618.PubMedGoogle Scholar
  32. Montero-Astúa, M., J.S. Hartung, E. Aguilar, C. Chacón, W. Li, F.J. Albertazzi, and C. Rivera. 2007. Genetic diversity of Xylella fastidiosa strains from Costa Rica, São Paulo, Brazil and the United States. Phytopathology 97, 1338–1406.PubMedCrossRefGoogle Scholar
  33. Pooler, M.R. and J.S. Hartung. 1995a. Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data. Curr. Microbiol. 31, 134–137.PubMedCrossRefGoogle Scholar
  34. Pooler, M.R. and J.S. Hartung. 1995b. Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis. Curr. Microbiol. 31, 377–381.PubMedCrossRefGoogle Scholar
  35. Qin, X., V.S. Miranda, M.A. Machado, E.G.M. Lemos, and J.S. Hartung. 2001. An evaluation of the genetic diversity of Xylella fastidiosa isolated from diseased citrus and coffee in Sao Paulo, Brazil. Phytopathology 91, 599–605.PubMedCrossRefGoogle Scholar
  36. Raju, B.C., A.C. Goheen, D. Teliz, and G. Nyland. 1980. Pierce’s disease of grapevines in Mexico. Plant Dis. 64, 280–282.Google Scholar
  37. Rich, P.V. and T.H. Rich. 1983. The central American dispersal route: biotic history and paleogeography, p. 12–34. In D.H. Janzen (ed.), Costa Rican Natural History. University of Chicago Press.Google Scholar
  38. Rodriguez, C.M., J.J. Obando, W. Villalobos, L. Moreira, and C. Rivera. 2001. First report of Xylella fastidiosa infecting coffee in Costa Rica. Plant Dis. 85, 1027.CrossRefGoogle Scholar
  39. Rosato, Y.B., J.R. Neto, V.S. Miranda, E.F. Carlos, and G.P. Manfio. 1998. Diversity of a Xylella fastidiosa population isolated from Citrus sinensis affected by citrus variegated chlorosis in Brazil. Syst. Appl. Microbiol. 21, 593–598.Google Scholar
  40. Schaad, N.W., E. Postnikova, G. Lacy, M. Fatmi, and C.J. Chang. 2004. Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei, subsp. nov., X. fastidiosa subsp. multiplex, subsp. nov., X. fastidiosa subsp. pauca, subsp. nov.. Syst. Appl. Microbiol. 27, 290–00.PubMedCrossRefGoogle Scholar
  41. Schuenzel, E.L., M. Scally, R. Stouthamer, and L. Nunney. 2005. A multigene phylogenetic study of clonal diversity and divergence in North American strains of the plant pathogen Xylella fastidiosa. Appl. Environ. Microbiol. 71, 3832–3839.PubMedCrossRefGoogle Scholar
  42. Shapland, E.B., K.M. Daane, G.Y. Yokota, C. Wistrom, J.H. Connell, R.A. Duncan, and M.A. Viveros. 2006. Ground vegetation survey for Xylella fastidiosa in California almond orchards. Plant Dis. 90, 905–909.CrossRefGoogle Scholar
  43. Simpson, A.J.G., F.C. Reinach, P. Arruda, F.A. Abreu, M. Acencio, R. Alvarenga, L.M.C. Alves, J.E. Araya, G.S. Baia, C.S. Baptista, M.H. Barros, E.D. Bonaccorsi, S. Bordin, J.M. Bové, M.R.S. Briones, M.R.P. Bueno, A.A. Camargo, L.E.A. Camargo, D.M. Carraro, H. Carrer, et al. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406, 151–157.PubMedCrossRefGoogle Scholar
  44. Solórzano, A., R. León, and M. Garbanzo. 2001. Determinacion del agent causal y evaluacion del efecto en la produccion causado por la crespera en el cultivo de cafe (Coffea arabica) en la zona de Los Santos, Costa Rica, p. 128–133. Protección de Cultivos, Dirección deInvestigaciones Agropecuarias. Ministerio de Agricultura y Ganadería.Google Scholar
  45. Sutula, C.L., J.M. Gillett, S.M. Morrissey, and D.C. Ramsdell. 1986. Interpreting ELISA data and establishing the positive-negative threshold. Plant Dis. 70, 722–726.CrossRefGoogle Scholar
  46. Van De Peer, Y. and R. De Wachter. 1994. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput. Applic. Biosci. 10, 569–570.Google Scholar
  47. Wells, J.M., B.C. Raju, H.Y. Hung, W.G. Weisburg, L. Mandelcoaul, and D.J. Brenner. 1987. Xylella fastidiosagen. nov., sp. nov: Gram-negative, xylem limited, fastidious plant bacteria related to Xanthomonas spp.. Int. J. System. Bacteriol. 37, 136–143.CrossRefGoogle Scholar
  48. Wendland, A., D. Truffi, R.P. Leite, Jr., and L.E.A. Camargo. 2003. Seqüenciamento e variabilidade do fragmento genômico de Xylella fastidiosa amplificado pelos iniciadores RST31/33. Fitopatol. Bras. 28, 298–301.CrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelber GmbH 2008

Authors and Affiliations

  • Mauricio Montero-Astúa
    • 1
  • Carlos Chacón-Díaz
    • 1
    • 2
  • Estela Aguilar
    • 1
  • Carlos Mario Rodríguez
    • 3
  • Laura Garita
    • 1
  • William Villalobos
    • 1
  • Lisela Moreira
    • 1
    • 4
  • John S. Hartung
    • 5
    Email author
  • Carmen Rivera
    • 1
    • 2
  1. 1.Centro de Investigación en Biología Celular y Molecular (CIBCM)Universidad de Costa RicaSan PedroCosta Rica
  2. 2.Facultad de MicrobiologíaUniversidad de Costa RicaSan PedroCosta Rica
  3. 3.Instituto del Café de Costa Rica (ICAFE)HerediaCosta Rica
  4. 4.Escuela de AgronomíaUniversidad de Costa Rica (UCR)San PedroCosta Rica
  5. 5.USDA-ARS Molecular Plant Pathology LaboratoryBeltsvilleUSA

Personalised recommendations