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

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Abstract

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.

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Authors and Affiliations

  1. Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica, San Pedro, 2060, Costa Rica

    Mauricio Montero-Astúa, Carlos Chacón-Díaz, Estela Aguilar, Laura Garita, William Villalobos, Lisela Moreira & Carmen Rivera

  2. Facultad de Microbiología, Universidad de Costa Rica, San Pedro, 2060, Costa Rica

    Carlos Chacón-Díaz & Carmen Rivera

  3. Instituto del Café de Costa Rica (ICAFE), Heredia, Costa Rica

    Carlos Mario Rodríguez

  4. Escuela de Agronomía, Universidad de Costa Rica (UCR), San Pedro, 2060, Costa Rica

    Lisela Moreira

  5. USDA-ARS Molecular Plant Pathology Laboratory, 10300 Baltimore Ave., Beltsville, MD, 20705, USA

    John S. Hartung

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  1. Mauricio Montero-Astúa
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  2. Carlos Chacón-Díaz
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  3. Estela Aguilar
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  4. Carlos Mario Rodríguez
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  5. Laura Garita
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  6. William Villalobos
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  8. John S. Hartung
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  9. Carmen Rivera
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Correspondence to John S. Hartung.

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Montero-Astúa, M., Chacón-Díaz, C., Aguilar, E. et al. Isolation and molecular characterization of Xylella fastidiosa from coffee plants in Costa Rica. J Microbiol. 46, 482–490 (2008). https://doi.org/10.1007/s12275-008-0072-8

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  • DOI: https://doi.org/10.1007/s12275-008-0072-8

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