Isolation and Molecular Characterization of Xylella fastidiosa from Coffee Plants in Costa Rica

Mauricio Montero-Astua; Carlos Chacon-Diaz; Estela Aguilar; Carlos Mario Rodriguez; Laura Garita; William Villalobos; Lisela Moreira; John S. Hartung; Carmen Rivera

doi:10.1007/s12275-008-0072-8

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Research Support, U.S. Gov't, Non-P.H.S.
Isolation and Molecular Characterization of Xylella fastidiosa from Coffee Plants in Costa Rica: Mauricio Montero-Astua ¹, Carlos Chacon-Diaz ^1,2, Estela Aguilar ¹, Carlos Mario Rodriguez ³, Laura Garita ¹, William Villalobos ¹, Lisela Moreira ^1,4, John S. Hartung ⁵, Carmen Rivera ^1,2; Journal of Microbiology 2008;46(5):482-490.
DOI: https://doi.org/10.1007/s12275-008-0072-8
Published online: October 31, 2008

¹Centro de Investigacion en Biologia Celular y Molecular (CIBCM), Universidad de Costa Rica, San Pedro 2060, Costa Rica, ²Facultad de Microbiologia, Universidad de Costa Rica, San Pedro 2060, Costa Rica, ³Instituto del Cafe de Costa Rica (ICAFE), Heredia, Costa Rica, ⁴Escuela de Agronomia, Universidad de Costa Rica (UCR), San Pedro 2060, Costa Rica, ⁵USDA-ARS Molecular Plant Pathology Laboratory, 10300 Baltimore Ave., Beltsville, MD 20705, USA

Corresponding author: John S. Hartung , Tel: 1-301-504-6374,

Received: 18 March 2008 • Accepted: 14 August 2008

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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 um in width and 1.5 to 3.0 um 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 272-1-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.

Keywords: citrus variegated chlorosis;coffee leaf scorch;crespera;Pierce's disease

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Isolation and Molecular Characterization of Xylella fastidiosa from Coffee Plants in Costa Rica

J. Microbiol. 2008;46(5):482-490. Published online October 31, 2008

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

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