Isolation and molecular characterization of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> from coffee plants in Costa Rica |
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Authors: | Mauricio Montero-Astúa Carlos Chacón-Díaz Estela Aguilar Carlos Mario Rodríguez Laura Garita William Villalobos Lisela Moreira John S Hartung Carmen Rivera |
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Institution: | (1) Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica, San Pedro, 2060, Costa Rica;(2) Facultad de Microbiología, Universidad de Costa Rica, San Pedro, 2060, Costa Rica;(3) Instituto del Café de Costa Rica (ICAFE), Heredia, Costa Rica;(4) Escuela de Agronomía, Universidad de Costa Rica (UCR), San Pedro, 2060, Costa Rica;(5) USDA-ARS Molecular Plant Pathology Laboratory, 10300 Baltimore Ave., Beltsville, MD 20705, USA |
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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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Keywords: | citrus variegated chlorosis coffee leaf scorch crespera Pierce’ s disease |
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