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

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 microm in width and 1.5 to 3.0 microm 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.

The Complex Biogeography of the Plant Pathogen Xylella fastidiosa: Genetic Evidence of Introductions and Subspecific Introgression in Central America

“…pauca in Brazil [19], may be more serious than the losses due to the “crespera” disease typical of infection by X. f. subsp. fastidiosa [16], [20].…”

“…The analysis was based 38 isolates from Costa Rica obtained from symptomatic plants as described in Montero-Astua et al [16]. Details of 24 of these isolates are provided elsewhere [11], [16]; the remainder were isolated by BO and others at the University of Costa Rica.…”

“…Details of 24 of these isolates are provided elsewhere [11], [16]; the remainder were isolated by BO and others at the University of Costa Rica. DNA was extracted from cultures derived from these isolates by us at the University of Costa Rica and shipped to the University of California Riverside where we typed the isolates using the MLST scheme developed for this species [14], [15], plus we sequenced the non-MLST pilU locus.…”

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

“…Since the first report in grape, X. fastidiosa was isolated and identified from an increasingly large number of plant hosts, with or without symptoms, and recognized to be the causal agent of different diseases(Moller et al, 1974; Hearon et al, 1980; Chang et al, 1993; Grebus et al, 1996; McElrone et al, 1999; Hopkins and Purcell, 2002; Montero-Astua et al, 2008). In many cases also wild plant species were found to carry this pathogen, but often in a latent stage only (Freitag, 1951; Raju et al, 1983; Hopkins and Adlerz, 1988; Blake, 1993; Hill and Purcell, 1997; Li et al, 2001).…”

First report of Iris yellow spot virus in Costa Rica

“…Small leaf segments (1 × 2 mm) were harvested around necrotic lesions on symptomatic onion leaves and fixed in Karnovsky solution (Karnovsky 1965) in 0.05 M cacodylate buffer pH 7 at 6-8°C. Fixed tissue was processed and embedded in Spurr's epoxy resin (Spurr 1969) as described by Montero-Astúa et al (2008). Thin sections were obtained with a diamond knife and an ultramicrotome (Power Tome PC, RMC) and stained for 5 min with 2% uranyl acetate aqueous and for 2 min with Sato's modified lead stain method (Hanaichi et al 1986).…”