Confirmation of Xylella fastidiosa infecting grapes (Vitis vinifera) in Costa Rica

Abstract

In 2003, symptoms of Pierce Disease (PD) were observed in two vineyards established in two different localities in San Jose province (Santa Ana and La Uruca) and in La Garita, Alajuela province. Twenty four symptomatic plants (15 from Santa Ana, five from La Uruca and four from La Garita) were analyzed by DAS-ELISA using antibodies against Xylella fastidiosa. Sixteen plants (nine from Santa Ana, five from La Uruca, and two from La Garita) were positives for the bacterium. Isolation attempts were made from petioles of these plants, and seven isolates were obtained in liquid PW. Aliquots of liquid media were inoculated on solid PW. Ten days after inoculation, circular and convex colonies, with entire margin, smooth and opalescent, of 0.5-1mm in diameter were observed. From each isolate three colonies were selected and considered as different clones. Every clone was analyzed by DAS-ELISA with positive results. Clones were Gram negative, catalase positive and oxidase negative. The bacteria were rod shaped, and showed a typically rippled cell wall. DNA of each clone was extracted and used as template in PCR with primers 272-1/272-2 and RST31/RST33; products of 500pb and 733pb were obtained, respectively. These results confirmed the presence of X. fastidiosa causing PD in grapevine plants in different vineyards in Costa Rica.

Durante el año 2003, se observaron síntomas del mal de Pierce (PD) en dos plantaciones de vid en la provincia de San José (Santa Ana y La Uruca) y en otra en La Garita, provincia de Alajuela. De un total de 24 plantas sintomáticas analizadas mediante DAS-ELISA usando anticuerpos para Xylella fastidiosa, se detectaron 16 plantas positivas (nueve de Santa Ana, cinco de La Uruca y dos de La Garita). A partir de diferentes intentos de aislamiento de los peciolos en medio líquido PW, se obtuvieron siete aislamientos. Alícuotas de medio líquido se inocularon en medio PW sólido. Diez días después de la inoculación, se observaron colonias circulares, cónvexas, lisas y opalescentes, con márgenes enteros, con diámetro entre 0.5-1mm. De cada aislamiento se seleccionaron tres colonias, las cuales fueron consideradas como clones diferentes. Todos los clones presentaron valores positivos mediante DAS-ELISA. Los clones presentaron bacilos Gram negativo, catalasa positivo y oxidasa negativo. Los bacilos al observarse al microscopio electrónico de transmisión presentaron la pared rugosa previamente informada para X. fastidiosa. Al amplificar el ADN extraido de cada clon mediante la técnica de la PCR con los imprimadores 272-1/272-2 y RST31/RST33, se obtuvieron productos de 500pb y 733pb, respectivamente. Estos resultados confirman la presencia de X. fastidiosa causando mal de Pierce en vid en Costa Rica.