During the spring of 1998, mature oleanders (Nerium oleander L.), pruned to form a 2-m-high hedge along an interstate highway in Orlando, FL, were observed declining and dying. Numerous plants along a 200-m section of highway were in various stages of decline. Symptoms began as chlorotic mottling along the edges of leaves and as the disease progressed, mottling became more severe and leaf margins became necrotic. Scorched leaves died, and symptoms spread throughout the plants, resulting in defoliation. New growth from the base of affected plants was stunted and severely mottled. Petioles and leaf midribs were taken from leaves with mottling symptoms and assayed for the presence of Xylella fastidiosa by polymerase chain reaction (PCR) and culturing on periwinkle wilt agar medium. For PCR assay, infected tissue from three plants was extracted by grinding in SCP buffer (1.0 g of trisodium citrate and 1.0 g of disodium succinate per liter, in 0.015 M phosphate buffer, pH 7.0) containing 0.02 M sodium ascorbate and 5% acid-washed polyvinylpyrrolidone. Amplification was performed with primers RST31 and RST33, as previously described, for specific detection of X. fastidiosa strains (1). A X. fastidiosa-specific amplification product was produced from all three extracts. For culturing, petioles and leaf midribs were cut into 0.5-cm sections, and sap was extracted from the tissue by squeezing with a forceps. Sap was blotted directly onto the medium and incubated at 28°C. Colonies typical of X. fastidiosa were observed after 10 to 14 days of incubation, and single colonies were transferred to fresh periwinkle wilt agar. The colonies were confirmed as X. fastidiosa by PCR assay. Two of the oleander strains were used to inoculate three red and three white 18-month-old oleanders by needle-puncture of the stem through a cloudy drop of bacterium in SCP buffer (108 CFU/ml). For controls, three red and three white oleanders were inoculated with SCP buffer alone. After 9 weeks in a screenhouse, marginal leaf mottling was observed in both the red and white oleanders inoculated with X. fastidiosa, and the bacterium was reisolated from leaves as described above, completing Koch's postulates. Control plants remained symptomless. Oleander leaf scorch caused by X. fastidiosa has been described previously in California and Texas (2). This is the first report of oleander leaf scorch in Florida and the eastern United States. References: (1) G. V. Minsavage et al. Phytopathology 84:456, 1994. (2) A. H. Purcell et al. Phytopathology 89:53, 1999.
Specific amplification of iron receptor genes in Xylella fastidiosa strains from different hosts