DETECTION OF XYLELLA FASTIDIOSA IN INSECT VECTORS IN CALIFORNIA

• Author(s): Cabrera, J.; Civerolo, H.; Francis, Marta;
• Abstract: Recent spread of Xylella fastidiosa (Xf) to several agricultural commodities and ornamental plants in California has prompted great interest in understanding the comparative interactions between Xf and native and recently introduced insect vectors. The generally low titer of Xf in insect vectors limits the use of serological techniques, such as ELISA, for qualitative and quantitative analyses of Xf associated with different insect vectors. Xf detection by molecular techniques, such as PCR, can potentially overcome this limitation. The objective of this study was to compare standard PCR for detection of Xf in fieldcollected insects as well as in greenhouse-reared insect vectors using primers RST31/RST33 with newly developed primers HL5/HL6 in standard PCR and in Real Time PCR using the system HL5/HL6 and a probe labeled with FAM. Two native species the green sharpshooter (Draeculacephala minerva) and the red-headed sharpshooter (Xyphon fulgida), and the recently introduced glassy-winged sharpshooter (Homalodisca coagulata) were included in this study. Field-collected insects were obtained from Xf-infected grapevines and almonds in the San Joaquin Valley, California. Greenhouse-reared green and red-headed sharpshooters were also obtained from cultures maintained on a non-host of Xf in Parlier, California. Five-10 Xf cells per microL of insect head DNA sample were detected with the HL5/HL6 primer pair-FAM system. Also, using this system, the number of Xf-cells detected in field-collected and greenhouse reared insect was between 10^2-10^3/microLiters sample/reaction. This concentration of Xf cells was detected by visualization of the Xf-specific amplicon (221 bp) in gels following standard PCR with the HL5/HL6 primers. This level of pathogen in insect heads was below the limit of detection in standard PCR with primers RST31/RST33. Using Real-Time PCR quantification with the system HL5/HL6-FAM, the total amount of Xfcells per insect head was estimated to be between 104-105. Implications of these results on the epidemiology of the disease are discussed.
• Publication Date: Dec 2004
• Journal: 2004 Pierce's Disease Research Symposium