Development of QRT-PCR protocols for rapid Xylella fastidiosa subspecies diagnostics

Xylella fastidiosa (Xf) is a plant pathogenic bacterium that is transmitted between hosts by the glassy-winged sharpshooter (GWSS) (Homalodisca vitripennis). Multiple subspecies of Xf occur and are host specific. Xf subsp. fastidiosa is the causal agent of Pierce’s disease. Xf subsp. multiplex and Xf subsp. sandyi are commonly found in North America but do not cause Pierce’s disease. Rapid diagnostics to determine presence of Xf and differentiation of these subspecies is necessary for effective management of Pierce’s disease. In this study, three methods by which the subspecies can be distinguished by QRT-PCR were compared. SYBR green^®, Eva Green^®, and Takara SYBR Green^® melt curve analysis of partial gyraseB amplicons, Zot gene amplicons, and five TonB amplicons were evaluated for consistency and quality. Multiple melts were performed to find the ideal conditions for distinguishing between the subspecies. Emphasis was placed on a Ragweed insertion in the Zot gene, for which a probe was designed to increase the reliability of rapid diagnostics and differentiation of Xf subspecies. These new methods provide a more reliable protocol by which the subspecies of Xf can be determined.