Development of Quantitative Real-Time Polymerase Chain Reaction protocols for rapid detection and differentiation of Xylella fastidiosa subsp. fastidiosa and Xylella fastidiosa subsp. multiplex

Abstract

Xylella fastidiosa is a gram-negative, xylem-limited, plant pathogenic bacterium that is transmitted between hosts by the glassy-winged sharpshooter ( Homalodisca vitripennis) . Multiple subspecies of X. fastidiosa occur, exhibiting some degree of host specificity. X. fastidiosa subsp. fastidiosa is the causal agent of Pierce’s disease of grapevine. X. fastidiosa subsp. multiplex and X. fastidiosa subsp. sandyi are commonly found in North America but do not cause Pierce’s disease. Rapid diagnostics to determine presence of X. fastidiosa and differentiation of these subspecies is necessary for effective management and prevention of Pierce’s disease. In this study, three methods to distinguish X.fastidiosa subspecies using Quantitative Real-Time Polymerase Chain Reaction were compared. SYBR ® green, Eva Green ® , and Takara SYBR Green ® melt curve analysis of partial gyraseB amplicons, zot1 gene amplicons, and five tonB amplicons were evaluated for consistency and quality. Diagnostic protocols based on TaqMan ® and Molecular Beacon ® hybridization probes were developed with an emphasis placed on a X. fastidiosa subsp. multiplex insertion in the zot1 gene. We found SYBR ® Green and TaqMan ® based diagnostic protocols did not provide the necessary resolution for accurate and consistent differentiation of X. fastidiosa subspecies. Diagnostic protocols we developed utilizing the Molecular Beacon ® probe allow for highly specific and reliable differentiation of X. fastidiosa subspecies, even in cases where subspecies were mixed in solution. These new methods provide a more reliable protocol by which the subspecies of X. fastidiosa can be rapidly identified for the purposes of laboratory study and sample diagnostics.