DEVELOPMENT OF QRT-PCR PROTOCOLS FOR RAPID XYLELLA FASTIDIOSA SUBSPECIES DIAGNOSTICS Principal Investigator:

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 (PD). Xf subsp. multiplex and Xf subsp. sandyi are commonly found in North America but do not cause PD. Rapid diagnostics to determine presence of Xf and differentiation of these subspecies is necessary for effective management of PD. 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. LAYPERSON SUMMARY Detection of the pathogen in an agronomic disease system is an important component of a management strategy. This is especially true in cases where incredibly mobile insects (like the glassy-winged sharpshooter) are the primary vectors of a pathogen (like Xylella fastidiosa; Xf). In short, it is important to know which plants are infected with the pathogen and which insects in the vineyards are carrying the pathogen. To further confuse our understanding of the system, three subspecies of Xf exist in our grape growing areas which are currently detectible by standard assays; however, only one of these subspecies will lead to Pierce’s disease if it is in a vine. So in this study, we have developed a detection assay that is inexpensive and more sensitive than any detection protocols available. Furthermore, we have designed the protocol to distinguish between strains of Xf. INTRODUCTION Xylella fastidiosa (Xf) is a plant pathogenic bacterium residing in the xylem vessels and is the causal agent of many plant diseases including Pierce’s disease (PD) of the grapevine (Hopkins and Purcell, 2002; Bextine et al, 2007). In the southwestern region of the United States, there are three common strains of Xf: Xf subsp. fastidiosa (PD), Xf subsp. multiplex (RW) (Schaad et al, 2004), and Xf subsp. sandyi (OLS) (Hernandez-Martinez et al, 2007). Previous methods of distinguishing Xf subspecies are slow and more prone to error. Initially, these previous methods, using SYBR Green, were evaluated and compared to new methods which used Takara SYBR Green® and Eva Green®. Melt analysis was used to analyze the strains. The gyraseB gene was used in the evaluation of subspecies determination protocols. In the second part of the experiment, new primers were designed for the Zot and TonB genes in order to attempt differentiation of the species based on melting temperature analysis. In the Zot gene of the Ragweed strain an insertion from base pair 379-426 (Figure 1) was the main focus of the Zot primers. Figure 1: Section of Zot gene in PD, OLS, and RW Xf subspecies showing Ragweed insertion.