IMPACT OF MULTIPLE-STRAIN INFECTIONS OF XYLELLA FASTIDIOSA ON ACQUISITION AND TRANSMISSION BY THE GLASSY-WINGED SHARPSHOOTER Project Leaders:

In this project, studies were conducted to 1) develop a method to detect and differentiate multiple strains of Xylella fastidiosa in individual glassy-winged sharpshooters (GWSS,) and 2) determine the relative ability of an individual GWSS to simultaneously retain or transmit two strains of X. fastidiosa. Strain-specific primers were developed that can detect and differentiate the Pierce’s disease strain (PD) and the oleander strain (OLS) present in extracts from individual insects fed on X. fastidiosa-infected grape and oleander plants. After feeding on infected grape and oleander source plants for one day each, 76% of surviving adults tested positive for one or both strains of X. fastidiosa. The majority of individuals tested positive for only one strain of the pathogen (29% PD, 41% OLS), and only 7% tested positive for both strains; 24 % tested negative. Overall, individual insects transmitted the pathogen 39% of the time (13% PD, 26%OLS). Thus, only about half the insects that tested positive for X. fastidiosa actually transmitted the pathogen to a susceptible host. Although each individual used in transmission studies was exposed to both strains of the pathogen and both types of test plants, in all cases an individual insect transmitted only one strain of the pathogen, never both. OBJECTIVES Assess the ability of glassy-winged sharpshooter exposed to two strains of X. fastidiosa to transmit either strain of the pathogen. INTRODUCTION Xylella fastidiosa is a bacterial plant pathogen that causes a variety of diseases in a broad range of plant hosts including Pierce’s disease of grapevines, almond leaf scorch, alfalfa dwarf, citrus variegated chlorosis, leaf scorch of live oak, pear leaf scorch, and oleander leaf scorch (Brlansky et al., 1982; Hopkins, 1989; Hartung et al., 1994; Purcell and Hopkins, 1996; Purcell et al., 1999). The genetic diversity of additional strains has been examined (Pooler and Hartung, 1995; Albibi et al., 1998; Chen et al., 1992; da Costa et al. 2000; Hendson et al. 2001). Two strains of this pathogen that are presently causing severe economic losses in California are the Pierce’s disease (PD) strain that infects grape and other hosts, and the oleander leaf scorch (OLS) strain that infects oleander (Blua et al., 1999; Purcell and Saunders, 1999, Purcell et al., 1999). The PD strain does not infect oleander and the OLS strain does not infect grape. The grape strain appears to have a broader host range than the oleander strain, however the complete host range of each strain is not really known. The pathogen is spread from plant to plant by leafhoppers. Several leafhopper vectors transmit this pathogen, but the dominant vector in Southern California is the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Blua et al., 1999; Sorensen and Gill, 1996, Purcell and Saunders 1999). This insect feeds on a very broad range of plant hosts and is capable of transmitting both the grape and oleander strain of the pathogen (Purcell, 1990; Purcell et al., 1999; Costa et al. 2000). The high mobility of this insect, and its utilization of large number of host plants provide this vector with a great opportunity to be exposed to more than one strain of the Xylella pathogen in its lifetime. In this project, studies were conducted to determine the relative ability of an individual glassy-winged sharpshooter to simultaneously retain or transmit two different strains of X. fastidiosa. RESULTS DNA extraction and amplification Both the commercial DNA extraction kit and immunocapture techniques we used effectively extracted enough bacterial DNA from plant and insect tissue to allow detection by PCR, however, the commercial kit extraction method was preferred because it was faster and less complicated than the immunocapture techniques. The mixing of strain specific primers in a single reaction minimized the number of samples needed and amount of handling required to get a strain specific identification. The use of the mesh-lined sample homogenization bags for extraction also greatly increased the speed of processing samples compared to using liquid nitrogen or grinding in microcentrifuge tubes. In addition, the use of the Ready-To-Go beads