PARATRANSGENESIS TO CONTROL PIERCE'S DISEASE: BIOLOGY OF ENDOPHYTIC BACTERIA IN GRAPE PLANTS AND BIOASSAY OF REAGENTS TO DISRUPT PIERCE’S DISEASE Project Leader:

Xylella fastidiosa (Xf), which causes Pierce’s disease (PD) in grapevines, is transmitted by the glassy-winged sharpshooter (GWSS). Paratransgenesis employs symbiotic bacteria to deliver anti-Xf compounds to disrupt transmission of the pathogen to new host plants. Alcaligenes xylosoxidans denitrificans (Axd) was identified as a potential agent for paratransgenesis because it inhabits the foregut of GWSS, as does Xf, and the xylem of plants. In this report, we describe the interaction of Axd with plants, GWSS predators, and alternate Xf-vectors and report preliminary data on inhibition of transmission by anti-Xf factors. Axd colonized and traveled within 6 host plants, reaching the highest titers in lemon. Axd colonized the foregut region of two alternate Xf-vectors, the blue green and smoke tree sharpshooters, and was not identified in predatory arthropods that fed on Axd-positive GWSS. Disruption of Xf-transmission by GWSS was demonstrated using two reagents, a single chained antibody fragment and an antibiotic peptide. INTRODUCTION The glassy-winged sharpshooter (GWSS) is the principle vector of the xylem-limited bacterium Xylella fastidiosa (Xf), which causes Pierce’s disease (PD) in grapevines. Limiting the spread of this pathogen by rendering GWSS incapable of pathogen transmission would control the disease. Paratransgenic approaches to pathogen control are currently being developed to deliver anti-pathogen strategies to disrupt Triatomid transmission of Trypanosoma cruzi (1), to prevent colitis in mammals (2, 7), and to interfere with transmission of HIV(4). Candidate microbes that live in close proximity to the pathogen in the vector insects and in host plant tissues would by ideal vehicles to control Xf. Alcaligenes xylosoxidans denitrificans (Axd), originally isolated from the cibarium of GWSS, has been described as a nonpathogenic plant endophyte and a non-pathogenic soil-borne microbe (5, 8). Axd, genetically marked with DsRed or EGFP protein, colonized the cibarium of GWSS for up to 35 days, the longest period tested (3). Two categories anti-pathogen reagents, single-chained antibodies (scFV) and antibiotic peptides, were tested for activity against Xf. Screening of scFV uncovered an antibody fragment that was specific to Xf and may be specific to the PD-causing strain of Xf. Four toxic peptides were identified that inhibited the growth of Xf, but did not inhibit the growth of Axd. OBJECTIVES 1. Identify environmental endpoints of Axd in plants, GWSS predators, and alternate insect vectors. 2. Test the ability of anti-pathogens to disrupt Xf disease cycle. RESULTS Axd movement within and colonization of six host plants. Plants treated with Axd showed no abnormalities (stunting, chlorosis, necrosis, etc.) two weeks after inoculation, despite relatively high titers in several hosts as compared to control plants. The melting temperature of the 199 bp Axd EGFP insert sequence is 92.1°C. Comparing PCR product melting temperature to that of the primer dimer (80.6°C), conformations were made by increasing the temperature from 72 to 99°C at a rate of 1°C/45s and measuring fluorescence every 45s, then plotting results on the melting curve. A standard curve and a regression line (R=0.983, R2=0.967, efficiency=0.73, M=0.239, B=10.362) was constructed based the amplification of known standards with plant background. Standards were tested in the presence of each host plant and no significant differences were found, therefore differences in quantification were not attributed to deleterious plant effects on PCR. Axd was found most consistently (44 of 45) and in the highest concentrations in lemon plants in both replications (Table 1). In both replications, lemon plants were found to have greater than 3 million Axd cells per 2 cm of tissue, almost one order of