DEVELOPING A MICROARRAY-PCR-BASED IDENTIFICATION AND DETECTION SYSTEM FOR XYLELLA FASTIDIOSA STRAINS IMPORTANT TO CALIFORNIA Project Leaders:

From the analysis of the 16S rDNA sequence of Xylella fastidiosa, we have identified four single nucleotide polymorphisms (SNPs). The combination of these four SNPs placed all of the known X. fastidiosa strains into four groups. With a few exceptions, the four SNP groups are very similar to those based on other genetic analyses such as RAPD analysis, whole 16S rDNA sequence analysis, and the combination of phenotypic characterization, particularly pathogenicity tests. Of particular interest is the PD group. All eight PD strain 16S rDNA sequences from different labs clustered into the same group characterized by two SNPs. Utilizing the SNP information, primer sets, Teme150fc-Teme454rg, specific to PD strain group, and Dixon454fa-Dixon1261rg, specific to non-PD almond leaf scorch group, were designed. More than 200 X. fastidiosa strains isolated from California have been tested for the specificity of these SNPs and the results were quite consistent. A microarray system, initially based on the characteristic SNPs from the 16S rRNA locus, is under construction. Coupled with PCR using universal 16S rDNA primers, the microarray-PCR based system has a high potential for quick and accurate X. fastdiosa strain identification. INTRODUCTION The need to accurately identify and differentiate X. fastidiosa strains is becoming more apparent considering the coexistence of different pathotypes in the same crop (Chen et al., 2004a, b). This prompted us to research on improvement of pathogen detection. Polymerase chain reaction (PCR) has been a common technique for X. fastidiosa identification. There are, however, technical problems limiting the application of PCR. False positive amplifications can occur among related organisms in the environment sharing similar genetic sequences. Specific primers may fail to amplify DNA from a particular isolate if there is a spontaneous mutation(s) in the primer-binding site, leading to a false negative result. The sensitivity and specificity of PCR amplification tend to be inversely related. The rationale of this project is to maximize the sensitivity of PCR technology. To increase pathogen detection specificity, microarray methodology based on the principle of DNA hybridization is applied to further confirm the accuracy of the amplified DNA fragments (Chen and Civerolo, 2003). Conceptually, the development of high-density oligonucleotide arrays allows massively parallel hybridizations to occur on the same surface, permitting high levels of probe redundancy and multiple independent detections of a diagnostic DNA sequence. Because of the taxonomic value and available large genomic sequence database, single nucleotide polymorphisms in the 16S rRNA gene are particularly useful. Other genes and intergenic regions could also be the targets due to the availability of complete genome sequences from four different X. fastidiosa strains. OBJECTIVES The overall goal of this project is to develop and evaluate a microarray-PCR-based system for accurate and quick identification of X. fastidiosa strains. A particular emphasis is on strains currently important in California. Two specific objectives are: 1. Using the complete and annotated genome sequence of X. fastidiosa Temecula strain as a guide, select appropriate DNA sequences and evaluate their potential for pathotype / genotype identification. Design and construct a DNA microarray; and 2. Evaluate the effectiveness of the constructed microarray through hybridization experiment. Using the microarray as a reference, analyze genomic variation of different pathotypes with multiple strains collected from broad geographical areas and hosts. RESULTS AND CONCLUSIONS Selected sequences in the genome of X. fastidiosa Temecula were used as preliminary queries to identify diagnostic sequences. Because of the sequence availability, most comparisons were made to the four complete genome sequences including PD-Temecula, citrus variegated chlorosis-9a5c, almond leaf scorch disease-Dixon and oleander leaf scorch diseaseAnn-1. In general, the tested genome DNA sequences showed high level of similarity as expected. However, single nucleotide polymrphisms were found in most cases. Yet, the number of SNPs varied from gene to gene. Genes of evolutionary importance were particularly emphasized because they could provide a more stable and, therefore, a more consistent base for strain identification. Thus, special efforts were made on DNA sequences from rrn operons. In addition,