TOWARDS THE DEVELOPMENT OF A SEQUENCE-BASED PCR SYSTEM FOR DETECTION AND GENOMIC STUDY OF XYLELLA FASTIDOSA STRAINS IMPORTANT TO CALIFORNIA Project Leader:

We report two of our recent efforts for improvement of PCR detection and genomic analysis of Xylella fastidiosa (Xf). We evaluated the use of PCR primers from a hyper-variable locus to monitor diversity within strains of the same pathotype. The pspB (PD1208) locus, encoding a serine protease, was selected and analyzed. It was observed that tandem repeat sequences in pspB locus were highly variable from strain to strain. The biological significance of this hyper-variation is unknown. We also evaluated a simple sample preparation method for template DNA. The pulverized freeze-dried tissue PCR (PFT-PCR) test was compared to the “gold standard” pathogen isolation method. Our results indicated that PFT-PCR had a high predictive value (90.8%) for true positive samples, but a low predictive value for true negative results (29.7%), indicating that a PFT-PCR result is best suited to confirm the presence of Xf in a sample. INTRODUCTION The complete sequencing of Xylella fastidiosa (Xf) genome and their availability allow easy access to every genomic locus of the pathogen. With the availability of many primer design softwares in the Internet, it becomes highly feasible for many plant pathology laboratories to design PCR primers and explore their applications. These form the foundation for developing a genome based detection system for Xf using an array of primers from different loci on the platform of polymerase chain reaction (PCR). PCR is theoretically a highly sensitive and versatile technique for pathogen detection. A PCR experiment can be illustrated in the following formula: PCR SUCCESS = PRIMER * SAMPLE PREPARATION (1) Where a PCR success is the function of utilization of primers designed based on genomic information and sample preparation that provides DNA template. Either factor is equally critical in affecting PCR outcome. The evolution and nucleotide variation rates of each gene or genomic locus vary. Therefore, primers designed from different genomic loci have different value in evaluating the bacterial population diversity and usage to define Xf strains. Several specific PCR primer sets are currently available for Xf detection including the most thoroughly tested RST31/33 primer set (Minsavage, et al., 1994), derived from the RNA polymerase genomic locus, and those derived from 16S rRNA gene (Chen et al., 2005), an important taxonomical character for the description of Xf (Wells et al., 1987). These primers target the conserved genes. Variations in these gene sequences are closely associated with pathogen pathotypes. For example, in San Joaquin Valley of California, the 16S rDNA G-genotype (G-genotype) strains cause both Pierce’s disease (PD) of grapevine and almond leaf scorch disease (ALSD). The 16S rDNA A-genotype (A-genotype) strains cause only ALSD. Few studied have been performed on the use of less conserved or highly variable loci to study Xf. Information from the more variable loci could facilitate our understanding of the bacterial pathogenicity and environmental adaptations. In contrary to PCR primers, sample preparation methods have been subjected to much less vigorous evaluation. The most common PCR detection procedures for Xf detection involve DNA extraction to generate template DNA. However, this severely reduces the high throughput capacity of a PCR procedure. Efforts were made to simplify or omit the DNA extraction procedure by using expressed plant sap as PCR DNA template. However, the results were inconsistent. To address this problem, we previously reported the development of a procedure using pulverized freeze-dried almond tissues for PCR detection of Xf (Chen and Civerolo, 2005). In this report, we present the results of our recent analyses on using primers from a hyper-variable locus to evaluate the population diversity of Xf strains within the same pathotype/genotype. We also evaluated the procedure using pulverized freeze-dried almond tissues for PCR (PFT-PCR) detection of Xf. These are part of our effort in developing a comprehensive genome sequence-based detection system for Xf strains important to California.