2007 Annual Report 1a.Objectives (from AD-416) To develop a microarray-based Xylella fastidiosa detection and identification system. 1b.Approach (from AD-416) A PCR-microarray-based system that combines the sensitivity of PCR and specificity of microarray DNA hybridization will be developed and rapid identification of X. fastidiosa strains. Strain specific DNA sequence information will be identified through genome sequence analyses of multiple X. fastidiosa strains. The PCR-microarray system will be extensively evaluated using X. fastidiosa strains collected from different genotypes/pathotypes from different geographical regions. Documents SCA with UC Davis. Formerly 5302-22000-005-26S (10/05) & 5302-22000-007-14S (5/07). 3.Progress Report This report documents research conducted under a Specific Cooperative Agreement between ARS and the University of California, Davis. Additional details of the research can be found in the report for the parent project 5302-22000-008-00D, Epidemiology and Management of Xylella fastidiosa(Xf)and Other Exotic and Invasive Diseases and Insect Pests. This study focuses on characterization of the intra-genotype variations between strains and to develop a sequence based PCR for rapid detection and genomic study of Xylella fastidiosa. Monthly collections Xf-infected almond leaf samples from 2 different locations in Fresno County were analyzed. The year-round symptom development of ASLD under field conditions was monitored. Isolation of X.fastidiosa was compared with plant sap and freeze dried samples by PCR in correlation of symptom development in the fields. When using freeze-dried powdered samples, we observed a 92% match with the gold standard cultivation method. Primers using several hyper-variable loci in the genome of X. fastidiosa were designed and information from the variable loci was used to analyze the variability within the same population in relation to the seasonal behavior of the pathogen and to evaluate simplified procedures for PCR detection of X. fastidiosa. Three groups of repeats were observed and the number of repeats varied from month to month in the same tree. The year-round sampling showed a constant change in the tandem repeats and this implies that the pspB locus could be under constant change. X fastidiosa strains were isolated from 17 different hosts from California, Georgia and Florida. The X. fastidiosa strains were characterized phenotypically in colony morphology and growth in different culture media. These strains were further characterized genotypically at the highly conserve 16S rDNA locus and the highly variable pspB tandem repeat locus. Single nucleotide polymorphisms (SNPs) were used to sub-group the strains. The SNPs clearly divided the strains into distinct sub-groups. For further confirmation, 8 housekeeping genes were identified from the complete genome sequence of X. fastidiosa Temecula strain and primers were designed for the PCR-array. The PCR products were sequenced and analyzed with the help of CLUSTAL W program. The SNPs distinctly divided the strains into sub-groups and supported the current subspecies system. The progress of the research was monitored on a regular basis through meetings, brief reports and analyses of results.