Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Proceedings
Publication Acceptance Date: 10/5/2005
Publication Date: 12/5/2005
Citation: Chen, J., Civerolo, E.L. 2005. Evaluation of snps and sample preparation procedures in the detection of xylella fastidiosa strains important to california. Proceedings of the 2005 Pierce's Disease Research Symposium. p.152-154.
Interpretive Summary: We are developing an accurate and quick identification/detection system for Xylella fastidiosa, a bacterial pathogen threatening the production of grapes and almonds in California. The system is based on the currently available information of whole genome sequences of X. fastidiosa strains. The strategy was to design PCR primers based on conserved regions of the genome containing genes critical for all bacteria to grow. Differentiation of strains or pathotypes/genotypes was achieved by identifying the single nucleotide variations or single nucleotide polymorphosms (SNPs) within these genes. The other issue in pathogen detection is sample preparation. We have developed two procedures that are simple but reliable enough to extract DNA from X. fastidiosa cells inside plant tissues. The procedures minimize the amount of inhibitors from plant tissues and allow successful PCR amplification of X. fastidiosa DNA. The SNP analysis and sample preparation procedures will significantly contribute towards the development of a X. fastidiosa identification/detection system.
Technical Abstract: The main objective of this project is to develop a PCR-microarray-based system for accurate and quick identification of Xylella fastidiosa strains important to California crops. The major part of the current work focused on identification of important DNA sequences based on single nucleotide polymorphisms (SNPs) for the detection of X. fastidiosa Pierce's disease and almond leaf scorch disease strains. We expanded the previous SNP analysis in 16S rDNA to other house-keep genes, such as those coding for TCA cycle enzymes. X. fastidiosa-specific primers were designed and real-time PCR was employed. Melting point analysis was used to confirm the presence of SNPs in the amplicons and to detect different genotypic strains. A second effort was to develop and improved procedure of sample preparation, the bottleneck of PCR detection of X. fastidiosa in planta. We developed two simple sample preparation procedures for PCR amplification of X. fastidiosa DNA from infected almond and grape petioles using freshly squeezed petiole sap and freeze-dried tissue. The detection efficiency of the two PCR methods was similar to that of the pathogen isolation procedure