P-198 - Whole genome sequence-based detection of Xylella fastidiosa and its subspecies

Xylella fastidiosa (Xf) is a bacterial pathogen with multiple subspecies causing crop diseases such as Pierce’s disease of grapevine (Xf subsp. fastidiosa, Xff) and phony peach disease (Xf subsp. multiplex, Xfm). Effective disease managements rely on sensitive and accurate Xf detection. Currently, Xf detections mostly depend on polymerase chain reactions (PCRs) targeting a gene or DNA region. Depending on primer sets used, PCR results could be difficult to interpret, particularly in determining subspecies. Whole genome sequence (WGS)-based techniques target multiple genes, if not all genes, to assure taxonomy accuracy. Many Xf genome sequences are now available in public databases, which can serve as resources for Xf detection. Taking the advantages of NGS (next generation sequencing) technology, we have developed a WGS-based pipeline for Xf detection: 1. Collect and freeze-dry symptomatic leaves ; 2. DNA  extraction from petiole and midrib; 3. qPCR with appropriate primer sets; 4. NGS of an Xf positive sample; 5. Read-mapping to Xff and Xfm reference genomes using Bowtie2; and 6. BLAST search using the top 5 and bottom 5 reads from the mapped-read data set against the NCBI database that contains >200 Xf genome sequences including all subspecies. The top hits are used for taxonomy determination. A successful example using this pipeline has recently been published, https://doi.org/10.1094/PDIS-09-24-1990-PDN. Our study demonstrated that WGS-based approach is highly effective in assisting plant disease diagnosis. Furthermore, NGS data could be used for future references.