Submitted to: National American Phytopathology Meetings
Publication Type: Abstract Only
Publication Acceptance Date: April 5, 2006
Publication Date: July 28, 2006
Citation: Hsu, C., Mccallister, J.E., Hartung, J.S., Turechek, W. 2006. A quantitative pcr assay for detection of xanthomonas fragariae. National American Phytopathology Meetings. 96:S51. Technical Abstract: Angular leaf spot is an important disease of cultivated strawberry worldwide. The EPPO considers <i>X. fragariae</i> as an A2 quarantine pathogen and in certain European markets, nurseries must certify plants pathogen free if they wish to export to their customers. PCR is the desired tool for certification because of its sensitivity, reliability and ease of use. Three sets of Q-PCR primers were developed from DNA sequences corresponding with the products amplified by the oligonucleotide primers 241, 245 and 295 designed by Pooler et al. (Appl. Env. Microbiol., 62:3121-27). The sensitivity of the assay for each primer set was determined with serial dilutions of extracted DNA, whole cell bacteria, and plant extracts spiked with bacteria for 4 representative isolates of <i>X. fragariae</i>. Operating under the assumption that the <i>X. fragariae</i> genome is ~5 Mb, the sensitivity of the procedure was greatest with extracted DNA, followed by whole cell DNA and then plant extracts as expected. The procedure was capable of detecting as few as 50 cells reliably with extracted DNA and often with whole cell DNA, although the dilution series was not always fully reproducible with whole cell tests, presumably due to cell clumping. Results with plant and bacteria mixtures were also quite sensitive but more variable, particularly with DNA extracted from crown tissue. This procedure will be useful in applications requiring reliable quantification of <i>X. fragariae</i>.