|Bhat, Ravi - UNIV OF CALIF, DAVIS|
Submitted to: Joint Meeting of the American Phytopathological Society and Society of Nematology
Publication Type: Abstract Only
Publication Acceptance Date: August 1, 2007
Publication Date: N/A
Technical Abstract: Specific and rapid plant pathogen detection methods can aid in strawberry disease management decisions. PCR-based diagnostics for Phytophthora cactorum and other strawberry pathogens are hindered by PCR inhibitors and lack of species-specific PCR primers. We developed a DNA extraction and purification protocol, which involved soaking of tissue pieces in a detergent for at least 8 h prior to extraction and separating inhibitors from the DNA after extraction by using a modified agarose-embedded technique. Our method reliably solved the problem of PCR inhibitors and facilitated amplification of target pathogen DNA from necrotic root, crown, and petiole tissues of naturally infected strawberry plants. Also, while optimizing PCR conditions, we determined that inclusion of 2% PVP40 and a relatively high concentration of Taq polymerase (2.5 units per reaction) improved amplification in the presence of residual PCR inhibitors. A P. cactorum-specific primer pair that amplifies a 335 bp fragment from the ITS1-5.8S-ITS2 region was designed and validated for nested PCR using target and non-target DNA, and the DNA from greenhouse, nursery, and fruiting field strawberry plants infected with P. cactorum and other pathogens. The newly developed PCR-based detection system was determined to be quicker and more sensitive than cultivation- and morphology-based assays for P. cactorum.