|FESSEHAIE, ANANIA - IOWA STATE UNIVERSITY
|SHEPHERD, LISA - IOWA STATE UNIVERSITY
Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/16/2006
Publication Date: 7/28/2006
Citation: Fessehaie, A., Shepherd, L.M., Block, C.C. 2006. A Diagnostic Real-Time Taqman PCR Assay for the Detection of Pantoea Stewartii Subsp. Stewartii in Corn Seed [abstract]. American Phytopathological Society Annual Meeting. 96:S35.
Technical Abstract: A sensitive and specific real-time PCR assay was developed for Pantoea stewartii subsp. stewartii (Pss), the causal agent of Stewart’s bacterial wilt and leaf blight of sweet corn and maize. Working in the same intergenic spacer (IGS) region of the ribosomal DNA as an existing conventional PCR assay, we developed a new primer and probe set suitable for a real-time PCR assay. The specificity of the primers and probe was tested against 21 Pss strains, several plant-associated bacteria of various genera and species, and maize genomic DNA. A 126 bp PCR product was amplified from all strains of Pss, but not from the non-Pss bacterial strains or from plant genomic DNA. The detection limits for Pss were 0.2-2 pg using purified DNA and 5-50 cells. The Pss-specific PCR assay was tested in a multiplex format by adding an internal reference based on the corn-specific alcohol dehydrogenase gene, adh-1. The multiplex real-time PCR assay was optimized by evaluating various combinations of primer/probe concentration ratios. The PCR assay which includes an internal amplification control was specific, highly sensitive and efficiently reproducible. The assay should be ideally suited for rapid and sensitive identification of bacterial isolates and may also allow the development of a multiplex PCR assay for simultaneous detection of Pss and other maize pathogens.