ARS | Publication request: Duplex TaqMan real-time PCR assay for quantitative detection of Pantoea stewartii subsp. stewartii and Stenocarpella maydis

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract
Publication Acceptance Date: May 4, 2007
Publication Date: August 1, 2007
Citation: Fessehaie, A., Block, C.C., Shepherd, L.M., Misra, M.K. 2007. Duplex TaqMan real-time PCR assay for quantitative detection of Pantoea stewartii subsp. stewartii and Stenocarpella maydis [abstract]. American Phytopathological Society Annual Meeting. 97:S35.

Technical Abstract: A new TaqMan real-time PCR assay was developed for the simultaneous quantitative detection of two seedborne maize pathogens in a single assay. Pantoea stewartii subsp. stewartii (Pnss) (syn. Erwinia stewartii) is the causal agent of Stewart's bacterial wilt and leaf blight of maize. Stewart's wilt is a disease of major phytosanitary importance in the U.S., as it affects the international movement of corn seed due to concerns about seed transmission of Pnss. Stenocarpella maydis (SM) (syn.Diplodia maydis), is an important ear and stalk rot pathogen in much of the eastern Midwest and is a common contaminant in maize crops worldwide, especially during humid seasons. We successfully combined monoplex TaqMan assays for Pnss and SM into a duplex PCR assay. Optimal primer-probe combinations for the duplex TaqMan PCR assay were determined to be 900nM primers and 200nM MGB probe for Pnss, and 250nM primers and 200nM DNA probe for SM. The detection limit of the duplex TaqMan PCR assay for purified Pnss and SM DNAs was 2pg and 2fg, respectively. The limit of detection on tenfold serial dilutions of Pnss cells and purified SM DNA was determined to be 104 CFU/ml and 2fg DNA, respectively. This novel duplex real-time PCR assay shows potential to be a simple, sensitive method for simultaneous identification of both pathogens.