ARS | Publication request: REAL-TIME PCR ASSAY DEVELOPMENT FOR MULTIPLE MAIZE PATHOGENS

Submitted to: American Seed Trade Association Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: December 6, 2006
Publication Date: December 6, 2006
Citation: Block, C.C., Fessehaie, A., Shepherd, L., Misra, M. 2006. Real-time PCR assay development for multiple maize pathogens [abstract]. American Seed Trade Association Conference Proceedings.

Technical Abstract: This talk presents updates on the development of real-time PCR assays for two seedborne pathogens of maize, Pantoea (Erwinia) stewartii, the causal agent of Stewart's bacterial wilt, and Stenocarpella (Diplodia) maydis, the causal agent of Diplodia ear rot. We developed primers and a real-time PCR probe set for P. stewartii by working in the same intergenic transcribed spacer (ITS) region of the 16S-23S ribosomal RNA genes as used for a conventional PCR assay. The sequence between the 16S and 23S rRNA genes is frequently used to differentiate bacterial strains at the species and intraspecies levels. The specificity of the primers and probe for P. stewartii was tested against 21 P. stewartii strains, several maize-associated bacteria (pathogens and non-pathogens) of various genera and species, and against plant genomic DNA. A 126 bp PCR product was amplified from all strains of P. stewartii, but not from the non-P. stewartii strains or from plant genomic DNA. A similar approach is being followed for S. maydis, working in the ITS region between the 18S-28S fungal rRNA genes. A primer and real-time PCR probe set is being tested against 22 strains of S. maydis. The two assays worked successfully, in initial tests, in a duplex real-time PCR. The next phase of the project will involve extraction of the pathogens from infected seed using paramagnetic beads coated with DNA-based capture probes specific to each pathogen. The goal is to develop an assay suitable for simultaneous detection of P. stewartii and S. maydis from infected maize seed.