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2011 APS Annual Meeting Abstract

 

Application of multiplex PCR to mixed populations of tomato bacterial pathogens
J. T. MIXON (1), A. L. Vu (1), B. H. Ownley (1), S. C. Bost (2)
(1) University of Tennessee, Knoxville, TN, U.S.A.; (2) University of Tennessee, Nashville, TN, U.S.A.
Phytopathology 101:S121

Diagnosis of foliar bacterial diseases of tomato has been enhanced with polymerase chain reaction (PCR) and primers for the detection of pathogens. Furthermore, specific primers have been developed to detect specific pathogens in mixed bacterial populations. Previous work in other laboratories has evaluated multiplex PCR reactions for simultaneous detection of multiple species of bacterial pathogens using pure cultures. The objective of this study was to optimize a multiplex PCR protocol for specific detection of Xanthomonas species, Pseudomonas syringae pv. tomato, and Clavibacter michiganensis subsp. michiganensis in field samples of tomato foliage. The primers selected were RST 65 and RST 69 (Xanthomonas spp.), MM5 and MM6 (P. syringae pv. tomato), and CMM5 and CMM6 (C. michiganensis subsp. michiganensis). Temperature parameters and concentrations of reaction components for single PCR reations with these pathogens and their respective primers were harmonized to allow for DNA amplification of the three pathogens in one reaction. The resulting multiplex PCR gave optimal results for all three primer pairs at an annealing temperature of 57.2 C. Pure cultures were used to develop the protocol. The sensitivity and specificity of the assay will be discussed. A sensitive multiplex PCR protocol for evaluation of field samples will allow for rapid identification of these pathogens, facilitate population studies, and provide a valuable diagnostic tool.

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