Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2009
Publication Date: 6/1/2009
Citation: Ha, Y., Fessehaie, A., Ling, K., Wechter, W.P., Keinath, A.P., Walcott, R.R. 2009. Simultaneous Detection of Acidovorax Avenae Subsp. Citrulli and Didymella Bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization and Real-Time Polymerase Chain Reaction. Phytopathology. 99:666-678.
Interpretive Summary: Watermelon is widely cultivated throughout the U.S. with annual value of $355 million. This source of revenue is constantly threatened by a number of plant diseases. In cases where seed is the primary source of inoculum, disease management is especially difficult. Molecular diagnostic technique (such as polymerase chain reaction, or PCR) could be a sensitive seed health assay for seed borne pathogens. Unfortunately, many seeds contain compounds that inhibit enzymatic reactions in PCR that leads to false-negative results. In this study, we developed a technique called magnetic capture hybridization to capture target nucleic acids and to eliminate potential PCR inhibitors from seed extract. The enriched target DNA was amplified and detected by Real-time PCR. Furthermore, we demonstrated the applicability of using Real-time PCR to simultaneously detect two different types of pathogens in cucurbit seeds causing Bacterial fruit blotch and Gummy stem blight. Development of the sensitive and reliable Real-time PCR detection of seed-borne pathogens in seeds is a major improvement in seed health test, thus an important step in managing these devastating diseases on watermelon and other cucurbits.
Technical Abstract: To improve the detection of plant pathogens in seeds magnetic capture hybridization and multiplex real-time PCR (MCH real-time PCR) was evaluated. Single stranded DNA hybridization capture probes targeting Acidovorax avenae subsp. citrulli and Didymella bryoniae DNA were covalently attached to magnetic particles and used to selectively concentrate template DNA from seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. MCH multiplex real-time PCR displayed a detection threshold of 10 A. avenae subsp. citrulli CFU /ml and 10e5 D. bryoniae spores /ml, which was ten-fold more sensitive than direct real-time PCR in both cases. While direct real-time PCR displayed a detection threshold of 100 fg/µl of A. avenae subsp. citrulli DNA in 25% of the attempts made, MCH real-time PCR demonstrated 100% detection frequency at the same DNA concentration. MCH did not significantly improve detection sensitivity for D. bryoniae relative to real-time PCR in conidial suspensions; but MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds) with 0.02% A. avenae subsp. citrulli and 0.02% D. bryoniae infestation.