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Title: Development of a real-time RT-PCR assay for squash mosaic virus useful for broad spectrum detection of various serotypes and its incorporation into a multiplex seed health assay

Author
item Ling, Kai-Shu
item Wechter, William - Pat
item WALCOTT, RONALD - University Of Georgia
item KEINATH, ANTHONY - Clemson University

Submitted to: Journal of Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2011
Publication Date: 9/7/2011
Citation: Ling, K., Wechter, W.P., Walcott, R.R., Keinath, A.P. 2011. Development of a real-time RT-PCR assay for squash mosaic virus useful for broad spectrum detection of various serotypes and its incorporation into a multiplex seed health assay. Journal of Phytopathology. 159:649-656.

Interpretive Summary: Seed-borne pathogens pose a serious threat to modern agricultural cropping systems as they can be disseminated to many geographical regions around the world. With trends of increasing global seed production and trade, seed-health testing is an important quality control step to prevent the introduction of harmful pathogens into agricultural production systems. Effective seed-health assays depend on tests that provide timely, sensitive and broad-spectrum detection of all genetic variants of a pathogen, or in some cases, of several different pathogens in a crop. In the present study, we developed a real-time polymerase chain reaction (real-time PCR) assay that would permit a simultaneous detection of three major seed-borne pathogens of cucurbits, including bacterium Acidovorax citrulli (the causal agent of bacterial fruit blotch), fungus Didymella bryoniae (the causal agent of gummy stem blight), and Squash mosaic virus (the causal agent of squash mosaic). To facilitate seed health testing, a general total nucleic acid extraction method was developed for cucurbit seeds. The ability to use a single nucleic acid extraction technique with subsequent PCR to detect three different pathogen types lends itself to a universal system for cucurbit seed health testing. The data presented in this study demonstrate applicability and the potential for this technique to significantly improve high-throughput testing of seeds for pathogenic bacteria, fungi and viruses.

Technical Abstract: Seed-borne pathogens pose a serious threat to modern agricultural cropping systems as they can be disseminated to many geographical regions around the world. With trends of increasing global seed production and trade, seed-health testing is an important quality control step to prevent the introduction of harmful pathogens into agricultural production systems. Effective seed-health assays depend on tests that provide timely, sensitive and broad-spectrum detection of all genetic variants of a pathogen, or in some cases, of several different pathogens. In the present study, we developed a real-time PCR system that would permit a simultaneous detection of three major seed-borne pathogens of cucurbits, including bacterium Acidovorax citrulli (AC, the causal agent of bacterial fruit blotch), fungus Didymella bryoniae (DB, the causal agent of gummy stem blight, and Squash mosaic virus (SqMV, the causal agent of squash mosaic). Using the primer and probe combination designed with consensus viral sequences for a range of isolates, a sensitive and broad spectrum real-time polymerase chain reaction (real-time PCR) was developed for SqMV. Subsequently, a multiplex real-time PCR assay was designed to allow detection of above mentioned three pathogens in a single tube. Converting the purified SqMV RNA into cDNA prior to multiplexing stabilized the viral template in the mixture of two other DNA templates (AC and DB). To facilitate seed health testing, a general total nucleic acid extraction method was developed for cucurbit seeds. Using this method, nucleic acids extracted from seeds yielded strong signals for each target pathogen in multiplex real-time PCR. The ability to use a single nucleic acid extraction technique with subsequent PCR to detect bacterial, fungal, and viral plant pathogens lends itself to a universal system for cucurbit seed health testing.