|WITHERS, SAUNIA - North Carolina State University|
|GONGORA-CASTILLO, ELSA - North Carolina State University|
|Gent, David - Dave|
|THOMAS, ANNA - North Carolina State University|
|OJIAMBO, PETER - North Carolina State University|
|QUESADA-OCAMPO, LINA - North Carolina State University|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/17/2016
Publication Date: 6/17/2016
Citation: Withers, S., Gongora-Castillo, E., Gent, D.H., Thomas, A., Ojiambo, P., Quesada-Ocampo, L. 2016. Using next-generation sequencing to develop molecular diagnostics for Pseudoperonospora cubensis, the cucurbit downy mildew pathogen. Phytopathology. doi: 10.1094/PHYTO-10-15-0260-FI.
Interpretive Summary: Advances in sequencing technology can enable molecular diagnostic assays for pathogens to be developed rapidly and with high levels of specificity. In this project, a team of scientist identified genetic markers uniquely present in the cucurbit downy mildew pathogen, which causes an important disease of multiple crops but can be difficult to diagnose conclusively. Extensive testing of candidate markers found that the markers were robust and specific. The markers developed could be used for pathogen diagnostics on infected tissue, or adapted for detection of the pathogen in near real-time. Further, this work demonstrates a process that should be useful for developing markers in other systems.
Technical Abstract: Advances in Next Generation Sequencing (NGS) allow for rapid development of genomics resources needed to generate molecular diagnostics assays for infectious agents. NGS approaches are particularly helpful for organisms that cannot be cultured, such as the downy mildew pathogens, a group of biotrophic obligate oomycetes that infect crops of economic importance. Unlike most downy mildew pathogens that are highly host-specific Pseudoperonospora cubensis causes disease on a broad range of crops belonging to the Cucurbitaceae. In this study, we identified candidate diagnostic markers for P. cubensis by comparing NGS data from a diverse panel of P. cubensis and Pseudoperonospora humuli isolates, two very closely related oomycete species. P. cubensis isolates from diverse hosts and geographical regions in the United States were selected for sequencing to ensure that candidates were conserved in P. cubensis isolates infecting different cucurbit hosts. Genomic regions unique to and conserved in P. cubensis isolates were identified through bioinformatics. These candidate regions were then validated using PCR against a larger collection of isolates from P. cubensis, P. humuli, and other oomycetes. Overall seven diagnostic markers were found to be specific to P. cubensis. These markers could be used for pathogen diagnostics on infected tissue, or adapted for monitoring airborne inoculum with real-time PCR and spore traps.