Using Next-Generation Sequencing to Develop Molecular Diagnostics for Pseudoperonospora cubensis, the Cucurbit Downy Mildew Pathogen

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 biotroph...

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Bibliographic Details
Published in:Phytopathology 2016-10, Vol.106 (10), p.1105-1116
Main Authors: Withers, S, Gongora-Castillo, E, Gent, D, Thomas, A, Ojiambo, P S, Quesada-Ocampo, L M
Format: Article
Language:English
Subjects:
Cucurbitaceae
Cucurbitaceae - parasitology
Genetic Markers - genetics
Genomics
High-Throughput Nucleotide Sequencing - methods
Host Specificity
Oomycetes
Oomycetes - genetics
Oomycetes - isolation & purification
Plant Diseases - parasitology
Plant Leaves - parasitology
Pseudoperonospora cubensis
Real-Time Polymerase Chain Reaction
Sequence Analysis, DNA
Species Specificity
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Summary: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 family Cucurbitaceae. In this study, we identified candidate diagnostic markers for P. cubensis by comparing NGS data from a diverse panel of P. cubensis and P. 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.
ISSN:0031-949X
1943-7684
DOI:10.1094/PHYTO-10-15-0260-FI