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 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.
First, second, fourth, fifth, and sixth authors: Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616; third author: U.S. Department of Agriculture-Agricultural Research Service, Forage Seed and Cereal Research Unit, and Oregon State University, Corvallis 97331; and fourth and fifth authors: Center for Integrated Fungal Research, North Carolina State University, Raleigh 27695-7567.
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Withers
First, second, fourth, fifth, and sixth authors: Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616; third author: U.S. Department of Agriculture-Agricultural Research Service, Forage Seed and Cereal Research Unit, and Oregon State University, Corvallis 97331; and fourth and fifth authors: Center for Integrated Fungal Research, North Carolina State University, Raleigh 27695-7567.
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Gongora-Castillo
First, second, fourth, fifth, and sixth authors: Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616; third author: U.S. Department of Agriculture-Agricultural Research Service, Forage Seed and Cereal Research Unit, and Oregon State University, Corvallis 97331; and fourth and fifth authors: Center for Integrated Fungal Research, North Carolina State University, Raleigh 27695-7567.
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Gent
First, second, fourth, fifth, and sixth authors: Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616; third author: U.S. Department of Agriculture-Agricultural Research Service, Forage Seed and Cereal Research Unit, and Oregon State University, Corvallis 97331; and fourth and fifth authors: Center for Integrated Fungal Research, North Carolina State University, Raleigh 27695-7567.
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Thomas
First, second, fourth, fifth, and sixth authors: Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616; third author: U.S. Department of Agriculture-Agricultural Research Service, Forage Seed and Cereal Research Unit, and Oregon State University, Corvallis 97331; and fourth and fifth authors: Center for Integrated Fungal Research, North Carolina State University, Raleigh 27695-7567.
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Ojiambo
First, second, fourth, fifth, and sixth authors: Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616; third author: U.S. Department of Agriculture-Agricultural Research Service, Forage Seed and Cereal Research Unit, and Oregon State University, Corvallis 97331; and fourth and fifth authors: Center for Integrated Fungal Research, North Carolina State University, Raleigh 27695-7567.
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0000-0002-9072-7531
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Quesada-Ocampo
D019662:Cucurbitaceae / Q000469:parasitology*; D005819:Genetic Markers / Q000235:genetics; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D058507:Host Specificity; D009868:Oomycetes / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000469:parasitology*; D018515:Plant Leaves / Q000469:parasitology; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D005819:Genetic Markers