Development, Validation, and Utility of Species-Specific Diagnostic Markers for Detection of Peronospora belbahrii

Peronospora belbahrii is an oomycete and the cause of basil downy mildew, one of the most destructive diseases affecting basil production worldwide. Disease management is challenging due to wind-dispersed sporangia and contaminated seed; therefore, identifying P. belbahrii in seed lots before sale or planting or in the field before symptoms develop could allow for timely deployment of disease management strategies. In this study, a draft genome assembly and next-generation sequencing reads for P. belbahrii, as well as publicly available DNA-seq and RNA-seq reads of several other downy mildew pathogens, were incorporated into a bioinformatics pipeline to predict P. belbahrii-specific diagnostic markers. The specificity of each candidate marker was validated against a diverse DNA collection of P. belbahrii, host tissue, and related oomycetes using PCR. Two species-specific markers were identified and used as templates to develop a highly sensitive probe-based real-time quantitative PCR (qPCR) assay that could detect P. belbahrii in leaf tissue and seed samples. Both markers were capable of reliably detecting as low as 500 fg/mu l of P. belbahrii genomic DNA and as few as 10 sporangia. The qPCR assay was then validated with seed samples collected from a basil cultivar experiment. In total, 48 seed samples were collected and tested; P. belbahrii was detected in samples of all cultivars at estimated concentrations of 600 fg/mu l up to 250 pg/mu l and at as few as 10 sporangia up to >1,000 sporangia. The markers and assays are valuable for diagnostics and identifying P. belbahrii-contaminated seed lots to mitigate the effects of future basil downy mildew epidemics.

Automated summary

In this study, a bioinformatics pipeline was developed to predict specific diagnostic markers for Peronospora belbahrii, the causative agent of basil downy mildew. These markers were validated and used to develop a highly sensitive qPCR assay for detecting P. belbahrii in leaf tissue and seed samples. The assay was able to detect low concentrations of P. belbahrii genomic DNA and a small number of sporangia. This research is valuable for diagnostics and identifying contaminated seed lots to mitigate the effects of basil downy mildew epidemics.