@article{standish_gongora-castillo_bowman_childs_tian_quesada-ocampo_2022, title={Development, Validation, and Utility of Species-Specific Diagnostic Markers for Detection of Peronospora belbahrii}, volume={7}, ISSN={["1943-7684"]}, url={https://doi.org/10.1094/PHYTO-09-21-0393-R}, DOI={10.1094/PHYTO-09-21-0393-R}, abstractNote={ 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/µl 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/µl up to 250 pg/µl 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. }, journal={PHYTOPATHOLOGY}, author={Standish, J. R. and Gongora-Castillo, E. and Bowman, M. J. and Childs, K. L. and Tian, M. and Quesada-Ocampo, L. M.}, year={2022}, month={Jul} }
 @article{rahman_gongora-castillo_bowman_childs_gent_martin_quesada-ocampo_2019, title={Genome Sequencing and Transcriptome Analysis of the Hop Downy Mildew Pathogen Pseudoperonospora humuli Reveal Species-Specific Genes for Molecular Detection}, volume={109}, ISSN={["1943-7684"]}, url={https://doi.org/10.1094/PHYTO-11-18-0431-R}, DOI={10.1094/PHYTO-11-18-0431-R}, abstractNote={Pseudoperonospora humuli is an obligate oomycete pathogen of hop (Humulus lupulus) that causes downy mildew, an important disease in most production regions in the Northern Hemisphere. The pathogen can cause a systemic infection in hop, overwinter in the root system, and infect propagation material. Substantial yield loss may occur owing to P. humuli infection of strobiles (seed cones), shoots, and cone-bearing branches. Fungicide application and cultural practices are the primary methods to manage hop downy mildew. However, effective, sustainable, and cost-effective management of downy mildew can be improved by developing early detection systems to inform on disease risk and timely fungicide application. However, no species-specific diagnostic assays or genomic resources are available for P. humuli. The genome of the P. humuli OR502AA isolate was partially sequenced using Illumina technology and assembled with ABySS. The assembly had a minimum scaffold length of 500 bp and an N50 (median scaffold length of the assembled genome) of 19.2 kbp. A total number of 18,656 genes were identified using MAKER standard gene predictions. Additionally, transcriptome assemblies were generated using RNA-seq and Trinity for seven additional P. humuli isolates. Bioinformatics analyses of next generation sequencing reads of P. humuli and P. cubensis (a closely related sister species) identified 242 candidate species-specific P. humuli genes that could be used as diagnostic molecular markers. These candidate genes were validated using polymerase chain reaction against a diverse collection of isolates from P. humuli, P. cubensis, and other oomycetes. Overall, four diagnostic markers were found to be uniquely present in P. humuli. These candidate markers identified through comparative genomics can be used for pathogen diagnostics in propagation material, such as rhizomes and vegetative cuttings, or adapted for biosurveillance of airborne sporangia, an important source of inoculum in hop downy mildew epidemics.}, number={8}, journal={PHYTOPATHOLOGY}, publisher={Scientific Societies}, author={Rahman, A. and Gongora-Castillo, E. and Bowman, M. J. and Childs, K. L. and Gent, D. H. and Martin, F. N. and Quesada-Ocampo, L. M.}, year={2019}, month={Aug}, pages={1354–1366} }