Location: Crop Improvement and Protection ResearchTitle: Development and application of qPCR and RPA genus and species-specific detection of Phytophthora sojae and Phytophthora sansomeana root rot pathogens of soybean Author
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2017
Publication Date: 3/8/2017
Citation: Rojas, A., Miles, T.D., Coffey, M.D., Martin, F.N., Chilvers, M. 2017. Development and application of qPCR and RPA genus and species-specific detection of Phytophthora sojae and Phytophthora sansomeana root rot pathogens of soybean. Plant Disease. doi: 10.1094/PDIS-09-16-1225-RE. Interpretive Summary: The manuscript describes two molecular diagnostic tests to determine if two species of the plant pathogenic genus Phytophthora are causing disease on soybean (P. sojae and P. sansomeana). One assay is a TaqMan real time PCR technique that can detect the pathogens in the laboratory and the other is an isothermal assay that can detect the pathogens directly in the field without the need for DNA extraction. Both assays should improve the effort to manage these important pathogens of soybean.
Technical Abstract: Phytophthora root rot of soybean, caused by Phytophthora sojae is one of the most important diseases in the Midwest US, causing losses of up to 44 million bushels per year. Disease may also be caused by P. sansomeana, however the prevalence and damage caused by this species is not well known, partly due to limitations of current diagnostic tools. Efficient, accurate and sensitive detection of pathogens is crucial for management, thus a multiplex real-time PCR and isothermal (RPA: Recombinase Polymerase Amplification) assay were developed using a hierarchical approach. Both assays consist of a genus-specific probe and two species-specific probes that target a mitochondrial region that is highly specific for the genus Phytophthora. The qPCR approach multiplexes the three probes and a plant internal control. The RPA assay runs each probe independently, obtaining a result in 20 mins. The multi-copy mitochondrial genome provides sensitivity with sufficient variability to discern among different Phytophthora species. The assays were highly specific when tested against a panel of 96 Phytophthora spp. and range of Pythium spp. The detection level of the assay is 100 fg for the qPCR assay and 10 pg for the RPA assay. The assay was validated on symptomatic plants collected during the 2013 field season, showing correlation with isolation. In 2014, the assays were validated with samples from nine soybean producing states in the U.S. The assays are valuable diagnostic tools for detection of Phytophthora spp. affecting soybean.