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POSTERS: Pathogen detection, quantification and diagnosis

Detection of viable Phytophthora spp. associated with Christmas trees in Michigan using qPCR combined with propidium monoazide (PMA)
Keumchul Shin - Michigan State University. Carmen Medina-Mora- Michigan State University, Monique Sakalidis- Michigan State University

Phytophthora root rot can cause widespread mortality in numerous species of plants. Conventional PCR and qPCR assays used to detect and quantify pathogen presence may overestimate the number of active pathogens due to the lack of discrimination between viable and non-viable propagules of the pathogens. Detection of a pathogen and determination of live or dead status is critical to evaluate the efficacy and success of pathogen mitigation treatments. A quantitative PCR (qPCR) assay using pre-treatment of propidium monoazide (PMA), a DNA-binding dye penetrating broken membrane of dead cells and inhibiting PCR amplification, was evaluated to test the viability of Phytophthora spp. in artificially inoculated and field samples. The PMA-qPCR (atp9-nad9 region) protocol developed in this study, was specific and multiplexed with genus and species-specific for the detection of Phytophthora spp. in the samples tested. Detection sensitivities for the genus and species-specific assays down to 10 and 100 fg/µl of genomic DNA extracted from pure culture, respectively. Artificially inoculated soil samples treated with 40 µM of PMA showed a signal reduction (3.03 cycles) in qPCR between live and heat-killed zoospores of Phytophthora spp. We demonstrated that our PMA-qPCR treatment is a suitable approach to distinguish between viable and non-viable populations of Phytophthora spp. Further studies will focus on the validation of the PMA-qPCR assay using environmental samples.