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POSTERS: Pathogen detection, quantification and diagnosis

Improved detection method for the spinach downy mildew pathogen, Peronospora effusa.
Steve Klosterman - USDA ARS. Mychele Batista da Silva- University of California- Davis, Armando garcia L-lamos- University of California-Davis, Juliana Osorio Marin- University of California-Davis, Frank Martin- USDA ARS, Young-Joon Choi- Kunsan National University, Allen Van Deynze-

Peronospora effusa is an obligate oomycete and the causal agent of spinach downy mildew. P. effusa results in losses of up to 30% or higher in organic spinach production. Control options for this disease in organic production are limited to the development of disease resistant cultivars since fungicides are not available to use in organic production. Different pathotypes of P. effusa have been identified using a list of differential cultivars. There is a continuous need to update the differentials nearly every year due to the quick evolution of the pathogen. Early detection of the pathogen, especially in the field, can contribute to improved management strategies in both conventional and organic spinach production. We currently are testing a recombinase polymerase amplification assay (RPA) to detect the pathogen in the field. The RPA assay process works at low constant temperature due to a stabilizing enzyme and the use of specific primers. To confirm the specificity of the targeted locus, primers and a qPCR probe based on a mitochondrial locus for specific detection of P. effusa was developed. The assay was tested on 15 different isolates of P. effusa and eight closely-related species. The qPCR assay was specific for P. effusa. However, using only the primers without the qPCR probe, nonspecific detection of DNA from the related species P. obovata and P. lepigoni was observed. The next step is to adapt the P. effusa specific probe to in-field RPA-based detection.