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Rapid diagnostic tools for soilborne pathogens of strawberry
Alyssa Burkhardt: USDA ARS; Marina Ramon: USDA ARS; Steven Koike: Univ of California; Frank Martin: USDA ARS
<div>The ability to rapidly identify and quantify soilborne strawberry pathogens in plants and soil allows growers to make decisions about what to plant and how to manage diseases in a field. Therefore, we are developing rapid assays using TaqMan real-time PCR and recombinase polymerase amplification (RPA) to diagnose pathogens that severely damage strawberry, including <i>Verticillium dahliae</i>, <i>Fusarium oxysporum </i>f. sp. <i>fragariae</i>, and <i>Macrophomina phaseolina</i>. These assays provide results in hours (TaqMan) or in as little as 20 minutes (RPA) using DNA extracted from either infected plant tissue or soil. For <i>M. phaseolina</i>, genomic sequencing, assembling, and comparing multiple isolates has identified a genotype-specific locus to detect isolates which have been found to only infect strawberry. This locus was used to develop an RPA assay with a sensitivity of 200 fg that has been shown to be specific on nearly 100 samples. A nested qPCR TaqMan assay is also being optimized to detect a low level <i>of M. phaseolina</i> microsclerotia in the soil. A published locus specific for a majority <i>of F. oxysporum f. sp. fragariae</i> isolates has been translated to an RPA assay (200 fg sensitivity) and is being developed into a TaqMan assay for soil quantification. Additional loci are being developed to detect all <i>F. oxysporum f. sp. fragariae isolates</i>. For <i>V. dahliae</i>, a published TaqMan assay for soil has been transitioned into an RPA for infected crown tissue with a sensitivity of 200 fg.</div>

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