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Rapid diagnostic and soil inoculum quantification tools for soilborne pathogens of strawberry

Alyssa Burkhardt: USDA ARS

<div>The ability to rapidly identify strawberry pathogens from diseased plants in the field or to quantify their soil inoculum levels allows growers to better manage diseases. Assays to diagnose pathogens that severely damage strawberry, specifically <em>Macrophomina phaseolina,</em> <em>Fusarium oxysporum </em>f. sp. <em>fragariae (Fof)</em> and <em>Verticillium dahliae,</em> have been developed. These assays provide results in a few hours using DNA extracted from soil (TaqMan) or in as little as 20 minutes using tissue macerate directly from field crops (isothermal recombinase polymerase amplification; RPA). Comparative genomics was used to identify a genotype-specific locus for <em>M. phaseolina</em> isolates that exhibit a host preference for strawberry as well as a locus for specific detection of <em>Fof</em>. An RPA assay for <em>M. phaseolina</em> with a detection limit of 200 fg of pathogen DNA was validated with field samples. A single tube nested qPCR TaqMan assay for this pathogen can accurately quantify as little as 3 ms/g soil. An RPA assay for<em> Fof </em>is in the final stages of field validation while a TaqMan assay to quantify <em>Fof </em>in soil samples is specific and has a detection limit of 50 fg of pathogen DNA. Future work will correlate Ct values from the qPCR assay to plate counts of <em>Fof </em>from infested soil. An RPA assay for detection and quantification of <em>V. dahliae </em>was developed from a published qPCR assay. These assays provide new time-effective tools for pathogen detection and assessment of risk prior to planting.</div>