Detection and distribution of Fusarium oxysporum f. sp. fragariae race 2 in California

Fusarium wilt of strawberry is a widespread disease controlled through genetic resistance to Fusarium oxysporum f. sp. fragariae (Fof) race 1 conferred by the FW1 locus. In 2022, a novel race called CA Fof race 2 was identified in Oxnard, California (CA) that can overcome FW1 resistance and cannot be identified using published molecular diagnostic assays. To understand the prevalence and enable early detection of the novel CA Fof race 2, this research developed strain-specific qPCR and recombinase polymerase amplification (RPA) diagnostic assays and applied them to survey CA Fof race 2 in strawberry. A candidate locus was identified through alignment of reads from off-target isolates to genome assemblies of two virulent CA Fof race 2 isolates. Diagnostic qPCR and RPA tests were validated using target and off-target DNA. The RPA was validated in two laboratories to confirm reproducibility and used to test 2,945 diagnostic samples collected in strawberry-growing areas of the United States of America, Canada, and Mexico. Both assays showed 100% specificity based on laboratory screening of DNA. The limit of detection was 100 fg of DNA for the qPCR assay and 1 pg for the RPA assay. The RPA assay identified 19 out of 2,945 samples as positive for CA Fof race 2, all from six fields around Oxnard, CA; these results were confirmed by subsequent isolations and qPCR. Outcomes of this research provide essential diagnostic tools for quantifying and identifying CA Fof race 2 and highlight the limited geographic distribution of this novel resistance-breaking strawberry pathogen.