From Genomics to Diagnostics: Combating the Spread of Vascular Streak Dieback in North America

ABSTRACT Vascular streak dieback (VSD) has emerged as a major threat to woody ornamental production in the United States (U.S.), currently affecting at least 50 tree and shrub species across 25 botanical genera in at least twelve U.S. states. The putative causal agent, here termed the VSD‐associated Ceratobasidiaceae lineage ( Csp ), presents diagnostic challenges due to its fastidious growth requirements and vascular colonization pattern. Here, we integrate genomics‐guided marker discovery with multi‐platform genome‐anchored tools (SSR, TaqMan qPCR, and LAMP that target unique genomic regions identified through comparative genomics) to deliver a field‐deployable, genome‐anchored toolkit for detection and genotyping of Csp across nurseries and landscapes. We evaluated the performance of these tools using 103 samples representing DNA isolated from 64 VSD‐symptomatic plants or their fungal isolates and 39 non‐target Ceratobasidiaceae outgroups. Genome assembly of Illumina reads yielded ten informative microsatellite markers with an overall amplification success rate of 95.8% (91.7%–100.0% across Csp samples). These markers demonstrated strong performance in distinguishing the Csp samples from the outgroups. Among the three TaqMan probes developed, VSD048 exhibited superior performance with 100% detection across Csp samples, characterized by cycle threshold (C T ) values of 26.8 ± 4.2, and no cross‐reactivity with C. theobromae or other Ceratobasidiaceae species. A multi‐laboratory ring trial confirmed the reproducibility of the VSD048 TaqMan assay across various testing platforms. The loop‐mediated isothermal amplification (LAMP) assay targeting the Csp ‐unique region achieved a sensitivity of 0.82 [95% CI 0.68–0.968] and specificity of 1.00 [95% CI 1.00–1.00], with a practical detection threshold of approximately 25 copies of the target region. Our molecular survey of the disease revealed a surprisingly broad host range beyond Cercis canadensis (Eastern redbud), encompassing eight additional genera, and confirmed its geographical distribution across the eastern and midwestern U.S. These validated molecular tools provide rapid, sensitive, and specific detection methods for Csp surveillance and management. The field‐applicable LAMP assay and laboratory‐based TaqMan/SSR protocols offer complementary approaches for different diagnostic scenarios, thereby enabling early detection and supporting efforts to limit the spread of this emerging pathogen in nursery and landscape settings.