Development of Molecular Markers for the Detection of <i>Ceratobasidium</i> sp. D.P. Rogers Associated With Vascular Streak Dieback of Woody Ornamentals in the United States

Vascular streak dieback (VSD) poses a significant threat to the sustainability of woody ornamentals in the United States, particularly <i>Cercis</i> spp. (redbud). This study aimed to develop reliable molecular diagnostic tools for detecting <i>Ceratobasidium</i> sp. D.P. Rogers (<i>Csp</i>), the fungus consistently detected from VSD-symptomatic redbuds and other woody ornamentals. Molecular markers can provide rapid and accurate detection for fastidious fungi such as <i>Csp</i> that are challenging to recover in culture media. However, current molecular tools for <i>Csp</i> detection lack sensitivity and specificity. To address this, specific PCR and quantitative PCR (qPCR) primers were developed, targeting the internal transcribed spacer (ITS) genetic region. The existing primers for the second-largest subunit of RNA polymerase II (<i>RPB2</i>), translation elongation factor 1-alpha (<i>TEF1-α</i>), and ATP synthase subunit 6 (<i>ATP6</i>) genetic regions of <i>Rhizoctonia</i> fungi were also redesigned to enhance the sensitivity and accuracy of detection for <i>Csp</i>. The CP-qP-Csp primer set, with a detection limit of 86.02 pg/μl, effectively detected <i>Csp</i> in VSD-symptomatic tissues, cultures, and soils and is recommended for molecular detection of <i>Csp</i> from woody ornamentals in the U.S. Cross-platform validation across seven laboratories confirmed the robustness of the assays. These findings provide nursery producers with critical early-detection capabilities, potentially preventing significant economic losses and contributing to the sustainability of woody ornamental production. Future research will focus on refining these assays for field applications and exploring the genetic characteristics of <i>Csp</i> across different hosts and pathogen interactions to better understand the epidemiology of VSD.