Rapid detection of the oak wilt pathogen (Bretziella fagacearum) using loop-mediated isothermal amplification (LAMP)

Oak wilt, caused by Bretziella fagacearum, is a destructive disease of Quercus species. Symptoms consist of leaf scorch, dieback, defoliation, vascular discoloration, and eventual death. Prompt diagnosis is crucial for timely management that involves laborious and expensive practices such as sanitation, cutting of infected trees, and proactive tree removal to create a buffer zone, root trenching, and/or fungicide injections. Due to these resource-intensive management tactics, laboratory test accuracy and speed are paramount. Diagnoses of oak wilt currently rely on culturing, nested PCR, or qPCR, but all these methods have limitations, ranging from ambiguous results to high barriers to application. We developed a rapid, robust, and sensitive loop-mediated isothermal amplification (LAMP) diagnostic test using primers and probes that target a section of the internal transcribed spacer region of the rDNA of the oak wilt pathogen, B. fagacearum. This assay can detect B. fagacearum from pure isolates and environmental samples, including branch sapwood, leaf petioles, nitidulid sap beetle vectors, and degraded fungal mats. The LAMP assay developed here is sensitive to a detection limit as low as 1 pg/μL and can detect the pathogen in under 20 min. This real-time LAMP-based diagnostic test is thoroughly validated to specifically detect the oak wilt pathogen, B. fagacearum, and is demonstrated to detect the fungus from a variety of routinely collected sample types. Our rapid molecular detection protocol can provide a useful tool for diagnostic labs, forest health practitioners, and researchers studying oak wilt.IMPORTANCEOak wilt is a deadly disease of oaks that represents a growing threat to natural and planted forests throughout eastern North America. To enhance the speed and accuracy of molecular diagnostics for this disease, we designed a molecular test using loop-mediated isothermal amplification (LAMP) to reliably and rapidly detect the oak wilt pathogen, Bretziella fagacearum. To optimize the practicality of this assay in real-world conditions, we developed a rigorous validation process that incorporated the known breadth of genetic diversity of the oak wilt pathogen, sample tissue range, and a panel of non-target fungi to assess the potential for false positives. The novel assay is demonstrated to be highly specific and can detect B. fagacearum from host sapwood, leaf petioles, and nitidulid sap beetles, serving as a versatile new tool for forest health surveillance. Our approach also represents a thorough framework for researchers interested in the development of rapid molecular methods for the detection of forest microbes more broadly.