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.