A Metabarcoding Amplicon Sequencing Approach for Reliable Early Detection and Surveillance of Oak Wilt (Bretziella fagacearum) from Trap-Collected Nitidulid Beetles

Early detection protocols for the oak wilt fungal pathogen (Bretziella fagacearum) are crucial for forest health practitioners on the boundaries of the growing disease front. Established protocols use oak wilt-specific primers and gel electrophoresis to amplify and detect oak wilt among DNA extracted from nitidulid vectors. However, these protocols are prone to inconclusive results due to the presence of off-target amplification products similar in size to positive control bands. Using sequence-adapted oak wilt primers, we employed a metabarcoding amplicon sequencing approach that resolved inconclusive results and validated true oak wilt positives. We found that these off-target amplification products are co-occurring taxa from natural forest and beetle microbiomes, further necessitating a sequencing approach for early surveillance of oak wilt.