Rapid and Sensitive Molecular Detection Tools for Oak wilt and Laurel Wilt Pathogen and Comparative Transcriptomics of Oak Wilt Hosts

Vascular wilt diseases caused by fungal pathogens represent major threats to forest health and agricultural systems across North America. Laurel wilt, caused by Harringtonia lauricola and vectored primarily by the redbay ambrosia beetle, has led to extensive mortality of Lauraceae species in the southeastern United States (US) and poses a significant risk to avocado production in Florida. Oak wilt, caused by Bretziella fagacearum, continues to spread through central and eastern regions of the United States, killing red oaks (Quercus rubra) within weeks of infection while white oaks (Q. alba) exhibit partial resistance. A critical challenge common to both diseases is the difficulty of detecting fungal DNA during early or low-titer infection, especially in host tissues or insect vectors.

To overcome these limitations, we developed highly sensitive, species-specific detection assays targeting informative SSR regions: the IFW TaqMan probe for H. lauricola and the BF45 TaqMan probe for B. fagacearum. Both probes function with gold standard quantitative PCR (qPCR) and conventional PCR systems. Using a simple blue-light flashlight visualization method, fluorescence in amplified products can be interpreted without specialized equipment or technical expertise. Detection limits achieved by qPCR were 0.32 pg/µL for H. lauricola and 0.008 ng/µL for B. fagacearum, while conventional PCR combined with blue-light detection identified 0.008 ng/µL and 0.04 ng/µL, respectively. Validation using symptomatic host tissue and insect vectors confirmed the robustness of these assays for early-stage diagnosis and field-oriented surveillance.

We also investigated the molecular basis underlying the differential susceptibility of oak species to B. fagacearum through a time-course RNA-seq experiment comparing Q. rubra and Q. alba following controlled inoculation. Transcriptomic profiles revealed that Q. alba rapidly transitions from growth to defense, activating salicylic acid and reactive oxygen signaling, reinforcing cell walls, and inducing vessel-occluding defenses. In contrast, Q. rubra displayed strong early perception and detoxification signaling but weaker downstream defense integration, suggesting a temporal mismatch that contributes to systemic fungal spread and rapid mortality. Together, these studies deliver practical, low-cost diagnostic tools and provide new insight into host defense dynamics. The sensitive detection assays and molecular understanding of disease response will support monitoring, management, and resistance-based strategies for laurel wilt and oak wilt.