Rapid Detection of the Laurel Wilt Pathogen in Sapwood of Lauraceae Hosts

Laurel wilt (LW), caused by Raffaelea lauricola (RL), is a vascular fungal disease affecting species in the Lauraceae that has rapidly spread across the United States. This disease has caused significant tree losses in natural forests and Florida’s commercial avocado orchards. RL spreads through ambrosia beetle vectors and root grafts. Early detection and eradication are recommended to contain outbreaks. Therefore, rapid diagnosis is key for the timely implementation of mitigation strategies. Current LW diagnosis can take up to 10 days and involves pathogen isolation and the amplification of two microsatellite regions. To reduce diagnosis time, we optimized the standard PCR-based detection technique and assessed its potential use in the testing of woody samples. We further screened the microsatellite primers IFW and CHK on a higher number of fungal taxa as well as 11 host genotypes. Sensitivity was evaluated using RL-DNA at different concentrations in pure and mixed solutions. There was no cross-amplification in non-RL species. Both primers amplified all tested RL strains (100); however, the IFW primers were more sensitive than the CHK primers. Using the IFW primers, we detected RL in 89% of sapwood samples (76/85). This protocol provides a rapid and effective molecular-based approach that reduces the diagnostic time from 10 days to 24 h. This method can be an important tool for diagnostic laboratories. Altogether, our data and fungal collection, represent a robust foundation for future transferability of this protocol to more sensitive detection technologies (qPCR, LAMP) and for its application to samples from diverse origin (beetles, roots).