Genome-Informed Development of a Highly Specific Loop-Mediated Isothermal Amplification Assay for Detection of the Laurel Wilt Pathogen in Avocado

Laurel wilt, caused by the ambrosia fungus Harringtonia lauricola (HL), threatens Lauraceae forests and avocado production. Rapid and accurate diagnosis is critical for disease management, but current methods are often slow or difficult to implement directly from field-collected wood samples. A published loop-mediated isothermal amplification (LAMP) assay targeting the HL β-tubulin ( β-tub ) gene enables rapid detection but lacks extensive validation for avocado tissues and may produce ambiguous amplification products when applied to non-target fungi and crude plant extracts. This study evaluated the specificity, sensitivity, and inclusivity of the β-tub assay and developed a novel HL-specific LAMP marker (HL_0354) through comparative genomic analyses. Both assays were validated using fungal isolates, diverse HL strains, and naturally and artificially infected avocado wood samples. The β-tub assay produced late nonspecific amplification products with several non-HL species and consistently detected HL at 5 pg DNA. In contrast, HL_0354 showed no reproducible amplification from non-target fungi and reproducibly detected 0.5 pg of HL DNA in crude avocado DNA matrices (0.05 ng/µl), with amplifications typically occurring within 5–12 min. Inclusivity testing confirmed detection of all HL strains evaluated despite minor annealing-temperature variation among Southeast Asian isolates. When applied to field samples, HL_0354 achieved a true-positive rate of 97% and an overall diagnostic accuracy of 98%, outperforming the β-tub assay (90% and 94%, respectively), without false-positive detections. Coupled with a simple Chelex-based extraction protocol, HL_0354 provides a rapid, sensitive, and field-deployable approach for detection of H. lauricola in avocado wood samples, supporting laurel wilt surveillance and management.