In-Field LAMP Detection of Flavescence Dorée Phytoplasma in Crude Extracts of the Scaphoideus titanus Vector

Abstract

One of the most destructive diseases affecting grapevine in Europe is caused by Flavescence Dorée phytoplasma (FDp), which belongs to the 16Sr-V group and is a European Union quarantine pathogen. Although many molecular techniques such as loop-mediated isothermal amplification (LAMP) are widely used for the rapid detection of FDp in infected grapevine plants, there is no developed isothermal amplification assay for FDp detection in the insect vectors that are fundamental for the spread of the disease. For this reason, a simple in-field real-time LAMP protocol was optimized and developed for the specific detection of FDp in the insect vector Scaphoideus titanus. The LAMP assay was optimized to work with crude insect extracts obtained by manually shaking a single insect in a buffer for 5 min. Such a simple, sensitive, specific, economic, and user-friendly LAMP assay allowed the detection of FDp in S. titanus in less than half an hour, directly in the field. The developed insect tissue preparation procedure, combined with the LAMP protocol, promptly revealed the presence of FDp in infected S. titanus directly in the vineyards, allowing for monitoring of the spread of the pathogen in the field and to apply timely strategies required for the mandatory control of this pathogen.