Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolarik) in Infected Walnut

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantationforestry for high-quality timber and nut production, as well as in urban greening as ornamentalplants. These species are currently threatened by the thousand cankers disease (TCD) complex, aninsect-fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, thebark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated inNorth America, little research has been carried out in Europe, where it was more recently introduced.A key step in research to cope with this new phytosanitary emergency is the development of effectivemolecular detection tools. In this work, we report two accurate molecular methods for the diagnosisof GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary toand integrated with similar recently developed assays. Our protocols detected GM DNA from puremycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, withoutcross-reactivity to a large panel of taxonomically related species. The precision and robustness of ourtests guarantee high diagnostic standards and could be used to support field diagnostic end-users inTCD monitoring and surveillance campaigns.