Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of <i>Geosmithia morbida</i> (Kolařik) in Infected Walnut
Domenico Rizzo,
Chiara Aglietti,
Alessandra Benigno,
Matteo Bracalini,
Daniele Da Lio,
Linda Bartolini,
Giovanni Cappellini,
Antonio Aronadio,
Cristina Francia,
Nicola Luchi,
Alberto Santini,
Santa Olga Cacciola,
Tiziana Panzavolta,
Salvatore Moricca
Affiliations
Domenico Rizzo
Laboratory of Phytopathological Diagnostics and Molecular Biology, Plant Protection Service of Tuscany, Via Ciliegiole 99, 51100 Pistoia, Italy
Chiara Aglietti
Department of Agricultural, Food, Environmental and Forestry Science and Technology (DAGRI), Plant Pathology and Entomology Section, University of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy
Alessandra Benigno
Department of Agricultural, Food, Environmental and Forestry Science and Technology (DAGRI), Plant Pathology and Entomology Section, University of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy
Matteo Bracalini
Department of Agricultural, Food, Environmental and Forestry Science and Technology (DAGRI), Plant Pathology and Entomology Section, University of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy
Daniele Da Lio
Department of Agricultural, Food and Agro-Environmental Sciences, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy
Linda Bartolini
Laboratory of Phytopathological Diagnostics and Molecular Biology, Plant Protection Service of Tuscany, Via Ciliegiole 99, 51100 Pistoia, Italy
Giovanni Cappellini
Laboratory of Phytopathological Diagnostics and Molecular Biology, Plant Protection Service of Tuscany, Via Ciliegiole 99, 51100 Pistoia, Italy
Antonio Aronadio
Plant Protection Service of Tuscany, c/o Interporto Toscano “Amerigo Vespucci”, Collesalvetti, 57014 Livorno, Italy
Cristina Francia
Laboratory of Phytopathological Diagnostics and Molecular Biology, Plant Protection Service of Tuscany, Via Ciliegiole 99, 51100 Pistoia, Italy
Nicola Luchi
Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy
Alberto Santini
Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy
Santa Olga Cacciola
Department of Agriculture, Food and Environment, University of Catania, 95123 Catania, Italy
Tiziana Panzavolta
Department of Agricultural, Food, Environmental and Forestry Science and Technology (DAGRI), Plant Pathology and Entomology Section, University of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy
Salvatore Moricca
Department of Agricultural, Food, Environmental and Forestry Science and Technology (DAGRI), Plant Pathology and Entomology Section, University of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy
Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect–fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North 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 effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.
