Rapid Detection of <i>Pityophthorus juglandis</i> (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method
Domenico Rizzo,
Salvatore Moricca,
Matteo Bracalini,
Alessandra Benigno,
Umberto Bernardo,
Nicola Luchi,
Daniele Da Lio,
Francesco Nugnes,
Giovanni Cappellini,
Chiara Salemi,
Santa Olga Cacciola,
Tiziana Panzavolta
Affiliations
Domenico Rizzo
Laboratory of Phytopathological Diagnostics and Molecular Biology, Plant Protection Service of Tuscany, Via Ciliegiole 99, 51100 Pistoia, 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
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
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
Umberto Bernardo
Portici Unit, Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), P. le Enrico Fermi 1, 80055 Portici, Italy
Nicola Luchi
Florence Unit, Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy
Daniele Da Lio
Department of Agricultural, Food and Agro-Environmental Sciences, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy
Francesco Nugnes
Portici Unit, Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), P. le Enrico Fermi 1, 80055 Portici, Italy
Giovanni Cappellini
Laboratory of Phytopathological Diagnostics and Molecular Biology, Plant Protection Service of Tuscany, Via Ciliegiole 99, 51100 Pistoia, Italy
Chiara Salemi
Department of Agricultural, Food and Agro-Environmental Sciences, University of Pisa, Via del Borghetto 80, 56124 Pisa, 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
The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect’s nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).
