Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method

Abstract

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).

Stilbocrea banihashemiana sp. nov. a New Fungal Pathogen Causing Stem Cankers and Twig Dieback of Fruit Trees

“…Members of Bionectriaceae are less known as canker pathogens. However, also this family encompasses species that were reported as causal agents of cankers on woody plants, such as Nectriella pironii Alfieri & Samuels [ 11 ], which causes gall and stem cankers on fig, and Geosmithia morbida M. Kolark, E. Freeland, C. Utley & Tissera, the causal agent of Thousand Cankers Disease (TCD) of walnut ( Juglans regia L.) [ 12 , 13 , 14 ]. Moreover, some species of Stilbocrea , a genus within this family, cause wood discoloration and trunk diseases, e.g., S. colubrensis Lechat & J. Fourn on Bambusa vulgaris (Schrad.…”

Alien Invasive Pathogens and Pests Harming Trees, Forests, and Plantations: Pathways, Global Consequences and Management

“…Preventing the introduction of dangerous IAPPs into new areas is the first step in their management, so this has become an important priority in many countries. Preventing introductions is carried out through effective surveillance activities, namely phytosanitary inspections at ports of entry (ports, airports, and custom barriers), as well as at vulnerable sites, i.e., those places where the plant material is in transit or processed, such as nurseries, loading and sorting stations, and wood processing companies [112,113]. Considering the enormous volume of commerce at present, and the incessant mobility of humans for work, tourism, etc., many IAPPs elude preventative measures, it being practically impossible to intercept all the species that transit on all commodities [114,115].…”

“…Furthermore, for an effective inspection system which can cover the ever-increasing volume of import-export material, simple, timely, and repeatable methods have to be developed. Modern molecular diagnostic tools, which detect DNA polymorphisms, ensure high accuracy and diagnostic sensitivity, allowing the detection of even minimal traces of nucleic acids, such as those remaining in the frass of xylophagous insects [113]. Some of these methods have the advantage of being able to process a large number of samples in a short time [121].…”

The Future of Forest Pathology in North America

“…Pathogen identification and detection have revolutionized how we diagnose tree diseases (Stewart et al, 2018). Plant health clinics now routinely use DNA-based methods (Martin et al, 2009;Wu et al, 2011;Lamarche et al, 2015;Yang and Juzwik, 2017;Oren et al, 2018;Parra et al, 2020;Rizzo et al, 2021;Stackhouse et al, 2021) that provide rapid and accurate diagnostics. Regulatory agencies have adopted the tools developed by forest pathologists for their day-to-day testing for invasive species.…”