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

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).

Dispersal and colonization risk of the Walnut Twig Beetle, Pityophthorus juglandis, in southern Europe

The Walnut Twig Beetle (WTB), Pityophthorus juglandis Blackman, is a small bark beetle native to Mexico and Southwestern USA recorded for the first time in Europe (NE Italy) in 2013. WTB attacks walnut (Juglans spp.) and wingnut trees (Pterocarya spp.) and is the vector of Geosmithia morbida Kolarík et al., a pathogen causing the thousand cankers disease (TCD). WTB and TCD represent a serious threat for walnut orchards in Europe. Spatiotemporal data of the WTB-TCD infestations recorded from an 8-year-long (2013–2020) monitoring conducted in 106 walnut orchards of NE Italy were used to develop a model in order to analyze: (i) the effective dispersal capacity of WTB, (ii) the factors affecting dispersal and (iii) the colonization risk of healthy walnut orchards. We registered a mean annual dispersal of 9.4 km, with peaks of about 40 km. Pest dispersal is affected by distance of suitable hosts from the nearest infested site, number of walnut orchards in the surroundings (both infested and healthy), orchard size and walnut species in the orchard. Using the model, it was also possible to calculate the colonization risk of a specific walnut orchard according to its characteristics showing, for instance, that a medium-size (5,000 trees) black walnut orchard located at 25 km from the nearest infested orchard has an infestation risk of about 50% of probability.

Geosmithia morbida (thousand cankers disease).

Thousand Cankers Disease is a disease complex native to the western United States that affects many Juglans and Pterocarya species, i.e. walnut and wingnut trees. It is caused by the fungus Geosmithia morbida, which is vectored by the walnut twig beetle (Pityophthorus juglandis), and possibly by other insects. The beetle carries fungal spores that are introduced into the tree during gallery construction, and the fungus then causes cankers in the inner bark that disrupt the flow of nutrients throughout the tree, often leading to its death. In recent years the disease has been reported in several eastern states, and also in Italy. Long-distance spread is thought to be a result of the movement of infected and infested wood.

Assessment of Semiochemical Repellents for Protecting Walnut Trees From Walnut Twig Beetle (Coleoptera: Curculionidae) Attack in a Commercial Orchard Setting in California

The walnut twig beetle, Pityophthorus juglandis Blackman, the vector of thousand cankers disease (TCD), poses a significant threat to North American walnut (Juglandaceae Juglans) trees. Despite discovery of TCD-related tree mortality over a decade ago, management options are lacking. This study represents the culmination of several years of investigating the chemical ecology of P. juglandis in hopes of developing a semiochemical repellent to disrupt the beetle’s host colonization and aggregation behaviors. Numbers of P. juglandis landing on semiochemical-treated Juglans regia L. trees in a commercial walnut orchard were compared based on captures on sticky traps. Two repellent combinations were tested: R-(+)-limonene and trans-conophthorin (LimeCon), and R-(+)-limonene, trans-conophthorin, and R-(+)-verbenone (LCV). Both repellents reduced P. juglandis aggregation (captures) equally; thus, we proceeded with the LimeCon combination to reduce potential treatment cost. Subsequent trials included a 2× dose (Dual) of LimeCon. Both LimeCon and Dual significantly reduced the number of P. juglandis caught compared with the baited control, however, only for the lower of two trap positions. Beetle landings were modeled by trap distance from repellent placement on each tree. Beetle responses to the pheromone lure were surprisingly localized and did not bring the whole tree under attack. LimeCon, LCV, and Dual treatments averaged fewer than a single beetle caught for all trap distances; however, performance of the repellents beyond 150 cm is not clear due to the localized landing response of P. juglandis to pheromone lures. Further testing is required to fully analyze the zone of inhibition of the LimeCon repellent.

Trapping Failure Leads to Discovery of Potent Semiochemical Repellent for the Walnut Twig Beetle

The walnut twig beetle, Pityophthorus juglandis Blackman, and its associated fungal pathogen that causes thousand cankers disease, currently threaten the viability of walnut trees across much of North America. During a 2011 assessment of seasonal flight patterns of P. juglandis with yellow sticky traps baited with the male-produced aggregation pheromone component, 3-methyl-2-buten-1-ol, dramatically reduced catches were recorded when Tree Tanglefoot adhesive was used to coat the traps. In summer 2011, two trap adhesives were tested for potential repellency against P. juglandis in a field trapping bioassay. SuperQ extracts of volatiles from the most repellent adhesive were analyzed by gas chromatography–mass spectrometry, and limonene and α-pinene were identified as predominant components. In field-based, trapping experiments both enantiomers of limonene at a release rate of ~700 mg/d conferred 91–99% reduction in trap catches of P. juglandis to pheromone-baited traps. (+)- and (‒)-α-Pinene reduced trap catch by 40 and 53%, respectively, at the highest release rate tested. While a combination of R-(+)-limonene and (+)-α-pinene resulted in a 97% reduction in the number of P. juglandis caught, the combination did not consistently result in greater flight trap catch reduction than individual limonene enantiomers. The repellent effect of limonene may be valuable in the development of a semiochemical-based tool for management of P. juglandis and thousand cankers disease.

Bursaphelenchus juglandis n. sp. (Nematoda: Aphelenchoididae), an associate of walnut twig beetle, Pityophthorus juglandis, the vector of thousand cankers disease

Summary Bursaphelenchus juglandis n. sp. was isolated from the walnut twig beetle, Pityophthorus juglandis, and walnut trees, Juglans spp. with symptoms of thousand cankers disease, in California, USA. Based on analysis of three rRNA genes and morphological features (three lines in lateral field, small arched vulval flap in female, broad spicule with two lines along blade and small cucullus, digitate dorsally bent condylus, male tail pattern of five papilliform papillae and one pair of glandpapillae (P5), and curved conical female tail), the new species belongs to the Abietinus group within Bursaphelenchus. It differs from similar species of this group by the presence of a cephalic disc with lateral labial sensilla at the disc border, and in having thick spicules with the capitulum surface almost parallel to a virtual direct line extending from the spicule end. An emended diagnosis, tabular polytomous identification key and compendium of species with the lists of their vectors, plant hosts, and distribution are provided for the Abietinus group. The diagnostics of the propagative developmental stages is given, including sex differences; the transmission dauer stage was identified as the third stage and its description given with sexual differences. A molecular phylogeny of Bursaphelenchus is provided based on partial 18S rRNA, ITS rRNA and the D2-D3 expansion fragments of 28S rRNA gene sequences. A PCR with a species-specific primer was developed for detection of B. juglandis n. sp.