Verbenone Affects the Behavior of Insect Predators and Other Saproxylic Beetles Differently: Trials Using Pheromone-Baited Bark Beetle Traps.

In our study, we assessed the effects of verbenone, the most widely studied bark beetle aggregation inhibitor, on saproxylic beetles in a Mediterranean pine forest in Tuscany. Verbenone pouches were devised in the laboratory and then applied to Ips sexdentatus pheromone traps so that their catches could be compared to those of traps containing just the pheromone. The trial was carried out in spring-summer 2023, and insect catches were collected every two weeks. A total of 9440 beetles were collected that belonged to 32 different families and 57 species. About 80% of the captures were bark beetles, mainly Orthotomicus erosus. Beetle predators accounted for about 17% of the captures, with a total of 12 species. Some of these predator species had not yet been studied in relation to verbenone effects, like other saproxylic beetles recorded in this study. A significant reduction in captures was recorded for some beetles (e.g., I. sexdentatus and O. erosus), while for other species, no differences emerged, and in some cases, captures increased significantly when verbenone was present in the traps (i.e., Hylurgus ligniperda, Corticeus pini, and Aulonium ruficorne). The diversity of caught saproxylic beetles increased significantly in the verbenone traps, highlighting possible implications of the use of verbenone when managing bark beetle outbreaks.

Thousand Cankers Disease in Walnut Trees in Europe: Current Status and Management.

Thousand cankers disease (TCD) is a new deadly disease in walnut trees (Juglans spp.), which is plaguing commercial plantations, natural groves, and ornamental black walnut trees (Juglans nigra) in their native and invasion areas in the US and, more recently, in artificial plantations and amenity trees in the newly-invaded areas in Europe (Italy). This insect/fungus complex arises from the intense trophic activity of the bark beetle vector Pityophthorus juglandis in the phloem of Juglans spp. and the subsequent development of multiple Geosmithia morbida cankers around beetles' entry/exit holes. After an analysis of the main biological and ecological traits of both members of this insect/fungus complex, this review explores the options available for TCD prevention and management. Special focus is given to those diagnostic tools developed for disease detection, surveillance, and monitoring, as well as to existing phytosanitary regulations, protocols, and measures that comply with TCD eradication and containment. Only integrated disease management can effectively curtail the pervasive spread of TCD, thus limiting the damage to natural ecosystems, plantations, and ornamental walnuts.

Quantitative Real-Time PCR Based on SYBR Green Technology for the Identification of Philaenus italosignus Drosopoulos & Remane (Hemiptera Aphrophoridae).

The use of molecular tools to identify insect pests is a critical issue, especially when rapid and reliable tests are required. We proposed a protocol based on qPCR with SYBR Green technology to identify Philaenus italosignus (Hemiptera, Aphrophoridae). The species is one of the three spittlebugs able to transmit Xylella fastidiosa subsp. pauca ST53 in Italy, together with Philaenus spumarius and Neophilaenus campestris. Although less common than the other two species, its identification is key to verifying which role it can play when locally abundant. The proposed assay shows analytical specificity being inclusive with different populations of the target species and exclusive with non-target taxa, either taxonomically related or not. Moreover, it shows analytical sensibility, repeatability, and reproducibility, resulting in an excellent candidate for an official diagnostic method. The molecular test can discriminate P. italosignus from all non-target species, including the congeneric P. spumarius.

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 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.

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).

Thousand cankers disease in Juglans: Optimizing sampling and identification procedures for the vector Pityophthorus juglandis, and the causal agent Geosmithia morbida.

Lindgren funnel traps were used to monitor Pityophthorus juglandis occurrence. Traps were placed directly on walnut trees, with the top tied to one of the lower branches (about 2m high). An 8-funnel model was used instead of a 4-funnel trap, with the specific pheromone bait positioned between the fourth and the fifth funnel. Traps were customized with a 5mm metal mesh which was placed inside the bottom funnel so that debris (mainly foliage) and larger non-target insects would not end up inside the collecting jar. Geosmithia morbida was isolated from beetle adults, larvae and necrotic woody tissue around beetle galleries. Contaminant-free colonies were subcultured in purity and identified by: a) colony phenotyping [morphology, texture and pigmentation; margin type (regular/irregular; lobed/non-lobed); mycelium compactness; surface bumpiness; growth/temperature relationships]; b) micromorphology: type, morphology and ontogeny of conidiophores, metulae and phialides; conidiogenesis; shape, dimension and pigmentation of conidia; c) DNA fingerprinting.•Our protocol was customized to prevent traps from swinging in the wind and to optimize beetle catches by transversely fixing the bottom of funnel traps to the tree trunk with wooden shafts for stability.•To enhance fungus isolation in purity, a semi-selective Potato Dextrose Agar (PDA) medium, enriched with the antibiotics Ampicillin (Policillin-N) and Rifampicin (Rifamycin), was devised to prevent contamination by Gram-positive and Gram-negative bacteria and by mycobacteria.

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis.

The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, and of its vector, Pityophthorus juglandis, in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.

Effects of climate on pine processionary moth fecundity and on its egg parasitoids.

Climate change may be affecting the fecundity of phytophagous insects as well as impacting their natural enemies. However, temperature impacts these two insect groups differently, disrupting population regulation mechanisms, and ultimately, possibly culminating in an outbreak of the host. The pine processionary moth (PPM) is one of the most harmful insects of the Mediterranean basin. Not only are PPM larvae harmful to plants, but they are also dangerous to humans because of their urticating hairs. Although some information is available on climate change effects on the PPM, little is known about its potential effects on PPM egg parasitoids, especially on their distribution range or on their role in controlling PPM populations. The aim of this article was to verify the effects of climate on PPM fecundity and on its egg parasitoids. Our results show that climate warming may affect the PPM positively, but not its egg parasitoids. Specifically, during our study mild winters directly favored the PPM, while increasing summer temperatures (over 30°C) also favored the PPM indirectly, by decreasing parasitism rates. We predict that ever-milder winters will not only favor PPM development, but also encourage it to spread in otherwise previously inhospitable environments.

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism. The objective of this study was to determine the genetic structure of populations of Pissodes castaneus from different areas and on different species of Pinus using the PCR-AFLP technique. Twenty samples were analyzed, representing 19 populations from Brazil and one from Florence, Italy, which is the region of origin of P. castaneus. The four combinations of primers generated a total of 367 fragments of DNA, and 100% of polymorphic loci, indicating high degree of molecular polymorphism. The dendrogram did not reveal trends for grouping the populations in relation to origin. The low genetic similarity (0.11 between the most distant groups) and genetic distances of 0.13 and 0.44 for 10 out of the 20 samples may indicate several founding events or multiple introductions of heterogeneous strains into Brazil. The allelic fixation index (Fst) was 0.3851, considered high, and the number of migrants (Nm) was 0.3991, indicating low gene flow among populations. The highest genetic distances were between the population from Irani, SC and Cambará do Sul, RS and Bituruna, PR, indicating an independent founding event or a particular allelic fixation in the former location. The high genetic diversity among populations points out that the populations are genetically heterogeneous with a diverse gene pool in the surveyed areas, what makes them to respond differently to control measures.

Cone and seed pests of Pinus pinea: assessment and characterization of damage.

Cone and seed insects have played a key role in the decline of stone pine nut production in Italy. To evaluate the impact caused by native Palearctic and exotic insects, a greater knowledge of pest symptoms is required. During 2008-2009, first and second-year stone pine cones, as well as the seeds produced, were examined in Tuscany (Italy) to assess viability. Insect damage was characterized based on external signs on the cones and seed endosperms, and the impact of recorded insect species on nut production was evaluated. In the current study, cones attacked by anobiid beetles and Dioryctria spp. were observed, as well as asymptomatic dead cones and cones with resin drops and patches, that could not easily be related to a damaging agent. As regards the anobiid beetles, adults of Ernobius parens (Mulsant and Rey) and E. impressithorax Pic emerged from cones in laboratory rearing. A low number of cones damaged by Dioryctria spp. was recorded whereas high percentages of cones showed resin exudates. The presence of resin cannot be definitely related to a damaging agent, although the feeding activity of Leptoglossus occidentalis Heidemann could be one of the reasons. Damage by L. occidentalis was assessed by seed observation. Most of the seeds displayed tissues that had been damaged by this pest.