Pityophthorus Juglandis Research Articles

Potential biological control agents of Geosmithia morbida restrict fungal pathogen growth via mycoparasitism and antibiosis

Thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut) spp. (Fagales: Juglandaceae) is caused by the fungal pathogen Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat (Hypocreales: Bionectriaceae) and bark beetle pest/vector, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae). To further the development of biological management strategies for thousand cankers disease, we assessed the ability of 14 endophytic Trichoderma (Hypocreales: Hypocreaceae) isolates and the commercially available isolate T. afroharzianum strain KRL-AG2 to inhibit the in vitro growth of three different G. morbida isolates via mycoparasitism and antibiosis. To identify factors that may affect field success of candidate biological control agents, we quantified the growth responses of Trichoderma spp. and the commercially available entomopathogenic fungus, Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) strain GHA, to the plant secondary metabolite and antimicrobial compound, juglone in vitro. A total of 12 Trichoderma isolates (from six different Trichoderma species) demonstrated antagonistic activity towards G. morbida in dual-plate assays. Juglone consistently reduced the growth of B. bassiana strain GHA and 14 of the 15 screened Trichoderma isolates in vitro. Additionally, one metabolite-producing Trichoderma isolate, TN4-47, completely inhibited the growth of all three G. morbida isolates across all tested metabolite concentrations and had comparatively greater tolerance to juglone compared to other Trichoderma isolates. Future lines of research should focus on characterizing the active antagonistic compound present in the metabolite filtrates, determine the mode of action of the active component(s), and elucidate how abiotic and biotic factors may influence the growth, persistence, and antagonistic activity of candidate biological control agents in planta.

Fungal, host andnon‐hostvolatiles modify attraction of the walnut twig beetle,Pityophthorus juglandis, to pheromone lures

AbstractThousand Cankers Disease (TCD) of walnut trees is caused by the pathogenic fungusGeosmithia morbidavectored by the walnut twig beetle (WTB)Pityophthorus juglandis. Monitoring efforts for WTB rely on pheromone‐baited traps, but lures are likely effective at attracting beetles only over short distances. Fungal‐derived kairomones may increase the efficacy of current lures, while additional volatiles may repel beetles from valuable trees.The objective of this study was to determine the extent to which fungal, host and non‐host volatiles modify the attraction of WTB to pheromone‐baited traps. A trapping study that combined fungal, host‐associated and non‐host compounds with WTB‐pheromone lures was conducted over three years in black walnut plantations experiencing a TCD outbreak in Walla Walla, WA.Traps baited with pheromone andG. morbidavolatiles (i.e., isoamyl and isobutyl alcohol) consistently attracted more WTB, while other fungal volatiles inconsistently increased attraction compared to those baited with pheromone lure alone. This is the first field study that demonstrates fungal volatiles can increase the attraction of a bark beetle to its pheromone in a hardwood system.One fungal (benzyl alcohol) and two additional volatiles (limonene, piperitone) repelled WTB from pheromone‐baited traps. Although limonene is known to repel WTB, this is the first demonstration that benzyl alcohol and piperitone repel a bark beetle.Fungal volatiles may increase the efficacy of monitoring efforts and may play an important role in management tactics for WTB, especially in detecting the introduction and establishment of nascent populations and protecting trees from colonizing beetles.

Functional roles of nematodes associated with the walnut twig beetle and eastern black walnut in the inland northwest

AbstractBeetles (Scolytinae) form intimate associations with a taxonomically and functionally diverse suite of nematodes that are phytopathogens, fungal feeders, and entomoparasites. Despite their ubiquity, the ecological significance of nematodes in the lifecycles of economically important bark and ambrosia beetle species (Curculionidae: Scolytinae) and associated plant diseases remains largely unexplored. Thousand cankers disease (TCD) is caused by the walnut twig beetle (WTB, Pityophthorus juglandis Blackman) and the fungus Geosmithia morbida (Kolařík, Freeland, Utley & Tisserat; Ascoymycota: Hypocreales) and causes foliar senescence, progressive crown dieback, and mortality in black walnut (Juglans nigra L.) throughout western North America. In this study, nematodes recovered from P. juglandis and J. nigra in Idaho (ID) and Washington (WA) were identified morphologically and by constructing multilocus phylogenies to infer taxonomic relationships to taxa for which molecular data were available. We conducted assays to determine the extent to which nematodes feed and reproduce on G. morbida and other fungi commonly found in galleries of P. juglandis. Inoculation experiments were conducted to determine the effect of nematodes on the area of subdermal necrotic lesions (cankers) caused by G. morbida in branches of mature J. nigra and stems of seedlings. The phoretic nematode Bursaphlenhus juglandis (Ryss, Parker, Alvarez‐Ortega, Nadeler & Subbotin) was frequently found under elytra of WTB in all locations, and a free‐living nematode (Panagrolaimus sp.) was also widespread and found in the bark of mature trees. Both B. juglandis and Panagrolaimus sp. reduced the size of cankers caused by G. morbida in seedlings and branches of mature trees, respectively. However, these species may play opposite roles as disease synergists and antagonists based on the observation that exudates and/or microbiota associated with Panagrolaimus sp., but not B. juglandis destroyed G. morbida colonies in culture. Furthermore, B. juglandis contributed to foliar symptoms in seedlings inoculated with G. morbida. An entomoparasitic nematode (Aphelenchoididae), most closely resembling an Ektaphelenchus sp., was also found in the haemocoel of WTB. Infection rates were positively related to beetle population sizes as inferred from emergence rates. Ditylenchus sp. was also found in incubated walnut wood in WA and Rhabtidolaimus sp. was phoretic on P. juglandis and found in incubated walnut wood in WA and ID. The community of nematodes in J. nigra in WA and ID differed substantially from what has been observed associated with J. nigra in its native range.

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.

First report of thousand cankers disease caused by the fungus Geosmithia morbida and its vector Pityophthorus juglandis on Juglans regia in France

Thousand cankers disease (TCD) of walnut trees is a disease complex involving the ascomycete fungus Geosmithia morbida and its insect vector, the walnut twig beetle Pityophthorus juglandis. The disease is named after the large number of cankers that appear on the tree, caused by the wounds made by P. juglandis when it feeds and builds galleries under the bark and the parasitic behaviour of the fungus growing in the tissue surrounding the galleries. Several Juglans species may be affected. High mortality of walnut trees caused by TCD has been observed in the USA since the mid-1990's, mainly on Juglans nigra planted in urban areas. In 2013, TCD was reported for the first time in Italy, in the region of Veneto, on a small number of Juglans nigra of different ages (Montecchio & Faccoli, 2014). As part of a national survey organised by the French plant protection organization, pheromone traps for P. juglandis have been installed in some urban areas where Juglans spp. are present. In August and September 2022, scolytids were trapped in parks in the cities of Bron and Lyon (region of Auvergne-Rhône-Alpes, 45°43'17.6“N 4°54'00.3”E, and 45°46'33.7“N 4°51'09.0”E respectively), and sent to the laboratory for identification. The morphological characters matched the features of Pityophthorus juglandis according to Bright (1981) (Fig. 1) and the identity was confirmed by sequence analysis of the mitochondrial cytochrome oxidase I gene. The barcode region was amplified using the primer cocktail from Germain et al. (2013). The 658-bp long amplicon was sequenced (GenBank Accession No. OP975730) and BLAST analysis showed 100% identity with P. juglandis (KF725084), confirming identification. Following this finding, an active survey was triggered on walnut trees around these traps, looking for symptoms of TCD. Foliar wilt, twig and branch diebacks, small brown bark cankers on the branch and trunk (Fig. 2), sometimes with a cracked appearance (Fig. 3), as well as exit holes were observed on several Juglans regia trees of different ages. Removal of the bark around the cankers disclosed a brown discolouration of the phloem and the presence of bark beetle galleries (Fig. 4) where some P. juglandis were collected. Sections of branch or bark showing cankers were sampled and sent to the laboratory for analysis. After surface sterilisation with 70% ethanol (v/v), pieces of diseased tissues around the necrotic margins of the cankers were cut and plated onto malt extract agar (MEA) medium supplemented with 100 ppm chloramphenicol, and incubated at 22°C in the dark. After five to seven days, whitish to beige and flat-growing colonies were observed. After subculturing on MEA in the same conditions, the culture was slow growing, lobate, plane, white to yellowish (Fig. 5). All the features observed matched the description of Geosmithia morbida in culture made by Kolařic et al. (2011), such as the roughened penicillate conidiophores bearing narrowly cylindrical to ellipsoid conidia, 4–5 μm long, sometimes organized in persistent chains. Total DNA was extracted from two cultures and tested positive in real-time PCR with the G. morbida-specific assay developed by Rizzo et al. (2020). In addition, the rDNA internal transcribed spacer region was amplified for the two cultures using ITS5 and ITS4 primers. The 584-bp long amplicon was sequenced (OP930845 and OP930846) and BLAST analysis showed 100% identity with G. morbida (FN434082). Since this first finding, the presence of both the insect and fungus was also confirmed on J. nigra in the same area. An intensive survey is underway to determine the extent of the disease. To our knowledge, this is the second report of P. juglandis and G. morbida in Europe and the first in France, where eradication measures have been put in place. Their potential spread may endanger the existence of walnut trees and the associated economy in the whole continent. The mycology research unit of the ANSES Plant Health Laboratory (LSV) is supported by a grant managed by the French National Research Agency (ANR) as part of the French government's “Investing for the Future” (PIA) programme (ANR-11-LABX-0002-01, Laboratory of Excellence-ARBRE). We would like to thank Massimo Faccoli (University of Padua) for exchanges on Pityphthorus juglandis identity and the situation of thousand cankers disease in Europe.

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolařik) 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.

Differential Virulence Among Geosmithia morbida Isolates Collected Across the United States Occurrence Range of Thousand Cankers Disease

Thousand cankers disease (TCD), first documented in the western United States in the early 2000s, has spread into nine western and seven eastern states in the United States and northern Italy. TCD incidence and severity differ between eastern and western United States outbreak localities. Black walnut (Juglans nigra) trees, introduced into both urban and plantation settings in the western United States, have been severely impacted as evident by the documented high disease incidence and mortality. However, in eastern United States localities, where J. nigra is native, host-pathogen-vector interactions resulted in two different outcomes: trees either die or partly recover followed by infection. Recent genetic studies on the TCD causal agent, Geosmithia morbida, indicate the spatial genetic structure and high levels of genetic diversity among United States populations. Using detached branch inoculation assays, we reported differential virulence among 25 G. morbida isolates collected across the current distribution range of the disease. As a proxy for virulence, the canker area was measured to 7 days after inoculation. Varying degrees of virulence were observed among tested G. morbida isolates, which was partly explained by their genetic provenance (genetic clusters). Isolates that grouped within genetic cluster 2 (n = 7 from the eastern United States and n = 6 from the western United States; mean = 210.34 mm2) induced significantly larger cankers than isolates that grouped within genetic cluster 1 (n = 12; all western United States isolates; mean = 153.76 mm2). Canker sizes varied among isolates within each genetic cluster and were not correlated with a geographic region (eastern vs. western United States) but rather to the isolated state of origin. Mean canker size also differed in response to isolates that originated from different tree host species. G. morbida isolates collected from Juglans major induced statistically smaller cankers when compared to isolates recovered from undetermined Juglans species but not from J. nigra. In sum, the increased mortality reported for western United States walnut tree populations cannot be explained by a higher virulence of local G. morbida. Plausible explanations for the observed disparity include environmental conditions, such as prolonged drought, greater population densities of walnut twig beetle causing a higher number of inoculation events to individual trees, and multiple introductions of G. morbida originating from multiple locations. Future experimental evaluation should be undertaken to quantify the influence of these factors on the local epidemics.

Managing Thousand Cankers Disease in High-value Plantings of Black Walnut (Fagales: Juglandaceae) in Washington State

Abstract The health and productivity of black walnut (Juglans nigra L.) is currently threatened by Thousand Cankers Disease (TCD), a pest complex comprised of an insect vector, the walnut twig beetle (WTB) (Pityophthorus juglandis Blackman) (Coleoptera: Curculionidae), and associated pathogenic fungi (Geosmithia morbida Kolařík, Freeland, Utley, and Tisserat) (Hypocreales: Bionectriaceae). There is an urgent need for tools to manage TCD and avoid catastrophic losses in high-value plantations of black walnut. In this case study, we evaluated the efficacy of IPM strategies in three TCD-infested black walnut plantations in Walla Walla, WA. At the beginning of our study, average weekly captures of WTB per trap varied across sites; low (~32), moderate (~111), and high (>1,300). Each site received a separate management tactic: 1) girdled trap trees alone; 2) the insecticide emamectin benzoate alone or in combination with the fungicide propiconazole to protect healthy crop trees with superior form; and 3) the combination of girdled trap trees and chemically-protected crop trees. Walnut twig beetle populations remained at consistently low levels at both plantations after girdled trap trees were deployed. In plantations where crop trees were treated, the crown condition of those that received emamectin benzoate alone matched or exceeded that of trees treated with the combination of emamectin benzoate and propiconazole and the untreated control trees. Our findings suggest that using both trap trees and emamectin benzoate in an integrated approach has the most potential to reduce WTB populations and protect valuable crop trees in black walnut stands threatened by TCD.

Evaluation of Thousand Cankers Disease-Symptomatic Juglans nigra in Southwestern Ohio Following Population Collapse of Pityophthorus juglandis

Mass attacks of Juglans nigra by Pityophthorus juglandis and its introduction of the canker pathogen Geosmithia morbida lead to branch dieback and, commonly, tree death in locations with outbreaks of thousand cankers disease (TCD). Following initial detection of TCD in Butler County, Ohio, in 2012, and trapping of thousands of P. juglandis, a dramatic decrease in insect population occurred. Dissections of “lingering” TCD-symptomatic J. nigra in 2014 and 2015 were conducted to document the presence of “typical” Geosmithia cankers and other types of damage on branches and main stems. Isolation and subsequent molecular identification of G. morbida and other selected fungi were attempted from representatives of the various canker-like and insect-associated damage observed. Of the fungi obtained, G. morbida was most commonly isolated, primarily from “typical” Geosmithia cankers, but it was also obtained at lower frequencies from other damage types. In addition, G. morbida commonly co-occurred with Fusarium solani ( F. solani species complex) but infrequently with Diplodia seriata. These other fungal species could contribute to the severity of canker development. These results suggest that the TCD pathogen persists in the landscape following population collapse of the primary insect vector and would be of interest to state regulatory agencies in the eastern United States.

Conventional Gel Electrophoresis and TaqMan Probes Enable Rapid Confirmation of Thousand Cankers Disease from Diagnostic Samples.

Thousand cankers disease (TCD) is caused by the fungal pathogen Geosmithia morbida and vectored by the walnut twig beetle Pityophthorus juglandis. In infected walnut and butternut (Juglans spp.) hosts and wingnut species (Pterocarya spp.) hosts, tree decline and death results in ecological disruption and economic losses. A rapid molecular detection protocol for TCD using microsatellite markers can confirm the presence of insect vector or fungal pathogen DNA, but it requires specialized expensive equipment and technical expertise. Using four different experimental approaches, capillary and conventional gel electrophoresis, and traditional polymerase chain reaction (PCR) and quantitative PCR (qPCR), we describe simplified and inexpensive processes for diagnostic confirmation of TCD. The improved and rapid detection protocols reported in this study reduce time and equipment costs associated with detection of molecular pest and pathogen DNA by (1) using conventional gel electrophoresis or TaqMan molecular probes to elucidate the detection limits for G. morbida and P. juglandis DNA and (2) identifying resources that allow visualization of positive test results for infected host plant tissue samples. Conventional gel electrophoresis and TaqMan molecular probe protocols detected presence of DNA from TCD-associated fungal and insect samples. These procedural improvements can be readily adopted by diagnostic end-users and adapted for use with other complex disease systems to enable rapid pest and pathogen detection.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Competitive Advantage of Geosmithia morbida in Low-Moisture Wood May Explain Historical Outbreaks of Thousand Cankers Disease and Predict the Future Fate of Juglans nigra Within Its Native Range

Bark beetles vector symbiotic fungi and the success of these mutualisms may be limited by competition from other microbes. The outcome of fungal competition is strongly influenced by the physical and chemical conditions of the wood they inhabit. These conditions are in turn subject to climatic variation. In particular, wood moisture content (MC) influences fungal competition and, therefore, could help determine environmental suitability for thousand cankers disease (TCD) caused by Geosmithia morbida and its vector Pityophthorus juglandis. We conducted competition experiments in Juglans nigra wood that was naturally or artificially colonized by G. morbida and other fungi over a range of wood MC expected across prevailing United States climatic conditions. G. morbida outcompeted antagonistic fungi Clonostachys and Trichoderma spp. at <5% equilibrium moisture content. Aspergillus spp. outcompeted G. morbida at low moisture in wood from Indiana. We fit a logistic regression model to results of the competition experiments to predict survival of G. morbida across the United States. Expected survival of G. morbida was highest in historical TCD epicenters and accounted for the low incidence and severity of TCD in the eastern United States. Our results also predict that under future climate scenarios, the area impacted by TCD will expand into the native range of J. nigra. Given its role in emergent forest health threats, climate change should be a key consideration in the assessment of risks to hardwood resources.

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.

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.

Eastern Black Walnut (Juglans nigra L.) Originating From Native Range Varies in Their Response to Inoculation With Geosmithia morbida

Thousand cankers disease (TCD) is caused by the walnut twig beetle (Pityophthorus juglandis) vectoring the fungal canker pathogen Geosmithia morbida, which can result in severe dieback and eventual death to species of walnut (Juglans spp.) and wingnut (Pterocarya spp.). This disease is most devastating to the highly valued species J. nigra (black walnut). This species is primarily grown and harvested for timber production in the Central Hardwood Region of the United States, which comprises part of its native range. Management options for TCD are limited; therefore, finding resistant genotypes is needed. Initial studies on black walnut susceptibility to G. morbida documented some genetic variation and suggested potential resistance. Furthermore, G. morbida is thought to be native to the United States, which may have allowed for co-evolution. To capture the representative genetic diversity and screen for resistance to G. morbida, J. nigra families were collected from across the native range. These wild trees, in conjunction with seedlings developed in a black walnut timber improvement program, were planted in a common garden in Fort Collins, Colorado and repeatedly inoculated with G. morbida over the course of four years and three growing seasons. Improved seedlings exhibited larger cankered areas than wild J. nigra of the same provenance. Cankers induced by G. morbida in wild germplasm were smaller on J. nigra collected from the western and central portions of the native range compared to those collected from the eastern portion. Although trees from the western and central part of the range still incurred cankers, our findings indicate that variation in genetic resistance to G. morbida is present in black walnut. This study was performed with G. morbida independent of the walnut twig beetle, but our results suggest the limited G. morbida resistance observed in J. nigra will prevent the full compromise of black walnut to TCD. Results from this study should be taken into consideration in future black walnut breeding programs.

Vacuum Steam Treatment Effectiveness for Eradication of the Thousand Cankers Disease Vector and Pathogen in Logs From Diseased Walnut Trees

Logs of high-value eastern black walnut (Juglans nigra L.) are commonly exported from the United States for production of veneer and lumber. Veneer logs are not debarked to minimize degradation of wood quality and reduce moisture loss. Thousand cankers disease (TCD) is caused by the walnut twig beetle (Pityophthorus juglandis Blackman) and the fungal pathogen, Geosmithia morbida M. Kolarik, E. Freeland, C. Utley and N. Tisserat sp. nov., which colonize the inner bark of Juglans species. Effective eradication of these organisms by heat or chemical fumigation treatment is required for walnut logs prior to export. Because vacuum steam is an effective and efficient means of heating round wood, its use in eliminating the TCD causal agents was evaluated using Juglans logs (12- to 44-cm small end diameter and 1.7- to 1.9-m length) from TCD-symptomatic trees in Oregon and Washington State. Five replicate trials with three logs per load were conducted in a portable vacuum chamber to test two treatment schedules: 60°C for 60 min and 56°C for 30 min. Complete elimination of P. juglandis and G. morbida was achieved when using a minimum of 56°C at 5-cm targeted depth from bottom of bark furrow into the sapwood and held for 30 min. Treatment cycle time ranged from 298 to 576 min depending on log diameter and initial log temperature. Artificial inoculation of J. nigra trees with G. morbida within the TCD range in Pennsylvania was minimally successful in producing adequately colonized logs for experimental trials.

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.

Trap Assays of the Walnut Twig Beetle, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae: Scolytinae), Reveal an Effective Semiochemical Repellent Combination.

Thousand cankers disease (TCD), is an invasive insect-disease complex caused by the walnut twig beetle, Pityophthorus juglandis, and fungal pathogen, Geosmithia morbida. Semiochemical interruption is a viable option for protecting walnut trees from P. juglandis attack. The goal of this study was to test beetle responses to potential repellent compounds. The results of five, flight-intercept assays are reported. Assays 1-3 tested four compounds at variable release rates: (S)-(-)-verbenone, (R)-(+)-verbenone, racemic chalcogran, and racemic trans-conophthorin. Trapping results indicated that the highest release rate tested for each compound was the most effective in reducing the number of beetles caught. (S)-(-)-Verbenone was the least effective, reducing P. juglandis trap catches by 66%. (R)-(+)-Verbenone reduced the number of P. juglandis by 84%. Neither enantiomer of verbenone performed as well as chalcogran or trans-conophthorin, which both reduced the number of beetles caught by ca. 98%. Following individual assays, the most effective compounds were tested in subtractive-combination assays. Combinations ofhigh release rates for (R)-(+)-verbenone, trans-conophthorin, and two stereoisomers of limonene (tested in a previous study) were tested in two assays. The subtractive-combination assays were inconclusive in that trap catches were similar across all treatments. All combination treatments were highly effective, achieving approximately 99% reduction in the number of beetles caught. Based on the trapping results, commercial availability, and cost of the semiochemicals tested, we conclude that a combination of (R)-(+)-limonene, trans-conophthorin, and (R)-(+)-verbenone constitutes an effective tool for reducing P. juglandis trap catches.

Walnut twig beetle landing rates differ between host and nonhost hardwood trees under the influence of aggregation pheromone in a northern California riparian forest

Abstract Host selection behaviour of the walnut twig beetle (WTB) among hardwood trees was investigated in a riparian forest in northern California by monitoring the landing rate of the beetle with sticky traps on branches baited with 3‐methyl‐2‐buten‐1‐ol, the male‐produced aggregation pheromone. The assay was conducted over 7 days (22 May to 29 May 2017) and compared landing rates on branches of six nonhost species paired with northern California black walnut, Juglans hindsii (the host). A total of 2242/1192 WTB were collected on branches of host/nonhost pairs, and more WTB landed on J. hindsii than on nonhosts in 42 of 58 instances. Female landing rate generally exceeded male landing rate, which underscores the influence of the male‐produced synthetic pheromone in this system. Landing rates of WTB males, females, and the combined sexes on boxelder, Acer negundo, and valley oak, Quercus lobata, did not differ significantly from the landing rates on J. hindsii, suggesting that these two nonhost riparian hardwoods do not repel WTB (in the context of the aggregation pheromone). Significantly fewer WTB landed on Oregon ash, Fraxinus latifolia, river red gum, Eucalyptus camaldulensis, Fremont cottonwood, Populus fremontii, and red willow, Salix laevigata, than on J. hindsii, which suggests that these four nonhosts may repel one or both sexes of WTB in the context of the aggregation pheromone. Future analysis of the volatiles from these four hardwood species may lead to the discovery of semiochemical repellents for WTB.

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.