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.

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.

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,a, and vectored by the walnut twig beetle, Pityophthorus juglandis. In infected walnut and butternut (Juglans 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 PCR and qPCR, we describe simplified and inexpensive processes for diagnostic confirmation of TCD. The improved and rapid detection protocols reported here 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 allows visualization of positive test results for infected host plant tissue samples. Both 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.

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.

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.

Regional Differences in the Structure of Juglans nigra Phytobiome Reflect Geographical Differences in Thousand Cankers Disease Severity

Thousand cankers disease threatens Juglans nigra (Eastern Black Walnut) in urban and natural landscapes. Incidence and severity of thousand cankers disease is higher in the host’s introduced range in the western United States. We hypothesized that these differences are driven partly by geographical variation in the host phytobiome due to its roles in host stress tolerance, nutrient acquisition, and defense. To evaluate the role of the phytobiome in mediating thousand cankers disease, we characterized the J. nigra phytobiome of diseased and healthy trees in portions of its native (Indiana and Tennessee) and introduced (Washington) ranges. Grafted clones present in each state and open-pollinated populations were sampled. DNA was extracted from soil and branch (caulosphere) tissues and internal transcribed spacer and 16s regions were sequenced for characterization of fungal and bacterial communities. We found that microbial communities in the caulosphere and soil differ between native and introduced ranges of J. nigra and harbor different mutualistic and pathogenic microorganisms. Additionally, caulosphere microbial communities were more species rich and diverse in the native range of J. nigra, suggesting greater levels of functional redundancy and multifunctionality in the native-range phytobiome compared with the introduced range. We also found higher network complexity in the caulosphere of trees in the introduced range and evidence for two alternative stable community states associated with diseased and healthy trees. Our results provide support for the hypothesis that geographical variation in thousand cankers disease incidence and severity is partially driven by differences in the phytobiome of J. nigra in its introduced and native ranges. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Assessment of Alternative Candidate Subcortical Insect Vectors From Walnut Crowns in Habitats Quarantined for Thousand Cankers Disease

Thousand cankers disease (TCD) results from the combined activity of the fungal pathogen, Geosmithia morbida Kolařík, Freeland, Utley, and Tisserat and its principle vector, Pityophthorus juglandis (Blackman) (Coleoptera: Curculionidae: Scolytinae) in Juglans L. spp. and Pterocarya Kunth spp. host plants. TCD has been reported from the eastern and western United States. To evaluate potential for other beetle species to vector the fungus in east Tennessee, specimens were collected using ethanol-baited traps that were suspended beneath crowns of TCD-symptomatic trees. Associations of G. morbida with insect species collected in traps were assessed in an unsuccessful, preliminary culture-based fungal assay, and then with a molecular-based detection method. For culture-based assays, rinsate from washed, individual insects was plated on nutrient media and growing colonies were subcultured to obtain axenic G. morbida cultures for identification. For the molecular-based method, G. morbida presence was detected by amplifying the previously developed, species-specific microsatellite locus GS004. Capillary electrophoresis was used to detect the amplified amplicons and representative reactions were validated using Sanger sequencing. Eleven beetle species were found to carry G. morbida, including Cnestus mutilatus (Blandford), Dryoxylon onoharaensum (Murayama), Hylocurus rudis (LeConte), Monarthrum fasciatum (Say), Monarthrum mali (Fitch), Xyleborinus saxesenii (Ratzeburg), Xylosandrus crassiusculus (Motschulsky), Xylosandrus germanus (Blandford) (all Coleoptera: Curculionidae: Scolytinae), Stenomimus pallidus (Boheman) (Coleoptera: Curculionidae: Cossoninae), Oxoplatypus quadridentatus (Olivier) (Coleoptera: Curculionidae: Platypodinae), and Xylops basilaris (Say) (Coleoptera: Bostrichidae). These findings raise concerns that alternative subcortical insect species that already occur within quarantined habitats can sustain incidence of introduced G. morbida and contribute to spread within the native range of black walnut, Juglans nigra L., in the eastern United States.