Research Project: Integrated Approach to Manage the Pest Complex on Temperate Tree Fruits

Location: Temperate Tree Fruit and Vegetable Research

2023 Annual Report

Objectives
The long-term objective of this project is to provide the basic and applied information needed for the development and transfer of safe and environmentally sustainable tools or methods for management of arthropod pests of temperate tree fruits. Specifically, during the next five years we will focus on the following objectives: Objective 1: Develop and improve tools and approaches for early detection, prediction, and monitoring of arthropod pests and their natural enemies in temperate tree fruits. • Sub-objective 1A: Determine if pre-harvest commercial pear is an acceptable host for apple maggot fly. • Sub-objective 1B: Identify volatile attractants from pear trees for early detection of orchard-colonizing winterform pear psylla. • Sub-objective 1C: Identify the primary non-orchard habitats that are sources of orchard-colonizing winterform pear psylla. • Sub-objective 1D: Identify the thermal tolerances of brown marmorated stinkbug and Trissolcus japonicus to more accurately determine potential establishment and spread. • Sub-objective 1E: Identify non-crop plants that are sources of orchard-colonizing vectors of the X-disease Phytoplasma. Objective 2: Provide basic and applied information of the physiology, biochemistry, behavior, and landscape ecology of arthropod pests and their natural enemies necessary to develop new or improve existing integrated pest management strategies in temperate tree fruits. • Sub-objective 2A: Determine the thermal kinetics of apple maggot. • Sub-objective 2B: Determine functions of proteins key to codling moth reproduction, development, physiology, and behavior. • Sub-objective 2C: Determine extent of cryptic species diversity in minute pirate bug fauna of the western U.S. in both orchard and non-orchard habitats. • Sub-objective 2D: Examine non-target effects of herbicides on key natural enemies of arthropod pests of apple and pear. • Sub-objective 2E: Identify genes and proteins that are key to acquisition and transmission of X-disease Phytoplasma by leafhopper vectors. Sub-objective 2F: Identify plant-based volatile attractants or repellents of leafhopper vectors of X-disease Objective 3: Develop new or improved integrated management strategies to control arthropod pests in temperate tree fruits. • Sub-objective 3A: Improve control strategies for apple maggot in organic waste. • Sub-objective 3B: Develop pest management strategies for cherry fruit fly that incorporate newer and less toxic insecticides. • Sub-objective 3C: Model the relationship between estimated populations of Trechnites adults and rates of parasitism of pear psylla. • Sub-objective 3D: Develop management practices that increase the retention time of released natural enemies of arthropod pests of apple. • Sub-objective 3E: Develop new or improved management tools to control pathogens and vectors of little cherry disease.

Approach
Objective 1. Sub-objective 1A: Behavioral and development bioassays will determine if pear is an acceptable host for apple maggot and whether pear fruit is a potential source for apple maggot introduction in export countries. Sub-objective 1B: Volatiles will be identified from pear trees using GC-MS, and electroantennogram, behavior assays, and field trials will determine whether isolated volatiles elicit responses from pear psylla. The resulting lure will be used to monitor pear psylla populations. Sub-objective 1C: Gut content analysis will be used to where pear psylla overwinter. Sub-objective 1D: Differential scanning calorimetry will be used to determine the thermal capacity of brown marmorated stink bug and its parasitoid, and to identify climates that are favorable to their establishment and spread. Objective 2. Sub-objective 2A: The thermal death kinetics of apple maggot will be determined using differential scanning calorimetry and micro-respiration. Results will be used to improve management of apple maggot in commercial composting facilities located in apple maggot free zones. Sub-objective 2B: Candidate codling moth genes will be silenced using CRISP-Cas9 and behavior assays will be used to identify the function of the silenced genes. Results will identify viable gene-targets for use in codling moth control. Sub-objective 2C: Morphology of Orius will be examined to identify diagnostic traits and to possible new species, and a diagnostic key will be developed to identify species belonging to the group of important natural enemies. Sub-objective 2D: Standard laboratory bioassays will be used to examine the effects of herbicides and additives on mortality and fecundity of the key orchard predators Galendromus occidentalis, Amblydromella caudiglans, and the European earwig. Results of this study will allow growers to adapt orchard floor management strategies that conserve key natural enemies. Objective 3. Sub-objective 3A: Laboratory experiments will be conducted to determine whether 100% of apple maggot flies are killed by exposure to 55 degrees, the temperature found in commercial composting operations. Sub-objective 3B: Standard bioassays will be used to determine whether the insecticides tolfenpyrad, pyrifluquinazon, azadirachtin, and pyrethrins kill or reduce fecundity of cherry fruit fly and can be used as an alternative to spinosad as an attract and kill approach to fruit fly management. Sub-objective 3C: Adult populations of Trechnites will be monitored using traps and psylla parasitism rates will be monitored by dissection of psylla or by PCR and the relationship between adult populations of Trechnites and actual parasitism rates will be modeled to allow growers to incorporate biological control provided by Trechnites in psylla management practices. Sub-objective 3D: Field studies will be conducted in commercial apple orchards to test whether nutritional supplements including Typha spp. pollen or brine shrimp cysts increase the retention of released natural enemies, Orius insidiosus, Chrysoperla rufilabris, or Cryptolaemus montrouzieri. Results will improve augmentative biological control in commercial orchards.

Progress Report
Substantial progress was made on all three objectives with the overall goal to develop biorational management of insect pests of temperate tree fruits. The goal of Objective 1, and its five sub-objectives, is to develop or improve tools and approaches for early detection, prediction, or monitoring of tree fruit pests with a focus on apple maggot, pear psylla, brown marmorated stink bug, and leafhopper vectors that transmit x-disease. Sub-objective 1A was previously completed. In support of Sub-objective 1B, volatiles were collected from pear tree cuttings, and individual compounds were identified from extracted volatiles using standard methods. A series of behavioral experiments were performed to determine whether pear psylla use plant volatiles to locate their pear hosts. Field experiments were conducted to determine whether winterform pear psylla could be trapped using abundant volatiles identified from different pear phenological stages. For Sub-objective 1D, research on thermal tolerance limits for brown marmorated stinkbug has been completed. Data on lower, optimal, and upper thermal limits will be used to recalculate ecological niche models and compare to previous models and current distribution. Collaborative research was initiated with scientists at Oregon State University in Hood River to obtain and rear Trissolcus japonicus for experiments on thermal tolerance limits in this species. Expansion of the current brown marmorated stinkbug colony is underway to support the requirements needed for production of T. japonicus. To achieve the aim of Sub-objective 1E, molecular gut content analysis was performed on leafhopper vectors of x-disease phytoplasma collected from diseased orchards throughout Washington State. Results showed that leafhoppers that fed upon dandelion and mallow were very likely to carry phytoplasma, indicating that presence of these common weeds in orchards increases risk of x-disease. Progress was also made on several subordinate projects related to Objective 1. Compounds previously identified from the venom reservoir of the invasive Northern Giant Hornet and their potential food sources were used to make lures to test for trapping hornets in Washington state, Japan, and South Korea. A useful pheromone lure was identified through preliminary field experiments in Japan. Field trials will continue to aid in the development of an effective trapping method that can help with the local eradication of Northern Giant Hornet in Washington State. In another subordinate project, field experiments were conducted to test the effect of trap modifications and trap type on the captures of brown marmorated stink bug in and around orchards. Data collected will provide insight into the best trap for adults and nymphs. The goal of Objective 2 and its six sub-objectives is to provide information on the basic biology of tree fruit pests and their natural enemies. The first component of Sub-objective 2A has been completed, and results identified the lower, optimal, and upper thermal limits of diapausing apple maggot pupae in relation to chill duration directly after removal from the cold. Additional experiments are underway, studying the effects of chilling duration on thermotolerance of the apple maggot as pupae complete diapause development. In support of Sub-Objective 2B, gene expression studies have been completed for odorant and taste receptor gene families in codling moth female abdomen tip. These findings will provide guidance on functional assays of specific receptor proteins. Additionally, transcriptomes have been sequenced for codling moth larvae to identify genes expressed in different larval stages. Analyses of these transcriptomes will identify genes that may be targeted to disrupt larval development. For Sub-objective 2C, the first draft of a dichotomous key to the minute pirate bug fauna (Orius spp.) written last year was updated as additional specimens of insects were collected, examined, and measured to verify that the key correctly identifies the specimens. Pertaining to Sub-objective 2D, assays measuring off-target effects of herbicides on earwigs were completed, with seven herbicides tested. Mortality, effects on movement, and predatory efficacy were examined. Data are currently being analyzed. This line of research will provide growers with knowledge to select pesticide spray practices that best conserve beneficial arthropods. For Sub-objective 2F, field experiments were conducted in cherry orchards to test a potential trap and lure that can be used for leafhoppers that transmit the x-disease pathogen; field tests on the other trap options are ongoing. Research activities were undertaken on several subordinate projects related to Objective 2. In one study, collaboration with scientists at Washington State University produced the first description of the acoustic duet between male and female pear psylla, which showed that pitch of the acoustic calls differed between seasonal forms of the insect and varied with ambient temperature. In another subordinate project, research has been initiated on novel virus formulations that are effective as biocontrol agents against codling moth larvae. The Cryptophlebia peltastica Nucleopolyhedrovirus (CrpeNPV) was recently discovered in false codling moth and reported to be effective against codling moth. Basic lethality rates have been established in our lab for CrpeNPV, compared to Cydia pomonella granulovirus (CpGV) that is commercially used against codling moth. Next, CrpeNPV formulations, with or without CpGV, will be tested against codling moth populations that are known to be resistant to the CpGV. Subordinate to Sub-objective 2D, non-target effects of common orchard insecticides and fungicides were tested on earwigs and the key spider mite predator, Stethorus. For the latter, both diapausing and non-diapausing individuals were tested concerning mortality, movement, and predatory efficacy. For a subordinate project of Sub-objective 2E, genomes have been sequenced for the two most prominent leafhopper species that transmit the x-disease phytoplasma pathogen in Washington State. Analysis of these genomes will facilitate identification of genes and proteins that are involved in the acquisition and transmission of the X-disease pathogen. The goal of Objective 3 and its five sub-objectives is to develop new or improved integrated pest management strategies to control arthropod pests in temperate tree fruits. Sub-objective 3A was previously completed. In support of Sub-objective 3B, further laboratory testing of organic pyrethrin insecticides mixed with a hydrophobic, organic coating was conducted to determine their efficacy in killing and preventing oviposition by western cherry fruit fly. The mix of the two materials eliminated fly oviposition in cherries in laboratory trials. Field trials are underway. Pertaining to Sub-objective 3C, artificial intelligence/machine learning was used to model and predict parasitism rates of pear psylla by the parasitoid Trechnites insidious using trap counts of adults of both species. Pear psylla degree days and pear psylla counts were the most important factors and “pear growing region” was minimally important, indicating that the model is likely broadly generalizable. Data from commercial orchards is now being collected for ground truthing the model. For Sub-objective 3D, field studies were conducted to study the impact lures combined with food on biocontrol. Collection of year 2 data is underway. The experiment is being conducted in three orchards: commercial apple, commercial pear, and research apple. Analysis of year 1 data indicated that lures, but not food supplements, increased abundance of some orchard natural enemies in apples. The releases in pears did not reduce pear psylla abundance. In all trials, pest thrips were reduced after releases. Data collected last year indicates that mealybug destroyers are not effective at mealybug control in apple orchards. Plots have been established in a commercial apple orchard this year to determine if released convergent ladybeetle is a more effective predator. In support of Sub-objective 3E, a field study was initiated to evaluate the role of cherry root suckers in spread of x-disease. The extent of cherry feeding will be compared using molecular gut content analysis between leafhopper nymphs captured in cherry plots with or without cherry suckers. Results will be used to develop horticultural practices, namely mechanical removal of suckers, to reduce risk of x-disease. Progress was also made on subordinate projects related to Objective 3. Subordinate to Sub-objective 3C, work is ongoing to describe the population genetics of T. insidiosus with comparisons made across regions and in relation to historical specimens from four sites in Asian/Europe and from central Washington. Present-day samples have been collected across the United States. Collaborators in England and Italy, are currently trapping to obtain present-day European samples. An isofemale line of T. insidiosus is also being established to conduct de novo genome assembly. Subordinate to Sub-objective 3D, three trials have been conducted comparing methods for releasing lacewings for aphid control. Results were mixed, with some trials showing reduced aphids, but others showing no reduction of aphids compared to the no-release control. Furthermore, data analysis was completed on a project examining the use of cardboard traps for moving earwigs from stone fruit orchards, where they are pests, into pome fruit orchards, where they reduce pest populations. It was found that the traps could be used to establish earwigs in areas where they were previously absent and moderate reductions in pest levels was observed after 1-2 years. Data is now being collected to determine if earwig releases alter orchard natural enemy communities and to describe the diet of collected earwigs using molecular gut content analysis.

Accomplishments
1. Comprehensive description of herbicide non-target effects on key orchard predators. Minimizing use of pesticides that harm natural enemies is a key component of conservation biological control in orchards. While the effects of insecticides on orchard natural enemies are fairly well-known, there was virtually no information on herbicide non-target effects. ARS researchers in Wapato, Washington, and scientists at Washington State University, characterized the effects of seven herbicides on predators, examining mortality, repellency, fecundity, movement, and predator efficacy on three species of spiders, two species of predatory mites, convergent ladybeetle, green lacewings, minute pirate bugs, and earwigs. Glufosinate, paraquat, and oxyfluorfen were consistently the most harmful to these predators. This information is being used by orchard consultants to improve spray programs to reduce harm to natural enemies and disruption of biocontrol.

2. Upper thermal limits of Rhagoletis indifferens (Diptera: Tephritidae) pupae and pteromalid parasitoids (Hymenoptera: Pteromalidae) inside fly puparia. Determining upper thermal limits of fruit fly pupae can have practical implications for disinfesting soils as well as for predicting differential impacts of global warming on flies and their parasites. ARS researchers in Wapato, Washington, determined the upper thermal limits of western cherry fruit fly pupae and a wasp parasitoid inside fly puparia. They found that all fly pupae were killed at 49.4 degrees C and that the upper thermal limit of survival for fly pupae was 47.8 degrees C while for wasps it was 51.1 degrees C. Information on heat kill of soil insects will be used by Washington State Department of Agriculture for developing guidelines to disinfest soils and organic waste of pest insects, to reduce chances of soils spreading the pests and threatening orchardists.

3. Mortality of western cherry fruit fly (Diptera: Tephritidae) eggs and larvae in cherries submerged in hypoxic water. Western cherry fruit fly is a quarantine pest of cherry in western North America. Determining the water tolerance of its maggots in cherries can help us understand fly adaptations to stressful environments and have practical value for sanitizing orchards. ARS researchers in Wapato, Washington, and APHIS researchers in Miami, Florida, determined the effects of submerging infested cherries in water for 4, 8, and 12 days on kill of fly eggs and maggots in the cherries. They observed that 8- and 12-day water submersions killed 100% of eggs and maggots. Results are being used as a basis for recommendations by APHIS for orchard sanitation options for managing fruit flies.

4. Bacterial endosymbionts of leafhopper pests. Many insects harbor bacteria called symbionts that provide them with nutrition, or with protection from natural enemies, insecticides, and plant defenses. It remains largely unknown how these bacterial symbionts alter the ability of for insect vectors to spread crop pathogens. ARS researchers in Wapato, Washington, and Kimberly, Idaho, and scientists from Washington State University, identified symbionts from leafhopper vectors of phytoplasma pathogens that cause plant diseases including cherry x-disease (aka, little cherry disease). They discovered several previously unknown symbionts that may be beneficial to the vectors and uncovered new interactions between symbionts and the phytoplasma pathogens. Notably, the presence of the symbiont Wolbachia was associated with a higher likelihood that leafhoppers also carried Phytoplasma. This information will be used by scientists to conduct more in-depth research on leafhopper symbionts and how they influence biology and management of leafhopper vectors of cherry x-disease.

5. Use of eDNA technology to find invasive species in green yard waste. As large cities begin to overrun their landfill capacities, they begin to look for alternative locations to handle the waste stream. Seeing an opportunity to bring in revenue, rural communities offer to handle municipal waste in their landfills. Unfortunately, many rural communities are also locations of agricultural production, which are vulnerable to attacks by invasive species. ARS researchers in Wapato, Washington, used a combination of ecological niche modeling and eDNA sequencing to determine whether green yard waste could be a pathway for invasive species to enter and establish in the landfill-receiving agricultural community. They detected, in the green yard waste, DNA from several quarantine-actionable pests that pose a threat to agriculture in Washington State. Reports of research results and positive identifications of potential invasive species were made to federal and state regulatory agencies to aid in risk management decision making as it relates to the potential for invasive species to be transported through municipal waste relocation programs.

6. Abundances, ecologies, and distributions of Rhagoletis flies (Diptera: Tephritidae) in central Washington inferred from fly trapping surveys. Pest and non-pest fruit flies trapped in surveys can provide new information on fly abundances in a region. ARS researchers in Wapato, Washington, and scientists at the Washington State Department of Agriculture in Yakima, Washington, analyzed data from surveys for pest cherry fruit flies and apple maggot flies in central Washington to determine relative abundances of fly species on traps and factors related to their abundances. They found that cherry fruit flies were common, but that apple maggot flies were very rare in central Washington. Walnut husk fly was the most abundant fly species. Abundance data suggests that apple maggot is less tolerant of arid central Washington summers than other fruit flies and that relative fly abundances depend on site, tree type, and seasonal period. Results can be used by tree fruit industry representatives and APHIS to show that pest apple maggot flies are unlikely to develop large populations in apple-growing regions, thus affecting Washington State Department of Agriculture guidelines on pest level tolerance, designation of areas as low pest prevalence, how the pest should be managed, and APHIS negotiations with export markets on movement of apples from these regions.

7. Evidence for adaptation of Rhagoletis pomonella (Diptera: Tephritidae) on large-thorn hawthorn, Crataegus macracantha, in Okanogan County, Washington. Apple maggot fly is a quarantine pest of apple in western North America. Determining whether a fly population has adapted to non-apple host plants can affect fly management, in that managing the fly in apple alone would be insufficient to reduce the threat of flies attacking apples. ARS researchers in Wapato, Washington and scientists at the Washington State Department of Agriculture in Yakima, Washington, and the University of Notre Dame in South Bend, Indiana, determined whether populations of flies attacking large-thorn hawthorn in north-central Washington have adapted to the plant. They found evidence that the flies have adapted to large-thorn hawthorn, as flies from large-thorn hawthorn emerged earlier than flies attacking apple and the flies preferred to attack and lay eggs in the hawthorn over apple. Discovery that this fly population has established in hawthorns has resulted in new quarantine boundaries around affected orchards created by apple industry representatives and the Washington State Department of Agriculture, as well as active control measures by the county pest board targeting the flies, which together will help growers export their apples to foreign markets.

8. First reported infestation of a native honeysuckle by a native Rhagoletis Fly (Diptera: Tephritidae) in North America. Host plant shifts have been hypothesized to be an important factor in initiating population divergence and speciation in fruit flies in the genus Rhagoletis. The apple maggot fly is a model organism for host race formation via host shifts, but its sibling species snowberry maggot fly has not been implicated as having shifted host plants since its evolution from apple maggot ancestors, with its only host being snowberry. ARS researchers in Wapato, Washington, and scientists at the Washington State Department of Agriculture in Yakima and Olympia, Washington, and the University of Notre Dame in South Bend, Indiana, determined that snowberry maggot also infests orange honeysuckle in Washington State. This represents the first reported case of a native honeysuckle infested by a native Rhagoletis in North America. This finding helps the academic and scientific community better understand the evolution of host plant switching by fruit flies and highlights the need of state agricultural officials to be constantly prepared for fly species shifting from wild host plants to economically important plants.

Review Publications
McCullough, C., Grab, H., Angelella, G.M., Karpanty, S., Samtani, J., Olimpi, E., O'Rourke, M. 2022. Diverse landscapes but not wildflower plantings increase marketable crop yield. Agriculture, Ecosystems and Environment. 339. Article 108120. https://doi.org/10.1016/j.agee.2022.108120.
Cattaneo, A.M., Witzgall, P., Kwadha, C., Becher, P.G., Walker III, W.B. 2022. Heterologous expression and functional characterization of Drosophila suzukii OR69a transcript variants unveiled response to kairomones and to a candidate pheromone. Journal of Pest Science. 96:1149-1171. https://doi.org/10.1007/s10340-022-01585-2.
Walker III, W.B., Mori, B.A., Cattaneo, A.M., Gonzalez, F., Witzgall, P., Becher, P. 2022. Comparative transcriptomic assessment of the chemosensory receptor repertoire of Drosophila suzukii adult and larval olfactory organs. Comparative Biochemistry and Physiology, Part D: Genomics and Proteomics. 45. Article 101049. https://doi.org/10.1016/j.cbd.2022.101049.
Cooper, W.R., Walker III, W.B., Angelella, G.M., Swisher Grimm, K.D., Foutz, J.J., Harper, S.J., Nottingham, L.B., Northfield, T.D., Wohleb, C.H., Stausbaugh, C.A. 2023. Bacterial endosymbionts identified from leafhopper (Hemiptera: Cicadellidae) vectors of phytoplasmas. Environmental Entomology. 52(2):243-353. https://doi.org/10.1093/ee/nvad015.
Looney, C., Carman, B., Cena, J., Cichorz, C., Iyer, V., Orr, J., Roueche, N., Salp, K., Serrano, J.M., Udo, L., van Westendorp, P., Wilson, T., Wojahn, R., Spichiger, S. 2023. Detection and description of four Vespa mandarinia (Hymenoptera, Vespidae) nests in western North America. Journal of Hymenoptera Research. 96:1-20. https://doi.org/10.3897/jhr.96.99307.
Harper, S.J., Northfield, T.D., Nottingham, L.R., Dupont, S.T., Thompson, A.A., Sollato, B.V., Serban, C.F., Shires, M.K., Wright, A.A., Catron, K.A., Marshall, A., Molnar, C., Cooper, W.R. 2023. Recovery plan for X-disease in stone fruit caused by 'Candidatus Phytoplasma pruni'. Plant Health Progress. 24(2):258-295. https://doi.org/10.1094/PHP-02-23-0016-RP.
Eigenbrode, S., Adhikari, S., Kistner-Thomas, E., Neven, L.G. 2022. Introduction to the collection: Climate change, insect pests, and beneficial arthropods in production systems. Journal of Economic Entomology. 115(5):1315-1319. https://doi.org/10.1093/jee/toac107.
Neven, L.G., Yee, W.L. 2022. Metabolic reserves of diapausing western cherry fruit fly (Diptera: Tephritidae) pupae in relation to chill duration and post-chill rearing conditions. Frontiers in Insect Science. 2. Article 989673. https://doi.org/10.3389/finsc.2022.989673.
Neven, L.G. 2023. Diapause of the Western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae): Metabolic rate and overwintering adaptations. Environmental Entomology. 52(3):436-445. https://doi.org/10.1093/ee/nvad030.
Beers, E.H., Beal, D., Smytheman, P., Abram, P.K., Schmidt-Jeffris, R.A., Moretti, E.A., Daane, K.M., Looney, C., Lue, C., Buffington, M.L. 2022. First records of adventive populations of the parasitoids Ganaspis brasiliensis and Leptopilina japonica in the United States. Journal of Hymenoptera Research. 91:11-25. https://doi.org/10.3897/jhr.91.82812.
Schmidt-Jeffris, R.A. 2023. Nontarget pesticide impacts on pest natural enemies: Progress and gaps in current knowledge. Current Opinion in Insect Science. 58. Article 101056. https://doi.org/10.1016/j.cois.2023.101056.
Yee, W.L., Milnes, J., Goughnour, R., Bush, M., Hood, G.R., Feder, J. 2023. Evidence for adaptation of Rhagoletis pomonella (Diptera: Tephritidae) on large-thorn hawthorn, Crataegus macracantha, in Okanogan County, Washington State, USA. Environmental Entomology. 52(3):455-464. https://doi.org/10.1093/ee/nvad026.