Research Project: Mitigation of Invasive Pest Threats to U.S. Subtropical Agriculture

Location: Subtropical Horticulture Research

2022 Annual Report

Objectives
1. Identify semiochemicals that mediate the behavior and physiology of exotic insect pests and develop semiochemical-based tools that lead to products for pest detection, behavioral disruption, and surveillance. [NP 304, Component 4, Problem Statement 4A] 1.A. Document behavioral responses and olfactory chemoreception to semiochemicals that have potential application in pest monitoring and control systems. 1.B. Identify and quantify insect semiochemicals; develop improved systems to collect, analyze and identify semiochemicals. 1.C. Develop synthetic and natural product lures based on host location and/or feeding cues that are formulated for effective use in IPM programs. 2. Evaluate ‘next generation’ technologies to advance methods for fruit fly detection and eradication, such as lasers, sonic methods, or nano-technologies. [NP 304, Component 4, Problem Statement 4A] 2.A. Compare available remote trapping systems that can automatically count and identify trapped insects; optimize automated detection systems for adult fruit fly pests. 2.B. Evaluate imaging techniques and non-destructive chemical analysis methods for detection of larval infestation within host fruit. 3. Evaluate essential oils for natural insect pest toxicants and elucidate the physiological mechanisms underlying resistance to conventional pesticides. [NP 304, Component 4, Problem Statement 4A] 3.A. Determine the molecular/physiological basis for development of pesticide resistance in adult fruit flies. 3.B. Identify plant essential oils and plant extracts that have insecticidal, repellent, or oviposition deterrent properties against adult fruit flies and other target pests. 3.C. Determine the specific chemical components responsible for the toxic/repellent effects of essential oils and plant extracts. 4. Integrate tools, technologies and management strategies to reduce the threat of pest establishment and mitigate the impact of exotic pest incursions. [NP 304, Component 4, Problem Statement 4A] 4.A. Develop new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field. 4.B. Develop improved IPM protocols for tephritid fruit flies, ambrosia beetles, and other target pests by integrating semiochemical-based technologies with other management options, such as biological control, judicious alternating use of insecticides to manage pesticide resistance, sanitation and other cultural practices.

Approach
Research will consist of field and laboratory experiments to investigate aspects of basic biology, physiology and chemical ecology that can be exploited to develop integrated pest management approaches for invasive insects that impact subtropical agriculture. Target species will include pests that threaten to invade or have recently established in south Florida, including fruit flies in the family Tephritidae (Oriental fruit fly, Medfly, and Anastrepha species) and ambrosia beetles that vector fungal pathogens (redbay ambrosia beetle and Euwallacea shot-hole borers). Strategies will include (1) identifying semiochemicals from natural product sources that can alter insect behavior, such as attractants, repellents, and oviposition deterrents; (2) developing new detection and delimitation tools that will include formulated lures, discrete attract-and-kill bait stations, next generation technologies like smart traps for automated pest surveillance, and non-destructive imaging techniques for detection of hidden infestation in agricultural commodities; (3) improving pesticide resistance management by identification of pesticide modes of action and physiological responses of fruit flies to toxicants; (4) evaluating plant essential oils as sources of natural toxicants (biopesticides) with less environmental impact; and (5) developing new approaches to assess efficacy of lures, repellents, and control practices based on spatial statistics and contour analysis of trap captures in the field.

Progress Report
Oriental fruit fly (OFF) is a destructive pest of tropical fruit crops worldwide. Current control programs rely on surveillance of male OFF, which is less effective for mitigating pest impact (caused by females). Under an Interagency Agreement with APHIS, ARS scientists in Miami, Florida, and Hilo, Hawaii, investigated the relationship between OFF olfactory preferences and oviposition. In lab bioassays using mated females, some preferred protein (torula yeast, the standard female lure) however, some preferred host fruit (guava volatiles). Females attracted to fruit odors laid 2.4x more eggs than females attracted to protein. Results suggest that host-based lures are better for trapping oviposition-ready female OFF. Parallel tests with Caribbean fruit fly, another pest of guava, are in progress to determine potential utility of host odors for management of this pest. Mediterranean fruit fly (medfly) is another major pest of fruit production globally. Trimedlure, a synthetic male attractant, is used to monitor medfly populations. However, due to cost and availability concerns, APHIS recently identified a need for new male attractants. ARS researchers in Miami, Florida, evaluated a series of essential oils (EOs), including leaf and fruit EO of Juniperus foetidissima and leaf EO of Origanum vulgare and Monarda didyma. Lab bioassays indicated that components of these EOs were attractive to medflies. Chemical analyses indicated the major constituents of J. foetidissima EO were a- and ß-thujone, and the O. vulgare and M. didyma EOs were rich in carvacrol and thymol, respectively. Tests are ongoing to determine the bioactivity of these novel compounds and their potential applications as alternative medfly lures. The collection, analysis and accurate identification of bioactive chemicals is a fundamental requirement for developing semiochemical-based tools for pest management. We previously identified tea tree oil (TTO) as an EO highly attractive to male medfly. Separation of TTO by thin layer chromatography (TLC) generated five major fractions, two of which were attractive. However, identification of specific attractants has been challenging since TTO is a complex mixture of terpenoids and TTOs vary based on the manufacturer. ARS-Miami has recently developed high performance TLC (HPTLC) protocols to evaluate variations in EOs. HPTLC provides a rapid screening method that improves separation, resolution, and isolation of the individual chemicals in TTO, facilitating identification of alternative attractants for medfly. Lychee erinose mite (LEM), recently established in Florida, is a high priority pest with potential to devastate the state’s lychee production. This microscopic mite attacks new leaves and flower buds, thereby preventing fruit development. No attractants are known for LEM; therefore, ARS researchers in Miami, Florida, in collaboration with the University of Florida (UF), initiated research to identify attractive host kairomones. Chemical analyses indicated that LEM infestation induces significant changes in host emissions, and lab bioassays demonstrated preferential attraction of LEM to particular host tissues. Research is in progress to identify the attractants used for host location, and to determine their application in monitoring and control systems for LEM. Hibiscus bud weevil (HBW) is emerging as a major pest in Florida. Females oviposit in flower buds, larvae feed within the buds, and buds drop prior to flowering. Lures are critically needed for early HBW detection. ARS researchers in Miami, Florida, in collaboration with UF, conducted two lines of research to identify potential attractants: host volatiles and species-specific pheromones. Volatile collections and chemical analyses found 42 compounds (primarily long-chain fatty acids) emitted from hibiscus plants and 4 male-produced aggregation pheromone components. Lab bioassays and electrophysiology studies (to evaluate attraction and olfaction, respectively) are ongoing to identify the most promising candidates for development of HBW lures. The giant African snail (GAS) is an invasive pest that causes feeding damage to a variety of agricultural, ornamental, and native plants. In recent years, GAS has established in Florida, prompting an extensive eradication program. In collaboration with APHIS-PPQ, ARS researchers in Miami, Florida, and Hilo, Hawaii, are conducting research to determine if signature volatiles are emitted from GAS mucus trails. If so, these chemical signals could provide the basis for trained canine detection. Initial research documented the odor profile emitted from GAS. Current research is identifying biomarkers from other land gastropods in Florida to determine which volatiles are unique to GAS and potentially diagnostic of pest GAS populations. This project recently received funding through the USDA Plant Protection Act Section 7721. Surveillance networks for pest fruit flies rely on a large number of static traps baited with synthetic lures. These traps require regular servicing to sort, count, and identify insect captures. Development of automated ‘smart traps’ could reduce program costs and optimize detection systems. ARS researchers in Miami, Florida, have developed a prototype trap entrance camera that is undergoing small-scale trials to determine its potential for image collection. However, the development and application of automatic identification algorithms will require large-scale data collection. Collaborations are being sought with researchers in China (Chinese Academy of Science, Beijing) and a private company that has assembled 15,000 images of tephritid fruit flies; access to these datasets would greatly facilitate development of automated traps. At U.S. ports of entry, inspectors check incoming produce shipments for fruit fly infestation by manually cutting open a small sample (= 2%) of fruit and searching for larvae. Consequently, there is a need for more sensitive, high-throughput screening methods. ARS researchers in Miami, Florida, are evaluating imaging techniques and non-destructive chemical analysis methods for detection of larval infestation. Initial experiments using an electronic sensor prototype, the Cyranose Electronic Nose (Sensigent Inc.), found that this system could differentiate chemicals produced by infested and damaged fruit. Experiments are ongoing to determine infestation thresholds required for consistent detection by either gas chromatography or electronic means. Caribbean fruit fly (CFF) is a quarantine pest of citrus and a production pest of guava in Florida. As with other pests, development of insecticide resistance is an ongoing challenge. ARS researchers in Miami, Florida, initiated a long-term project to document resistance levels of CFF in Miami-Dade County, FL. Initial work established a susceptible lab colony in 2020 and determined baseline susceptibility to methomyl. In 2022, wild CFF were assayed to determine the median lethal dosage (LD50) of methomyl, which was compared to baseline susceptibility to determine the resistance ratio. Results indicated that CFF populations are beginning to develop low levels of resistance to carbamate insecticides. CFF resistance levels will be monitored over time; however, to prevent further resistance, alternative control strategies will be required. In recent years, many essential oils (EOs) have been shown to possess insecticidal properties. These biopesticides provide environmentally sound alternatives to conventional pesticides. We evaluated 9 EOs extracted from plants in the Cupressaceae, Myrtaceae, Zygophyllaceae, Lamiaceae, Asteraceae, and Apiaceae for toxicity against adult CFF. Of the EOs tested, 6 were effective toxicants, particularly 3 from species of chamomile (Asteraceae): German, Roman, and Chinese chamomile. EO of Chinese chamomile, rich in oxygenated sesquiterpenes, showed the highest level of toxicity and thus the most promise for development as a biorational insecticide. Additional research is needed to determine the specific chemical components that confer toxicity to CFF. With the impending loss of methyl bromide, alternative fumigation strategies are needed for protection and disinfestation of postharvest commodities. ARS researchers in Miami, Florida, and Hilo, Hawaii, in collaboration with APHIS, developed fumigation protocols using ethyl formate (EF) to control OFF and CFF. We also evaluated combination treatments using EF with anisole (a volatile plant oil) for control of CFF and false spider mite. Results showed that addition of anisole improved efficacy of EF fumigation; the combination achieved complete control of both pests at lower concentrations than fumigation with either fumigant alone. Further testing will optimize EF and anisole concentrations to achieve synergistic fumigation protocols for postharvest pests. Laurel wilt (LW), a lethal disease of avocado and other U.S. laurel trees, is caused by a fungal symbiont of redbay ambrosia beetle. However, this pathogen has since transferred to at least 9 other beetle species, contributing to the loss of ~200,000 avocado trees in Florida. Sanitation practices (e.g., pruning, stumping, removal of infected trees) are used by growers to reduce the incidence of LW. To evaluate efficacy of cultural practices, scientists from UF, in collaboration with ARS researchers in Miami, Florida, investigated the effect of avocado canopy cover on ambrosia beetle abundance. Monitoring for 12 months indicated that groves with full canopy cover exhibited the highest number of beetles and the lowest light intensity. The opposite was found for topworked and newly planted groves. In addition, solar radiation had a significant effect on beetle dispersal flight. The results indicate that thinning canopy cover, which increases light intensity, suppresses ambrosia beetle abundance in commercial groves, thereby reducing the spread of LW.

Accomplishments
1. Repellents for pest ambrosia beetles. Redbay ambrosia beetle and tea shot-hole borer are vectors of laurel wilt and Fusarium dieback, respectively, two fungal diseases of avocado, woody ornamentals and native forest trees. Incorporating a repellent into pest management programs may reduce the incidence of these diseases. ARS researchers in Miami, Florida, identified piperitone as a new beetle repellent and compared its efficacy to two other repellents, verbenone and a-farnesene. Beetle captures in traps baited with lures were compared to captures in traps containing lures plus a repellent (a push-pull design). Farnesene was ineffective; however, piperitone and verbenone were equally effective, reducing captures by 50-70% for 10-12 weeks. Since piperitone is less expensive than verbenone, the standard beetle repellent, these studies identify an economical alternative for management of both pests in commercial avocado groves.

2. Risk assessment of Hass avocado and Mexican laurels for attack by redbay ambrosia beetle. Redbay ambrosia beetle vectors the pathogen that causes laurel wilt, a lethal disease of avocado and other U.S. trees in the laurel family. First detected in Georgia in 2002, the wood-boring pest has since spread to 12 southeastern states. With time, it will likely enter Mexico, threatening native forest species and the Mexican avocado industry based on the Hass cultivar. In advance of an incursion, ARS researchers in Miami, Florida, and the Instituto de Ecología (Veracruz, Mexico) conducted research to assess the potential risk. Freshly cut wood from Hass avocado and 8 native laurels (collected from Veracruz) was shipped to Florida to determine the beetle’s boring preferences in lab bioassays and relative attraction in field tests. Results indicated that Hass avocado, Persea schiedeana, and Ocotea sp. are highly attractive species susceptible to beetle attack. These findings emphasize the need for effective, early detection systems for redbay ambrosia beetle to prevent severe economic and ecological impact of laurel wilt in Mexico.

3. Functional roles of tea shot-hole borer symbionts. Tea shot-hole borer (TSHB) vectors a fungus that causes Fusarium dieback, a destructive disease of avocado trees. Current lures contain quercivorol, an attractant produced by the beetle’s fungal symbionts. The lure contains a mixture of compounds, so the exact attractant is unknown. The beetle also carries multiple symbionts, and the function of each symbiont is unknown. ARS researchers in Miami, Florida, in collaboration with the University of Florida, conducted research with pure cultures of six symbionts to address these questions. A study of the fungal volatile emissions revealed that trans-p-menth-2-en-ol and limonene are beetle attractants. Another study identified which symbionts provide nutrition for beetle development, and which ones were most pathogenic to Florida avocado trees. This information will direct future research in development of improved management programs for TSHB and Fusarium dieback disease.

4. Nitric oxide (NO) fumigation for postharvest pest control. Postharvest pest control needs safe and effective alternative treatments due to the phase out of methyl bromide. NO is a newly discovered fumigant that has been demonstrated efficacious against a variety of insect pests and safe to fumigated products. ARS researchers in Miami, Florida, and Salinas, California, in collaboration with Kansas State University, developed fumigation protocols using NO to control ham mites, Tyrophagus putrescentiae, on ham meat under ultra-low oxygen conditions. The results showed that complete control was achieved for all mite life stages; eggs were the most tolerant stage, but 100% mortality was obtained after 48 and 24 h treatments at 0.5 and 1.0% NO concentrations, respectively. NO fumigation, a safe and effective treatment, provides a potential alternative to methyl bromide fumigation for postharvest pest control.

5. Insecticide resistance mechanisms of Diamondback moth. The diamondback moth (DBM), Plutella xylostella, is a destructive pest of cruciferous crops. Due to extensive use of chemical controls, DBM has evolved resistance to diamide insecticides, including chlorantraniliprole. To better understand the resistance mechanisms of DBM, ARS researchers in Miami, Florida, in collaboration with the Guangdong Academy of Agricultural Science (Guangdong, China) used a comparative transcriptomic approach to analyze the genes associated with insecticide resistance. The study identified 21 metabolism-related genes associated with resistance development to chlorantraniliprole and Bacillus thuringiensis insecticides. This research provides a better understanding of DBM resistance mechanisms, facilitating design of new insecticide combinations for improved pest management.

Review Publications
Shabbir, M.N., Yang, X., Yin, F., Batool, R., Kendra, P.E., Li, Z. 2021. Bacillus thuringiensis and Chlorantraniliprole trigger the expression of detoxification-related genes in the larval midgut of Plutella xylostella. Frontiers in Physiology. 12:780255. https://doi.org/10.3389/fphys.2021.780255.
Revynthi, A.M., Cruz, L.F., Cannon, M.A., Crane, J.A., Kendra, P.E., Mannion, C., Carrillo, D. 2022. Evaluation of abamectin as a potential chemical control for the lychee erinose mite (Acari: eriophyidae), a new invasive pest in Florida. Florida Entomologist. 105(1):1-5. https://doi.org/10.1653/024.105.0101.
Kurtca, M., Tumen, I., Keskin, H., Tabanca, N., Yang, X., Demirci, B., Kendra, P.E. 2021. Chemical composition of essential oils from leaves and fruits of Juniperus foetidissima and their attractancy and toxicity to two economically important tephritid fruit fly species, Ceratitis capitata and Anastrepha suspensa. Molecules. 26(24):7504. https://doi.org/10.3390/molecules26247504.
Yang, X., Liu, Y., Singh, R., Phillips, T.W. 2022. Nitric oxide fumigation for control of ham mite, Tyrophagus putrescentiae (Sarcoptiformes: Acaridae). Journal of Economic Entomology. 115(2):501-507. https://doi.org/10.1093/jee/toac014.
Revynthi, A.M., Velazquez Hernandez, Y., Canon, M.A., Greene, D.A., Vargas, G., Kendra, P.E., Mannion, C.M. 2021. Biology of Anthonomus testaceosquamosus (Coleoptera: Curculionidae): a new pest of tropical hibiscus. Insects. 13(1):13. https://doi.org/10.3390/insects13010013.
Kendra, P.E., Cruz, L.F., Tabanca, N., Menocal, O., Schnell, E.Q., Carrillo, D. 2022. Volatile emissions and relative attraction of the fungal symbionts of tea shot hole borer (coleoptera: curculionidae). Biomolecules EISSN 2218-273X. 12(1):97. https://doi.org/10.3390/biom12010097.
Menocal, O., Kendra, P.E., Padilla, A., Chagas, P.C., Chagas, E.A., Crane, J.H., Carrillo, D. 2022. Influence of canopy cover and meteorological factors on the abundance of bark and ambrosia beetles (coleoptera: curculionidae) in avocado orchards affected by laurel wilt. Agronomy. 12(3):547. https://doi.org/10.3390/agronomy12030547.
Yusufoglu, H.S., Alqarni, M.H., Salkini, M.A., Tabanca, N., Demirci, B., Kendra, P.E. 2021. Chemical composition of essential oils of Pulicaria species growing in Saudi Arabia and activity for Mediterranean fruit fly, Ceratitis capitata. Phytochemistry Letters. 46:51-55. https://doi.org/10.1016/j.phytol.2021.08.021.
Sen-Utsukarci, B., Kessler, S.M., Akbal-Dagistan, O., Estep Iii, A.S., Tabanca, N., Kurkcuoglu, M., Demirci-Kayarian, S., Eroglu-Ozkan, E., Gul, Z., Bardacki, H., Becnel, J.J., Kiemer, A.K., Mat, A., Husnu Can Baser, K. 2021. Chemical composition and biological activities of valeriana dioscoridis sm. roots. South African Journal of Botany. 141:306-312. https://doi.org/10.1016/j.sajb.2021.05.007.
Kilic, M., Orhan, I., Eren, G., Okudan, E., Estep Iii, A.S., Becnel, J.J., Tabanca, N. 2021. Insecticidal activity of forty-seven marine algae species from the Mediterranean, Aegean and Sea of Marmara in connection with their cholinesterase and tyrosinase inhibitory activity. South African Journal of Botany. https://doi.org/10.1016/j.sajb.2021.06.038.
Roh, G., Kendra, P.E., Cha, D.H. 2021. Preferential attraction of oviposition-ready oriental fruit flies to host fruit odor over protein food odor. Insects. 12(10). Article 909. https://doi.org/10.3390/insects12100909.
Wheeler, G.S., Kendra, P.E., David, A.S., Lake, E.C., Sigmon, J., Palacios, J.N. 2021. Community of bark and ambrosia beetles (Coleoptera: Curculionidae) infesting Brazilian peppertree treated with herbicide and the volatile tree response. Environmental Entomology. 50(6):1311–1321. https://doi.org/10.1093/ee/nvab096.
Park, M., Lee, B., Yang, J., Kim, B., Roh, G., Kendra, P.E., Cha, D.H. 2021. Ethyl formate as a methyl bromide alternative for fumigation of citrus: Efficacy, fruit quality, and workplace safety. Journal of Economic Entomology. 114(6):2290-2296. https://doi.org/10.1093/jee/toab175.
Demiray, H., Estep III, A.S., Tabanca, N., Becnel, J.J., Demirci, B. 2022. Chemical constituents from Rheum ribes shoots and its insecticidal activity against Aedes aegypti. Revista Brasileira de Farmacognosia. https://doi.org/10.1007/s43450-021-00224-8.
Tsikolia, M., Tabanca, N., Kline, D.L., Demirci, B., Yang, L., Linthicum, K., Bloomquist, J.R., Bernier, U.R. 2022. Studies on the volatiles composition of stored sheep wool, and attractancy toward Aedes aegypti mosquitoes. Insects. 13(2):1-9. https://doi.org/10.3390/insects13020208.