2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The citrus, ornamental and vegetable industries are major economic businesses in the United States and particularly in Florida. The sustainability and profitability of these industries is continually challenged by subtropical arthropod pests. The major goal of this project is to increase sustainability of these industries by reducing economic losses to subtropical arthropod pests and by eliminating or reducing adverse environmental impacts associated with managing these pests. Research on invasive pest problems is emphasized. The southeastern U.S. and Florida in particular, are subject to introductions of exotic pest organisms due to geographic position, recent expansion of regional and global trade in agricultural products, and greatly increased tourist travel within and among areas of the U.S., Asia, Central and South America, and the Caribbean. Many of these exotic pests spread unabated because there are no natural enemies and usually no effective control methods have previously been developed to deal with the pests. Current research under this project is directed toward invasive pests such as the Diaprepes root weevil, pink hibiscus mealybug, Asian citrus psyllid, brown citrus aphid, leafminers and whiteflies. Research goals of the project include providing growers with pest management technologies to reduce damage by pests; to decrease dependence on chemical insecticides; to lower production costs; to increase yields; and to reduce adverse effects of pest management strategies on the environment and human health. The project is intended to provide new biological control agents including insect parasitoids, predators and pathogens; plant resistance to insect pests through traditional and transgenic approaches; novel approaches to disrupting insect-plant interactions and insect vectoring of plant diseases; new detection and sampling methods; better understanding of insect-plant interactions, genes/gene products that could lead to new control strategies, and information on the economic importance of subtropical insect pests. The Diaprepes root weevil is the leading invasive pest problem of citrus and certain horticultural crops in Florida. The Asian citrus psyllid is an economic threat to Florida citrus because the psyllid vectors citrus greening, a devastating disease not yet observed in the United States but found for the first time in the Americas during 2004. Another disease vector, the brown citrus aphid, has significantly increased the incidence of an important citrus disease in Florida, leading to yield losses, tree decline, and death of trees. More severe isolates of this citrus disease occur in other countries and could be devastating if introduced into the United States. The silverleaf whitefly has caused more than a billion dollars of production losses to growers throughout the U.S. since its introduction into Florida in 1986. This whitefly is the key pest of tomatoes in southern Florida, vectoring virus diseases and promoting an irregular ripening disorder that greatly reduces profitability of fresh tomatoes. A new biotype of this whitefly from Europe that has resistance to insecticides growers use for whitefly control in the United States has been found in Arizona and California. This new biotype could become established and spread into other states, which could be devastating to the tomato industry. The invasive pink hibiscus mealybug was discovered for the first time in the United States in Florida during June, 2002. Estimates indicate this mealybug could cause $750 million of damage annually over a range of horticultural and ornamental crops. The mealybug was initially restricted in Florida to small geographical areas but is spreading and proving difficult to manage on ornamental plants. An infested ornamental plant nursery unknowingly shipped plants potentially infested to 15 other states during 2004. This program falls within Component VI (Integrated Pest Management Systems and Areawide Suppression) of NP 304. The project focuses on developing economical and sustainable management of important subtropical insect and mite pests of horticultural plants using environmentally appropriate strategies including biological control with insect parasitoids, predators and entomopathogens; host plant resistance; and disrupting chemical and biological aspects of plant-insect interactions. The project specifically addresses NP 304 goals 2.2.2.1 (Insect/Mite Biology & Biosystematics) and 2.2.2.4 (Insect and Mite Control) as described in the National Program Action Plan. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2005) 1. Identify sources of plant resistance and elucidate the underlying mechanisms of resistance to the Diaprepes root weevil (DRW) in citrus, near-citrus taxa of the Aurantiodeae, and potential cover crops including plants with toxic and/or antifeedant activity towards DRW. 2. Initiate development of a transgenic citrus rootstock with resistance to the Diaprepes root weevil. 3. Global identification of tomato genes associated with SLW feeding will be made using gene array technology. Evaluations of the systemic nature of SLW feeding induced changes in tomato will be made in laboratory and greenhouse tests. Begomovirus induced changes in viruliferous SLW will be identified. 4. Discover, import, release, and monitor establishment and impact of parasitoids for control of the Diaprepes root weevil. 5. Describe basic biology of the Lobate lac scale alone and in association with entomopathogenic fungi. 6. Explore methods for monitoring, detecting, and disrupting mating of pink hibiscus mealybug (PHM) using a synthetic sex pheromone. Seasonal phenology of pink hibiscus mealybug will be described using weekly trap catches of males over multiple years. 7. Seasonal activity of psyllid infestations in citrus and on biological control of the psyllid by Tamarixia will be researched at two locations in the Indian River citrus growing area. Year 2 (FY 2006) 1. Continue identifying sources of plant resistance and elucidate the underlying mechanisms of resistance to the Diaprepes root weevil (DRW). Characterize elite citrus rootstock germplasm for reaction to DRW before varietal release. Biochemical basis for antibiotic resistance in roots of Tephrosia candida will be isolated and identified. 2. Continue development of a transgenic citrus rootstock with resistance to the Diaprepes root weevil. 3. Global identification of tomato genes associated with SLW feeding will be completed using gene array technology. Evaluations of the systemic nature of SLW feeding induced changes in tomato will be continued in laboratory and greenhouse tests. Begomovirus induced changes in viruliferous SLW will be identified. Protein assays will be performed to validate gene expression changes. Experiments will be performed to identify gene expression changes in SLW that influence begomovirus transmission. 4. Discover, import, release, and monitor establishment and impact of parasitoids for control of the Diaprepes root weevil. - Continue releases and evaluations. 5. Describe basic biology of the Lobate lac scale alone and in association with entomopathogenic fungi. - Continue gathering qualitative and quantitative information on the scale’s biology including development, sex ratio, reproduction, fecundity, and longevity. 6. Continue to explore methods for monitoring, detecting, and disrupting mating of pink hibiscus mealybug (PHM) using a synthetic sex pheromone. - Optimal trap designs will be expplored. Research on the effective range and residual activity of pheromone-baited traps will be conducted. Seasonal phenology of pink hibiscus mealybug will be described using weekly trap catches of males over multiple years. Deployment strategies will be described for applications in ornamental plantings. 7. Assessments will be concluded of psyllid infestation densities and biological control in the Indian River area. Evaluations of methods of trapping and monitoring psyllid begin. A collaborative project will be initiated to investigate regional differences in Florida with respect to infestation densities and biological control. Year 3 (FY 2007) 1. Identify sources of plant resistance and elucidate the underlying mechanisms of resistance to the Diaprepes root weevil (DRW) in citrus, near-citrus taxa of the Aurantiodeae, and potential cover crops. – Continue characterizing elite citrus rootstock germplasm for reaction to DRW before varietal release. Research on the biochemical basis for antibiotic resistance in roots of Tephrosia candida and other non-host plants will be continued. 2. Develop a transgenic citrus rootstock with resistance to the Diaprepes root weevil. - Bt-toxin activity will be verified using toxin purified from an E. coli expression system. A model plant system will be used (tobacco or alfalfa) for rapid analysis of transgenic expression of the Bt-toxin construct. Transformants expressing Bt-toxin will be identified and expression in root tissue studied. The transformants accumulating high levels of toxin in roots will be propagated and tested against DRW larvae. Transformation of citrus will be initiated. 3. Reduce silverleaf whitefly (SLW) mediated crop losses in tomato through molecular analysis of plant resistance and virus transmission. - Global identification of tomato genes associated with SLW feeding will be made using gene array technology. Evaluations of the systemic nature of SLW feeding induced changes in tomato will be made in laboratory and greenhouse tests. Begomovirus induced changes in viruliferous SLW will be identified. Genetic changes discovered in FY05-FY-07 will be verified using alternative techniques (real time RT-PCR and/or northern blots) and comparisons of virus tolerant and susceptible varieties will be done. Protein assays will be performed to validate gene expression changes. Experiments will be performed to identify gene expression changes in SLW that influence begomovirus transmission. 4. Discover, import, release, and monitor establishment and impact of parasitoids for control of the Diaprepes root weevil. - Continue releases and evaluations. For parasitoids with incomplete establishment, investigate reasons and possible steps to achieve complete establishment. Assess spread of parasitoids from release locations. 5. Describe basic biology of the Lobate lac scale alone and in association with entomopathogenic fungi. - Develop entomopathogenic assay techniques. Screen commercially available entomopathogenic fungi formulations in the laboratory and greenhouse. Field test promising formulations. 6. Continue exploring methods for monitoring, detecting, and disrupting mating of pink hibiscus mealybug (PHM) using a synthetic sex pheromone. - Formulation of best management practices and recommended integrated control tactics based on accumulated knowledge of behavior, response to pheromone traps, capacity for mating disruption, and integration with classical biological control agents. 7. Statistics associated with spatial distribution and sampling parameters for the psyllid will be investigated. Studies on the dispersal of adult psyllids and on the potential of plant resistance on psyllid management will be initiated. Year 4 (FY 2008) 1. Identify sources of plant resistance and elucidate the underlying mechanisms of resistance to the Diaprepes root weevil (DRW) in citrus, near-citrus taxa of the Aurantiodeae, and potential cover crops. - Final determination of the structure and activity of natural products from Tephrosia spp. and other species with activity towards DRW. 2. Develop a transgenic citrus rootstock with resistance to the Diaprepes root weevil. - Transgenic citrus will be screened and plants with satisfactory Bt expression will be propagated for use in DRW challenge experiments. Testing of DRW larvae performance on transgenic model plant will be continued. Transgenic citrus plants will be tested for resistance to DRW larvae. Transformed citrus will be used as a rootstock in grafting experiments, and the ability of the Bt-toxin to move through the graft union to the scion will be determined. Permits will be obtained and experiments initiated to field test Bt-toxin expressing citrus plants. 3. Reduce silverleaf whitefly (SLW) mediated crop losses in tomato through molecular analysis of plant resistance and virus transmission. - Gene regulation information from comparison of plant response in resistant and susceptible tomato lines will be used to develop transgenic methods for production of whitefly/virus resistant tomato. This information will also be used to identify molecular markers associated with resistance genes for use in tomato breeding. Important molecular steps necessary for begomovirus transmission in whitefly will be described, and information will be used to develop control strategies. 4. Discover, import, release, and monitor establishment and impact of parasitoids for control of the Diaprepes root weevil. - Continue releases and evaluations. For parasitoids with incomplete establishment, investigate reasons and possible steps to achieve complete establishment. Assess spread of parasitoids from release locations. 5. Describe basic biology of the Lobate lac scale alone and in association with entomopathogenic fungi. - Develop entomopathogenic assay techniques. Screen commercially available entomopathogenic fungi formulations in the laboratory and greenhouse. Field test promising formulations. 6. Conclude exploring methods for monitoring, detecting, and disrupting mating of pink hibiscus mealybug (PHM) using a synthetic sex pheromone. - Best management practices for PHM will be formulated and integrated control tactics recommended based on accumulated knowledge of behavior, response to pheromone traps, capacity for mating disruption, and integration with classical biological control agents. 7. Detection, monitoring and sampling methodologies will be made available through tech transfer. Studies on the dispersal of adult psyllids and on the potential of plant resistance on psyllid management will be concluded. 4a.What was the single most significant accomplishment this past year? A plastic version of the glass McPhail trap baited with a two-component lure consisting of ammonium acetate and putrescine was shown to be superior to the standard glass McPhail trap baited with torula yeast in water for required protocol monitoring of Caribbean fruit fly, Anastrepha suspensa, in fresh grapefruit for the export market. The new trap with the two-component lure is easier to use and less expensive.Impact: The Florida Department of Agriculture and Consumer Services is interested in adopting the new trapping approach pending approval within the foreign market. 4b.List other significant accomplishments, if any. Molecular phylogenetic analysis of the Caribbean fruit fly Anastrepha suspensa was used to clarify the relationship among different geographic strains of this economically important agricultural pest and to other Anastrepha species. From this analysis, a model was developed to describe the movement of this fly among regions of Florida and the Caribbean. These procedures are being refined and will be transferred to regulatory agencies, notably the Florida Department of Agriculture and Consumer Services for rapid distinction between A. suspensa, A. obliqua and A. ludens. These techniques will also be applied in collaborative tests to determine if a genetic factor contributes to observed differences in bait attraction. Furthermore, mitochondrial COI DNA analyses did not support previously predicted host specific segregation of A. suspensa individuals in Florida. 4c.List any significant activities that support special target populations. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. · Seasonal ecology of the pink hibiscus mealybug and the efficacy of a new synthetic sex pheromone as a tool for monitoring the pest were clarified. Infestation levels of the pest were shown to be highest during the months of July and August, thus IPM tactics may be strategically timed just prior to or during this time period. The synthetic attractant was shown to be effective and had an effective life of greater than 7 months when impregnated in rubber septa. · Advances were made in biological control of the Diaprepes root weevil. Two Caribbean parasitoid species have been established in south Florida for control of Diaprepes root weevil, but these have not adapted to other citrus areas in Florida. Foreign exploration for additional parasitoids has resulted in two new promising species that are currently in quarantine. · A particle film, kaolin, applied as a foliar spray was demonstrated to suppress infestations of Diaprepes root weevil in citrus and to enhance tree growth. This possible new IPM tool for the pest could have significant impact pending development of a rain-fast formulation. · The phylogeny of the Caribbean fruit fly Anastrepha suspensa in relation to different geographic strains of the pest and to other Anastrepha species was clarified. Molecular procedures were developed for distinguishing Anastrepha species. These procedures are being refined and will be transferred to regulatory agencies, notably the Florida Department of Agriculture and Consumer Services for rapid distinction between A. suspensa and A. ludens. Suspect host shifts among A. suspensa individuals in Florida could not be confirmed using mitochondrial dna analyses based on the CO1 region. A plastic version of the glass McPhail trap baited with a two-component lure consisting of ammonium acetate and putrescine was shown to be superior to the standard glass McPhail trap baited with torula yeast in water for required protocol monitoring of Caribbean fruit fly, Anastrepha suspensa, in fresh grapefruit for the export market. The new trap with the two-component lure is easier to use and less expensive. Impact: The Florida Department of Agriculture and Consumer Services is interested in adopting the new trapping approach pending approval within the foreign market. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? • Research by the Subtropical Insects Research Unit on this project prompted collaborative research within ARS toward successful development of an attractant for the invasive pink hibiscus mealybug. Although not yet commercially available, USDA-APHIS and the Florida Department of Agriculture are using traps baited with the attractant for surveillance purposes. • A plastic version of the glass McPhail trap baited with a two-component lure consisting of ammonium acetate and putrescine was shown to be superior to the standard glass McPhail trap baited with torula yeast in water for required protocol monitoring of Caribbean fruit fly, Anastrepha suspensa, in fresh grapefruit for the export market. The new trap with the two-component lure is easier to use and less expensive. The trap and lures are available commercially. The adoption of the trap and lure, and The Florida Department of Agriculture and Consumer Services is interested in adopting the new trapping approach pending approval within the foreign market. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Boykin, L., Shatters, R., Hall, D.G., Hibbard, K., Fritz, A. Mitochondrial and microsatellite data for anastrepha suspensa (Caribbean fruit fly): implications for Caribbean biogeography [abstract]. Evolution 2005, 6/14/2005, Fairbanks, AL. Boykin, L., Shatters, R., Hall, D.G., Hibbard, K., Fritz, A. Mitochondrial and microsatellite data for anastrepha suspensa [abstract]. 88th Annual Meeting of the Florida Entomological Society, July 27, 2005, Ft. Myers, FL. Hall, D.G., Alessandro, R. 2005. Some chemical and morphological comparisons of laboratory-reared and field-collected adult Diaprepes root weevil. [abstract]. Annual Meeting of the Diaprepes Task Force, May 12, 2005, Immokalee, FL. Hall, D.G., Lapointe, S.L. Seasonal Activity of pink hibiscus mealybug in east central Florida based on pheromone trapping of males [abstract]. 88th Annual Meeting of the Florida Entomological Sociey, July 27, 2005, Fort Myers Beach, FL, Paper No. 50. Katsar, C.S., Hunter, W.B., Lapointe, S.L. 2005. Delta-9 Desaturases in the Diaprepes root weevil [abstract]. 88th Annual Meeting of the Florida Entomological Society. July 24-27, 2005, Ft. Myers, FL. Katsar, C.S., Hunter, W.B., Lapointe, S.L. 2005. Delta-9 Desaturase from the Diaprepes root weevil [abstract]. Annual Meeting of the Diaprepes Task Force, May 12, 2005, Immokalee, FL. Lee, L., Hunter, W.B., Dang, P.M., Hunnicutt, L.E. 2005. An expressed sequence tag (EST) cDNA Library of Aphis gossypii alates [abstract]. The American Phytopathological Society Annual Meeting. July 30-Aug. 3rd. Austin, Texas. McKenzie, C.L., Albano, J.P. Does time of whitefly infestation affect the development of tomato irregular ripening disorder? [abstract]. 88th Annual Meeting of the Florida Entomological Society, July 27, 2005, Fort Myers Beach, FL. Shatters, Jr., R.G. The How's and Why's of Developing Transgenic Citrus with diaprepes root weevil Resistance [abstract]. Diaprepes Task Force Annual Meeting, May 12, 2005, Immokalee, FL. Shatters, Jr., R.G., McKenzie, C.L., Czosnek, H., Brown, J. Whitefly functional genomics: Bemisia tabaci transcriptome responses to plant pathogenic geminiviruses [abstract]. XLV Congreso Annual de la Sociedad Americana de Fitopatologia-Division Caribe. June 28, 2005, San Jose, Costa Rica. Ulmer, B.J., Pena, J.E., Duncan, R.E., Lapointe, S.L., Hall, D.G. Update on diaprepes egg parasitoid research. Diaprepes Task Force, Annual Meeting, May 12,Immokalee, FL. Weathersbee III, A.A., Boykin, L.M. An extracellular Cu Zn superoxide dismutase from Lysiphlebus testaceipes [abstract]. Paper No. DSP 19. 88th Annual Meeting of the Florida Entomological Society, July 24-27, 2005, Fort Myers Beach, FL. Review Publications McKenzie, C.L., Weathersbee III A.A., Puterka, G.J. 2005. Toxicity of sucrose octanoate to egg, nymphal, and adult Bemisia tabaci (Hemiptera: Aleyrodidae) using a novel plant-based bioassay. Journal of Economic Entomology. 98(4):1242-1247. Lapointe, S.L. 2005. Response of Diaprepes abbreviatus (Coleoptera: Curculionidae) to application rates of a particle film. Florida Entomologist. 88:222. Hall, D.G., Burns, R.E., Jenkins, C.C., Hibbard, K.L., Harris, D.L., Sivinski, J.M., Nigg, H.N. 2005. A field comparison of chemical attractants and traps for Caribbean Fruit Fly, Anastrepha suspensa (Loew)(Diptera: Tephritidae), in Florida citrus. Journal of Economic Entomology. 98(5):1641-1647.