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United States Department of Agriculture

Agricultural Research Service

2006 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? Why does it matter?
The corn rootworm pest complex (CRW) is a major threat to corn production in the United States, as well as a pest on cucurbits, peanuts, and soybeans. Crop losses and control costs attributed to CRW approach $1 billion annually. Growers prophylactically apply highly toxic soil insecticides, thus imposing health risks to humans and animals. In spite of the extensive use of these insecticides, growers continue to sustain significant yield losses due to CRW. Many of these insecticides will be reviewed under the federal re-registration program and some registrations may not be renewed. Because of these problems and the public's concern for food safety, it is critical to develop pest management strategies that will reduce the amount of insecticides applied in corn, cucurbits, peanuts, and soybeans. Adult CRW are developing resistance to insecticides in current use, in particular organophosphates. Although a rootworm-resistant transgenic corn incorporating beetle-specific Bt toxin has recently been registered by the U.S. EPA, relying on a single tactic for control can promote the development of resistance. A bait that can be formulated with a variety of insecticides at reduced rates will have positive impact on agribusiness where the corn rootworm plays a major role affecting profit margins of farmers. The flexibility offered to farmers to use different insecticides with the bait will slow the development of insect resistance. Also, these baits may be useful in high-value crops affected by rootworms, such as cucurbits and peanuts.

The Colorado potato beetle (CPB) is the single most destructive insect pest of potatoes, tomatoes, and eggplants in the United States, causing major losses when control measures fail. Current management practices rely heavily on the use of insecticides. Estimates of combined pesticide costs and crop loss can reach 28% of crop value, and CPB evolution of resistance to over 25 pesticides makes it one of the most difficult pests to control in row crops. To minimize the risk of negative environmental effects associated with the widespread use of insecticides, and to slow development of resistance of the CPB, we are developing pest management strategies that rely on sustainable tactics such as cover crops, vegetative barriers, predator evaluation and enhancement, microbial insecticides, and novel transgenic approaches.

This research is conducted as a part of National Program 304, Crop Protection and Quarantine. The research components supported are: biology of pests and natural enemies; plant, pest, and natural enemy interactions and ecology; pest control technologies; integrated pest management systems and area-wide suppression programs. The goal of this research is to develop insect pest management strategies that are compatible with conventional, organic, and non traditional agricultural systems, cost effective, environmentally safe, and part of a sustainable agriculture system.

2.List by year the currently approved milestones (indicators of research progress)
Year 1 (FY2006) • Complete development of CPB primers and PCR protocols for predator gut analysis; determine DNA half-lives for model predators fed CPB. • Parasitoids collected for primer development. • Complete lab bait and antifeedant trials with CPB and CRW; complete phytotoxicity tests. • Test avidin and JHE for toxic effects on CPB. • Complete characterization of tomato wound-induced genes, complete test of poplar promoters in Arabidopsis. Year 2 (FY2007) • Complete pilot study to optimize field sampling protocols; complete initial assessment of cover crops effects on CPB in potatoes. • Complete development of alternate prey primers. • Predators collected and identified to species. • Primers and PCR protocols for parasitoid detection developed. • Complete exploration of high-elevation areas for diabroticine natural enemies. • Complete lab bait and antifeedant tests with natural enemies, and small-scale field tests with antifeedants. • Complete cloning of avidin into PVX and synthesize and clone JHE genes into PVX vector; complete testing in insect bioassay. • Complete potato microarray experiments and further characterize gene expression; complete cloning of putative promoters for tomato wound-induced genes and testing in Arabidopsis. Year 3 (FY2008) • Determine DNA half-lives for field-collected predators fed CPB and alternate prey; complete and analyze first year field sampling and predator gut analysis. • Complete development of primers and PCR protocols for predator identification. • Complete exploration for diabroticine natural enemies in center of diversity (Central America and Mexico). • Complete assessment of best cucurbitacin-based baits for CRW (including South and North American sources). • Complete candidate gene cloning of putative promoters and testing in Arabidopsis. Year 4 (FY2009) • Complete studies of effects of release method and mulch treatment on Podisus retention and survival. • Complete Lebia assessment in potatoes. • Complete exploration for Leptinotarsa and Zygogramma natural enemies in South and Central America. • Complete plot tests with South and North American cucurbitacin-based baits; complete large-scale CPB antifeedant tests. • Complete cloning of insecticidal genes into tomato or potato and generate plants. Year 5 (FY2010) • Complete field sampling and gut analysis, and analysis of data. • Complete identification of field-collected predators; analyze 3-year data set. • Complete analysis of field parasitism.. • Complete plan for natural enemy introductions to North America. • Complete large-scale tests of cucurbitacins-based baits at three locations in USA. • Complete testing of other putative insecticidal genes using PVX. • Complete cloning and characterization of infestation-induced promoters in tomato or potato.

4a.List the single most significant research accomplishment during FY 2006.
The rate of decay in detectability of prey DNA in the guts of predators, called the DNA half-life, is an essential quantity for determining the importance of different predators feeding on the same prey species. We showed that the half-life for the DNA of a single egg of the Colorado potato beetle is seven hours for the pink lady beetle and almost fifty-one hours for the spined soldier bug, two of the most important predators of this pest. Knowing that the half-life is more than seven times longer in the bug than the lady beetle prevents us from overestimating the effect of the bug relative to the lady beetle in reducing Colorado potato beetle numbers. This finding has been submitted for publication. Use of molecular methods to identify larval or nymphal generalist predators in agricultural systems (NP304 Research Component 2 Insects and Mites: Biology of pests and natural enemies).

4b.List other significant research accomplishment(s), if any.
• A recombinant Pir toxin from Photorhabdus luminescens that is orally toxic to the diamondack moth was tested on larvae of CPB and other economically important pests and found to have no effect on mortality or growth. This finding has been submitted for publication. Discovery and testing of insecticidal toxins from the nematode symbiont Photorhabdus, and apparently related toxins from hemolymph of CPB and related beetles (NP304 Research Component 2 Insects and Mites: Biology of natural enemies, including microbes). • A project was initiated to determine if a toxic CPB hemolymph protein, leptinotarsin, is responsible for a phenotypic shift in the bacterium Photorhabdus luminescens to a form that does not support the growth of its nematode symbiont, Heterorhabditis marelatus. It has been suggested that leptinotarsin is responsible for the phenotypic change in the bacterium. Recent evidence suggests that leptinotarsin has a high degree of similarity to the Pir toxins of P. luminescens. Discovery and testing of insecticidal toxins from the nematode symbiont Photorhabdus, and apparently related toxins from hemolymph of CPB and related beetles (NP304 Research Component 2 Insects and Mites: Biology of natural enemies, including microbes). • A toxic factor from CPB hemolymph that is lethal to gypsy moth larvae upon injection has been partially purified. The toxic factor appears to be a small molecule, and is not the previously reported toxin leptinotarsin. Discovery and testing of insecticidal toxins from the nematode symbiont Photorhabdus, and apparently related toxins from hemolymph of CPB and related beetles (NP304 Research Component 2 Insects and Mites: Biology of natural enemies, including microbes). • Feeding studies with the important CPB generalist predator, the pink lady beetle Coleomegilla maculata, have shown that CPB eggs are inferior food for development of predator larvae, particularly young larvae, and this has important implications for there conservation and augmentation in agricultural systems, especially where CPB is an important target pest. This finding is under further study in cooperation with collaborators, for expected publication in 2007. Food choice and quality influence on larval development of lady beetle predators. (NP304 Research Component 2 Insects and Mites: Biology of pests and natural enemies). • We have identified and cloned three genes that are induced in potato by infestation, two of which are not induced by wounding, suggesting genes that are specific to the plant/insect interaction. The regulatory regions will be cloned and fused to a marker gene. Transformation of Arabidopsis with these transgenes will be performed so that we identify the regulatory regions of these genes and prove that they are sufficient for induction by infestation. This should ultimately allow the construction of a resistance gene that, when cloned into potato, creates a plant that expresses this transgene when CPB feeding occurs, thus diminishing the overall amount of genetically engineered plant material produced. Pest-induced potato genes of use in development of resistant crop plants (NP304 Research Component 2 Insects and Mites: Pest Control Technologies).

4c.List significant activities that support special target populations.
This project does not support special target populations.

4d.Progress report.
The BARC Multi-Lab Research Project, “Alternatives for Colorado potato beetle management,” 2003-2006, continued emphasis on sustainable practices and cover crops. This project was coordinated by IBL with five other laboratories, especially the Sustainable Agriculture Systems Lab. No-till potato planting with rye cover crops and mulch, as well as rye-legume cover crops and combinations both living and killed, continued to show significant reductions in 1st-generation CPB numbers and comparable reduction in mid-season defoliation. We are examining closely the role of plant foliage quality based on cover crop and cultivation, in slowing CPB larval development. The project also is examining (with HNRC-FCL) the composition of potatoes planted in different mulch systems, including organic versus conventional culture, and using cultivars and planting times, to determine which factors influence the nutrient and phytochemical composition of the harvested crop.

5.Describe the major accomplishments to date and their predicted or actual impact.
• Corn rootworm bait, derived from bitter Hawkesbury watermelon, in combination with a variety of insecticides, is being used by many growers and applicators throughout the corn belt. The use of a bait for control of corn rootworm adults reduces insecticide application for this adult insect by 90%. The product is commercialized by Florida Food Products (Invite®) under USDA license. (NP304 Research Component 2 Insects and Mites: Pest Control Technologies). • A semi-synthetic diet for CPB was developed and has been tested for 13 generations. A semi-synthetic diet has long been sought by scientists for use in mass rearing of CPB, its natural enemies, and to develop pathogens for biocontrol. Also, a new freeze-dried diet greatly facilitates microbial and heat-labile toxin testing on target insects. (NP304 Research Component 2 Insects and Mites: Biology of natural enemies) • A novel method to screen for insect-active genes has been developed. This method drastically shortens the time from a transformation event to actual testing in the plant making the screening process more efficient and economical. (NP304 Research Component 2 Insects and Mites: Pest Control Technologies). • We have determined the CPB DNA half-lives for the spined soldier bug and pink lady beetle, two of the key predators of CPB. Publication of the primer sequences and half-life data will provide other biocontrol scientists with the necessary tools and a model for how to assess the impact of predators on this pest. This will eventually answer the question of whether the reduction of defoliation by mulching is due to predators or some other mechanism. (NP304 Research Component 2 Insects and Mites: Biology of natural enemies) • A novel system for the biological control of Colorado potato beetle by in-field rearing of the spined soldier bug, Podisus maculiventris, shows promise for small potato plantings. (NP304 Research Component (A) Insects and Mites: Biology of natural enemies)

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?
• The corn rootworm bait, derived from Hawkesbury watermelon, has been awarded two patents. Corn rootworm bait is being used by a manufacturer of juice concentrates who has been selling the product throughout the corn belt since 2001. They expect to increase and improve production with continued success of the product, which has been well received. • The freeze-dried diet developed for the Colorado potato beetle has wide applicability to many diets for insects. The impediment to development is to mechanize the steps now performed by hand. • The use of commercially available spined soldier bug lures, traps and nursery cages will facilitate the transfer of this biocontrol technology to end users. • Genetic sequences for spider and carabid beetle species (25 and 13 species respectively) have been deposited in GenBank for use by other scientists in identifying and associating these agriculturally significant biocontrols with other collections. These are all mitochondrial cytochrome C Oxidase I sequences, so-called barcodes.

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).
• Greenstone, M. Invited seminar "Molecular Methods in Predation Research," Department of Entomology, Michigan State University, September 2005. • Weber, D.C., Rowley, D.L., Thorpe, K.W., and Aldrich, J.R. “Improving knowledge and management of native natural enemies for biological control of Colorado potato beetle.” Second International Symposium on Biological Control of Arthropods, Davos, Switzerland, September 2005 (poster). • Greenstone, M.H., Torres, J.B., Ruberson, J.R., and Rowley, D.L. “Immature Predators: an Underappreciated Resource for Biological Control.” Second International Symposium on Biological Control of Arthropods, Davos, Switzerland, September 2005 (poster). • Weber, D.C. “The carabid Lebia grandis and its host the Colorado potato beetle: French connection for a unique predator-parasitoid.” Invited seminar, USDA-ARS European Biological Control Laboratory, Montpellier, France, September 2005. • Weber, D.C. Co-organizer and Moderator of Symposium, “Diversity of trophic relationships in the Carabidae,” Entomological Society of America annual meeting, Fort Lauderdale, December 2005. • Greenstone, M. and Lundgren, J. “Carabid predation: How important are the immatures?” Entomological Society of America National Meeting, Fort Lauderdale, December 2005. • Weber, D.C. and Chaboo, C. “Ectoparasitoid carabids and their beetle hosts.” Entomological Society of America National Meeting, Fort Lauderdale, December 2005. • Alford, R., Alyokhin, A., Weber, D.C., and Dickens, J.C. “Towards developing a Colorado potato beetle attracticide: importance of inert ingredients.” Entomological Society of America National Meeting, Fort Lauderdale, December 2005 (poster). • “I.D.-ing Insects at Different Life Stages.” Science Update on Matthew Greenstone’s barcoding research. Agricultural Research 54(4): 23 (April 2006). • Weber, D.C. Short course on Ground beetles (Carabidae), Midwestern Biocontrol Institute, Oak Lake Field Station, Brookings, South Dakota. • Greenstone, M. "Diagnostic DNA barcoding for spiders: utility, variability, and choice of sequence". American Arachnological Society national meetings, Baltimore, June 2006. • Greenstone, M. Lecture for ENT770, Molecular Tools in Entomology: "Barcoding in Insect Ecology and Biocontrol", University of Kentucky, Department of Entomology, September 2006. • Greenstone, M. Invited Department Colloquium, "Molecular Biology for the Entomologist: Using Proteins and Nucleic Acids to Diagnose Natural Enemy-Host Interactions", University of Kentucky, Department of Entomology, September 2006.

Review Publications
Lawrence, S.D., Novak, N.G. 2006. Expression of poplar chitinase in tomato leads to inhibition of development in colorado potato beetle.. Biotechnology Letters. Vol. 28, no.#8 pg. 593-599

Greenstone, M.H. 2006. A review of molecular methods for assessing insect parasitism. Bulletin of Entomological Research. 96:1-13.

Weber, D.C., Rowley, D.L., Greenstone, M.H., Athanas, M.M. 2006. Lebia grandis (coleoptera: carabidae), a predator and parasitoid of leptinotarsa (coleoptera: chrysomelidae): prey preference and host suitability.. Journal of Insect Science. V.6;09

Last Modified: 5/23/2015
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