Substance That Gives Grapefruit Its Flavor and Aroma Could Give Insect Pests the Boot

Article ID: 606537

Released: 26-Aug-2013 7:00 AM EDT

Source Newsroom: American Chemical Society (ACS)

Wednesday, Sept. 11, 2013, 9 a.m. Eastern Time

Note to journalists: Please report that this research was presented at a meeting of the American Chemical Society.

A press conference on this topic will be held Wednesday, Sept. 11, at 1:45 p.m. in the ACS Press Center, Room 211, in the Indiana Convention Center. Reporters can attend in person or access live audio and video of the event and ask questions at

Newswise — INDIANAPOLIS, Sept. 11, 2013 — The citrus flavor and aroma of grapefruit — already used in fruit juices, citrus-flavored beverages, and prestige perfumes and colognes — may be heading for a new use in battling mosquitoes, ticks, head lice and bedbugs thanks to a less expensive way of making large amounts of the once rare and pricey ingredient, a scientist said here today.

A report on the new technology for making the ingredient, nootkatone, which previously had to be harvested from tons of grapefruit, was part of the 246th National Meeting & Exposition of the American Chemical Society (ACS), the world’s largest scientific society. The meeting, which includes almost 7,000 reports on new advances in science and other topics, continues here through tomorrow.

“A new product based on nootkatone would have multiple advantages over existing mosquito repellants based on DEET,” said Richard Burlingame, Ph.D., who presented the report. “Nootkatone is a broad-spectrum ingredient that has been shown to be effective as a control agent for mosquitoes, ticks and bedbugs. Nootkatone has been used for years to give beverages a grapefruit flavor. It is safe to eat, has a pleasant citrus flavor, is not greasy, both repels and kills insects, and should not have the toxicity concerns that exist for DEET.”

Burlingame, who is with Allylix, Inc., a renewable-chemical firm in Lexington, Ky., spoke at a symposium entitled “Biopesticides: State of the Art and Future Opportunities.” It includes presentations (abstracts appear below) on progress in developing new pesticides isolated from natural sources, or patterned closely after natural products that are effective in pest control.

“The goal of the symposium is to discuss the science behind these products, many of which are effective at lower doses and are less toxic to humans than conventional pesticides,” said James N. Seiber, Ph.D., of the University of California, Davis. He co-organized the symposium with Aaron Gross and Joel Coats, Ph.D., both of Iowa State University, and Stephen Duke, Ph.D., of the U.S. Department of Agriculture-Agricultural Research Service.

Burlingame cited nootkatone as an excellent example of the potential for developing new pesticides based on natural sources. Nootkatone is a component of the oil in grapefruit, and has been on the U.S. Food and Drug Administration’s list of substances generally recognized as safe for use in food. It has been in commercial use for years as a flavoring for foods and beverages and as a fragrance ingredient in perfumes. Those applications require only tiny amounts of nootkatone, and price — $25 per ounce when extracted from grapefruit — was not a major concern. It was slightly less expensive when produced from a substance called valencene, extracted from oranges.

The need for a more economical source of nootkatone intensified after scientists at the U.S. Centers for Disease Control and Prevention (CDC) discovered nootkatone’s effectiveness in controlling ticks, mosquitoes and other insects. Nootkatone extracted from grapefruit, however, would be too expensive for development of a consumer product. That use would require larger amounts of nootkatone. Allylix is now working with scientists at CDC to develop nootkatone for commercial use as an insect-control agent.

Burlingame described how Allylix used proprietary technology to develop a way of producing valencene from yeast growing in industrial fermentation vats. Technicians harvest the valencene and use a chemical process to convert it into nootkatone. Allylix said the process made it possible to market nootkatone at a competitive price.

“The effects of nootkatone last much longer than those of repellents currently on the market,” he said. “And nootkatone shows promise for being the most effective agent for the ticks that cause Lyme disease.”

Nookatone also works in a new way, so it can be used against insects that develop resistance and shrug off conventional pesticides, and yet would be very unlikely to harm people or pets.

Allylix currently sells nootkatone only for use in flavor and fragrance applications. The next step involves getting approval from the U.S. Environmental Protection Agency to sell nootkatone for insect control. “They haven’t approved it yet, so no products currently on the market in the U.S. include nootkatone as an active ingredient to control pests,” noted Burlingame. “But in the future, it could be a key ingredient in repellents for use on clothing or on skin as a spray, or even as a soap or shampoo.”

The scientists acknowledged funding from the National Science Foundation through the SBIR program and the Department of Energy.

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Biopesticides: State of the art and future opportunities
Seth D Goldblum, Craig B Warren, Richard P Burlingame, Allylix, Inc., Lexington, KY, United StatesTerpenes are the most diverse class of biomolecules, with over 50,000 described compounds. Fifteen carbon sesquiterpenes are ubiquitous in nature and are known to be effective in a wide range of high value applications including flavors and fragrances, cosmetic products, food ingredients, pharmaceutical products, and insect control. There is rich literature on the use of sesquiterpenes for insect control. Work at the US Centers for Disease Control has demonstrated that nootkatone, the defining flavor and fragrance of grapefruit and also found in Alaskan yellow cedar, is a very effective tick acaricide and repellent. In those studies, it was further demonstrated that low concentrations of nootkatone have the ability to maintain effective acaricidal activity for up to six weeks. In addition to nootkatone, numerous other sesquiterpenes have been shown to control effectively insects such as mosquitoes, ants, fleas, and bedbugs. In spite of their proven efficacy, there has been little commercial development of sesquiterpenes as insect control agents due to their lack of availability at commercially viable prices. Allylix has developed proprietary technology for the production of terpenes using synthetic biology and metabolic engineering of Saccharomyces cerevisiae yeast. This technology provides the basis for sustainable production of sesquiterpenes and other terpenes from abundant inexpensive raw materials, allowing reliable cost-effective production of these compounds at consistent quality. The production process for nootkatone has been practiced at commercial scale, and nootkatone is currently sold in the marketplace. Allylix technology can also serve as a platform for discovery of new biopesticide products. Using straightforward chemical modification of various terpene scaffolds produced by fermentation, libraries can be generated and tested for insect control and other applications. These technologies provide mechanisms to discover and to produce terpene-based biopesticide products that can compete with conventional pesticides on both and efficacy and an economic basis.

SPLAT: A delivery technology for attractants and repellentsAgenor Mafra-Neto,, ISCA Technologies, Riverside, CA 92507, United StatesISCA Technologies specializes in the development of semiochemical-based products for pest management. Invariably semiochemicals tend to be volatile, labile compounds that require a specialized formulation to protect and to modulate their release over long periods of time. Despite decades of successful research, the bottlenecks to widespread adoption of semiochemical control techniques still remain. Current techniques 1) require high doses of semiochemicals per area to protect crops which may be cost-prohibitive, 2) require companion insecticide treatments, 3) require manual application, and 4) shown lack of consistency in control. SPLAT® is a biologically-inert matrix developed for pest management in agricultural, urban, and forest ecosystems to provide long-term release of insect pheromones, plant volatiles, insecticides, phagostimulants, and other compounds used for pest control. The amorphous and flowable properties of SPLAT allow it to be applied using a wide range of manual and mechanical methods such as electric and pneumatic grease guns, syringes, caulking guns, tractors, and even aerial applications. SPLAT formulations can be designed to provide species-specific control through mating disruption, repellency, attract and kill, and mass trapping techniques. We discuss three effective insect pest management systems that use SPLAT: 1) SPLAT EC, a mating disruption system that uses a mimic of the sex pheromone of the Carob Moth, a pest of dates, carob, pomegranates, almonds, and palms; 2) SPLATverb formulated with an anti-aggregation pheromone that protects individual trees and forest stands from mountain pine beetle attack; and 3) SPLAT MAT ME, formulated with an attractant combined with an insecticide and a phagostimulant that has resulted in an optimal bait-and-kill strategy for a number of Tephritid fruit flies.

Discovery and development of repellents
R Michael Roe1,, Robert D Mitchell1, Andrew Wallace2, Ernest Hodgson2, Brooke Bissinger1, Ann Carr1, Charles Apperson1, Coby Schal1, Daniel Sonenshine3, Anirudh Dhammi1, Jaap van Kretschmar1, Jiwei Zhu1. (1) Entomology, North Carolina State University, Raleigh, NC 27695, United States, (2) Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695, United States, (3) Biological Sciences, Old Dominion University, Norfolk, VA 23529, United StatesThe synthetic compound, DEET, has been the gold standard for insect and tick repellents for over 50 years, and common thought was that it would be difficult to find something better than DEET. Since about one-third of the population in the US each year applies DEET to their skin or clothing at levels as high as 98% DEET, we should not be surprised of the public concern about its safety and demands for a green alternative. We have new data on the effect of DEET on total gene expression in primary human liver cells. A new, natural repellent, was discovered, BioUD, in a university laboratory. BioUD is more effective than DEET against mosquitoes and ticks, does not melt plastics like DEET, is non flammable, and is approved by the US EPA for use on children. The work on BioUD provides many lessons about technology discovery and commercialization in the University environment of “publish or perish”. Tick attractants will be discussed with activities as high as 18,000 times that of carbon dioxide. The work on BioUD and tick attractants has led unexpectedly to a new herbicide, a new fumigant, and basic new information on how ticks taste and smell.

Discovery of mosquito attractants and attraction-inhibitors
Ulrich R Bernier,, Maia Tsikolia, Natasha M Agramonte. Center for Medical, Agricultural, and Veterinary Entomology, USDA-Agricultural Research Service, Gainesville, FL 32608, United StatesThe United States Department of Agriculture (USDA) has developed repellents and insecticides for the U.S. military since 1942. A small component of this research program has aimed at the discovery of attractants that can be used to produce potent lures for haematophagous arthropods, with a primary focus on medically-important biting flies. Research on attractants in the late 1960s led to the discovery of L-lactic acid as one of the attractants for Aedes aegyptimosquitoes. In the mid and late 1990s, research involving multiple subjects led to the discovery of 277 compounds present on skin, and the results of this work produced lures that are highly effective at trapping Ae. aegypti in laboratory bioassays. As a result of these studies, it was discovered that some compounds on human skin inhibit the ability of mosquitoes to find hosts. These compounds are present on human skin at trace levels; however, when larger quantities of these attraction-inhibitors are presented with human odors to mosquitoes, they produce anosmia and hyposmia in test mosquitoes. This presentation will cover the USDA research to discover better mosquito attractants and novel means to deter mosquitoes from finding hosts.

Semiochemicals to control orchard pests
John J Beck,, Plant Mycotoxin Research, USDA-Agricultural Research Service, Albany, CA 94710, United StatesTree nuts – almonds, pistachios, and walnuts – are a significant agricultural commodity of California and supply a large portion of the world's consumption. The navel orangeworm (Amyelois transitella)is a major insect pest that inflicts significant monetary damage to California tree nuts. During their development, larvae of A. transitella feed upon the nut meat, which causes physical damage and ultimately lowers kernel quality. Moreover, infestation of A. transitella in tree nuts represents a serious food safety concern since the larvae have been purported to vector aflatoxigenic fungi into the food product. Aflatoxins are toxic metabolites produced by Aspergillus flavus and Asp. parasiticus – ubiquitous fungi in tree nut orchards. Semiochemicals play a large role in efforts to control or monitor A. transitella moths. For instance, a blend of almond host plant odors has recently been reported to attract both male and female navel orangeworm during field trapping studies. The origin of many of the components within this host plant blend appears to be from the almond host. However, new reports regarding the blend component, conophthorin, imply a fungal origin for this particular volatile. Discussed will be current applications for host plant semiochemicals to attract navel orangeworm as well as future opportunities.

Botanical insecticides: A global perspective
Murray B Isman,, Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC V6T1Z4, CanadaAccording to the CAB Direct database of scientific publications, there has been an enormous growth in research on botanical insecticides over the past 30 years. In 1980, less than 3% of all journal papers on insecticides dealt with botanicals , whereas that proportion reached 20% in 2011. In particular, there has been explosive growth in studies on insecticidal properties of plant essential oils; over half of the 2,000 papers on essential oils as insecticides have been published since 2006. In contrast, commercialization of botanical insecticides has continued to proceed at a relative snail's pace, indicating a big disconnect between theory and practice. This is certainly the case in the jurisdictions with the most rigorous regulatory standards – the EU, USA, and Japan. Using California as an example, use data for botanical insecticides also suggests a very modest market presence. According to Cal DPR data from 2010, botanicals constituted only 5.6% of all biopesticides used and less than 0.05% of all pesticide use; however, some recently introduced products have seen modest success. On the other hand, there appears to be increasing commercialization of botanical insecticides in China, Latin America, and Africa, regions where socio-economic conditions have led to some of the worst examples of human poisonings and environmental contamination. Arguably, botanicals should be of greater value in developing countries where the useful plant species are often locally abundant, accessible, and inexpensive. In many tropical countries, semi-refined plant preparations are likely to be relatively safe for users and more cost-effective than imported conventional crop protection products. In G20 countries, botanical insecticides will probably remain niche products for use in public health, urban pest control, and organic food production, but with considerable market opportunities.

Market opportunities for biopesticides
Pamela G Marrone,, Phyllis Himmel, Alison Stewart. Marrone Bio Innovations, Inc., Davis, CA 95618, United StatesThe biopesticide market is reported to be approximnately $1.7 billion annually out of a $45 billion total global pesticide market. Biopesticides are reported to be growing at 10-15% per year compared to chemical pesticide growth of 5-6%. When used in integrated pest management programs, biopesticides offer value to customers with higher yields and quality than chemical-only programs. Regulatory restrictions, residue/MRL issues on exported crops, pest resistance, and worker and environmental safety are added benefits contributing to their increased adoption. These factors have fueled robust deal making where large agrichemical companies are licensing biopesticides for their portfolios and also acquiring entire companies. Trends and factors that will further increase biopesticide adoption will be discussed.

Chemical ecoprospecting and biopesticides
May R Berenbaum,, Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801-3795, United StatesDespite the fact that plant-derived natural products have historically been the basis for multiple useful biopesticides, screening efforts over the last quarter-century have been far more likely to focus on identifying novel pharmaceuticals than on biopesticides. A hallmark of both enterprises, however, is their relatively low success rates, with associated low return on investment. Efforts to increase the success rate of screening for drugs have included conducting ethnopharmacognostical and zoopharmacognostical surveys and examining ancient classical texts for guideposts; more recently, ecological theory has been applied to guide screening and to predict general pharmacological activity. Ecological theory can be applied far more productively, though, to predict biopesticidal activity. Among theories predicting community-wide distribution patterns of bioactive chemicals, the majority (including the carbon/nutrient balance hypothesis and the growth-differentiation hypothesis) predict only allocation patterns of defense chemicals rather than modes of action or spectra of activity. Both optimal defense theory and apparency theory can be applied to predict with some success different types of bioactivity as well as different types of allocation patterns. Moreover, recent modifications of theories that incorporate new knowledge relating to multitrophic interactions and molecular mechanisms underlying plant responses have improved the prospects for predicting sources of new biopesticides.

Bringing new biologicals to market
Marcus Meadows-Smith,, Bayer Crop Science, RTP, NC, United StatesThis presentation will weave together three critical considerations in the development and commercialization of new biologic pest control products: the economics driving agricultural production, the regulatory environments in which biologics are measured, and the market drivers influencing farmers' agronomic decisions both today and into the future. As the Head of Biologics within Bayer CropScience and the former CEO of AgraQuest Inc., the presenter will present a unique view of the position biologics play in emerging offerings from leading multi-national agchem companies. Through this presentation, the presenter will demonstrate that biologics must meet performance, price, and production hurdles in order to justify R&D investment and grower acceptance. The presenter will show that sustainable agricultural relies on an integrated approach to biologics and conventional agchem technologies.

Microbially-derived pesticides: Challenges and opportunities
B. Clifford Gerwick,, Crop Protection Discovery Research, Dow AgroSciences, Indianapolis, Indiana 46268, United StatesMicrobially-derived pesticides include living or killed microbes with enriched levels of pesticidal agents, such as, proteins or natural products, natural products purified from microbes, semi-synthetic modifications of microbial natural products, and fully synthetic pesticides inspired by microbial natural product starting points. Each of these approaches marks a different set of challenges and opportunities, including efficacy, production, formulation, and a likely path to registration. Among those microbial natural products produced by fermentation, only abamectin and spinosad are major products with sales in excess of $100 million annually, and similarly, only spinetoram and emamectin benzoate are major semi-synthetically, modified natural products of microbial origin. Challenges to discovery of microbial-derived pesticides such as these will be discussed.

USDA-Agricultural Research Service: Perspectives on biopesticides
Edward B Knipling,, USDA-Agricultural Research Service, Washington, DC, United StatesThe United States Department of Agriculture, Agricultural Research Sevice has a long history of research on the discovery and development of biopesticides, particularly in the area of biocontrol agents. Examples of some of these will be discussed, as well as our current research in this vital area of pest management.

Chemical ecology and biorational pesticide design
John A Pickett,, Department of Biological Chemistry, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United KingdomBiorational design of pesticides has been an elusive target, not because predicting intrinsic activity has eluded agrochemistry more than pharmacochemistry but because application is so distant from the target pest compared with application of human or veterinary medicines. Chemical ecology has given us a range of lead compounds that may not represent formally biorational design, but certainly biorational discovery in that natural phenomena, such as impairment of pest, pathogen or weed development, can be used in bioassay guided fractionation to provide the chemical structure involved. We have examples where valuable pesticides can then be designed and even where the natural product itself is utilised directly. However, many such leads are too weakly active and too broadly toxic for successful exploitation. Now that the provenance of such compounds in nature means that we can potentially use GM and other approaches for synthesis in plants, greater opportunities exist for exploitation, but we may for the long term consider more those compounds such as pheromones and other semiochemicals that act by sophisticated non-toxic regulatory mechanisms. This approach will be exemplified in detail from the laboratory to the farm.

Outlook for biopesticides in agriculture and public health
James N Seiber1,, Joel R Coats2, Aaron Gross2, Stephen O Duke3. (1) Department of Environmental Toxicology, University of California, Davis, Davis, California 95616, United States, (2) Department of Entomology, Iowa State University, Ames, Iowa 50011-3140, United States, (3) Natural Products Utilization Research Unit, USDA-Agricultural Research Service, University, Mississippi 38677, United StatesThe authors will lead discussion on the status of biopesticide research, discovery, effectiveness, regulation, economics, and future outlook in the U.S. and internationally. The appropriateness of terms such as biopesticide, biorational pesticide, naturally-occurring, and 3rd generation pest control agents will be discussed, as well as, exploited targets and opportunities for biopesticides in integrated pest management.

Fascinating science, frustrating reality: Five barriers to broad utility of biopesticides
Daniel Kittle,, Research and Development, Dow AgroSciences, Indianapolis, IN 46268, United StatesFor more than a century, growers have understood the potential of biological control agents as effective tools for crop protection. The agriculture industry has yet to realize, however, many predictions about the pace of growth and adoption of biopesticides. Advances in science have increased the breadth of understanding about the potential of biopesticides while simultaneously deepening the realization of the challenge inherent in converting that potential into a robust solution. Learnings from the past decades of agricultural chemistry research have built a clearer understanding of the five key barriers to broad utility of biopesticides and the magnitude of improvements required.

Registration of biopesticides
Keith A Matthews,, Office of Pesticide Programs, US Environmental Protection Agency, Washington, DC 20468, United StatesFederal law requires that all pesticides sold and distributed in interstate commerce in the United States be registered. The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) requires that the EPA determine that a pesticide, when used consistent with label directions, will not cause unreasonable adverse effects to human health or the environment. The US EPA, Office of Pesticide Programs, Biopesticides and Pollution Prevention Division is responsible for the registration and registration review of biopesticide products. Biopesticides include naturally occurring substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesticides), and transgenic plant pesticides, known as plant-incorporated protectants or PIPs. Biopesticides typically are distinguished from conventional chemical pesticides by increased specificity to target species, natural occurrence, low human toxicity, or low volume of use. This presentation will focus on the biopesticides regulatory scheme. Definitions and legal framework, key steps, and recent improvements will be reviewed.

Growing need for bioherbicides
Stephen O Duke,, P.O. Box 8048, USDA-Agricultural Research Service, University, MS 38677, United StatesSeveral factors have converged to make the need for bioherbicides perhaps more urgent than the need for other biopesticides. First, there is a critical need for new herbicide modes of action, as a new mode of action has not been introduced in more than 20 years, and new modes of action are needed to manage growing resistance to herbicides with old modes of action. Phytotoxic natural products that might be considered bioherbicides are a major source of novel modes of action. For example, many unused sites of action are known for microbial phytotoxins. Second, the biggest pest management need of organic farmers is new, effective tools for weed control. Thus, new bioherbicides that might be accepted by the organic farming community are a critical need for improving the economics and effectiveness of pest management in organic systems. Lastly, greener weed management with bioherbicides is highly desirable in order to reduce the environmental footprint of weed management in conventional crops. For example, the possibilities of RNAi as a bioherbicide in eliminating pesticide residues in the environment and in harvested crops, as well as eliminating non-target species effects are huge. There is tremendous potential to solve all three of these needs with greater bioherbicide discovery and development efforts.

IR-4 Project: A public sector program to facilitate the registration of biopesticides
Michael Braverman,, Daniel Kunkel, Jerry Baron. IR-4, Rutgers University, Princeton, NJ 08540, United StatesThe IR-4 Project was established 50 years ago to help to register pesticides in specialty crops or minor crops in addition to minor uses. IR-4 is a public sector organization, primarily funded by USDA-NIFA. Most biopesticides fill small but important segments of crop production, so like minor uses, it is difficult for small companies to justify investment in the registration process. Often researchers and small companies lack the expertise on how to prepare the information required by EPA. The biopesticide program was established in 1982 as part of IR-4 to assist in the federal registration of biopesticides with EPA. IR-4 is involved in assisting the registration of biochemical biopesticides such as plant extracts, pheromones, and minerals in addition to microbial and biotechnology products. This presentation will provide an overview of the IR-4 Biopesticide Program and recent registrations as well as current projects.

Setting the stage for future transgenic insect control products
Mark E Nelson1,, Gusui Wu2, J Lindsey Flexner1. (1) Trait Characterization and Development, Dupont Pioneer, Wilmington, DE 19805, United States, (2) Trait Discovery, Dupont Pioneer, Johnston, IA 50131, United StatesTransgenic insect control (IC) has important health and environmental benefits and provides a useful pest management option to growers. The success of transgenic crops has resulted in ever increasing adoption of this technology, but the possibility for resistance to IC traits remains. To be sustainable, future IC products will depend on the discovery of new modes of action to provide effective insect resistance management (IRM) features. This need drives considerable investment in research and development efforts aimed at identifying actives with novel modes of action. Appropriate characterization of these leads is necessary to understand how they can be best utilized in IRM strategies and includes the application of a variety of functional and biochemical approaches. Several of these approaches will be discussed.

Discovery of new insect resistance traits for control of insect pests in transgenic crops
Thomas Meade,, Kenneth E Narva, Nicholas Storer. Dow AgroSciences, Indianapolis, Indiana 46268, United StatesThe first transgenic crop was deregulated in 1995 and the power of this technology to increase agricultural productivity is reflected in the fact that in 2012, transgenic crops were cultivated on 170 million hectares in 28 countries. First generation products containing transgenic insect resistance (IR) traits typically contained a single mechanism of action (MOA) targeting a specific pest complex, while the most recent products utilize pyramided MOAs. While there is an increased cost to the trait provider in pyramiding MOAs, a benefit is the opportunity to simplify grower production practices and increase product lifespan by incorporating the resistance-mitigating “refuge” seed (containing no IR traits) as a seed mixture with IR trait-containing seed. Pyramiding of traits, emergence of new pests, incorporation of IR traits into new crops, and the finite lifespan of commercialized IR traits, all drive the search for novel IR traits. Insecticidal protein genes from Gram-positive bacteria like Bacillus thuringiensis remain a significant focus of new IR trait discovery. Engineering these proteins to alter specific attributes has been used successfully to create novel IR traits. The advent of Next Generation Sequencing technologies and associated bioinformatics capabilities have enabled rapid sequencing of bacterial genomes and dramatically increased the rate of discovery of novel IR genes. These same technologies have enabled the creation of RNAi-based IR traits for some pests. Engineering plants to produce insecticidal or behavior-modifying small molecules also holds promise as a means to create novel IR traits. A challenge to sustaining innovation in the discovery and commercialization of novel IR traits remains creating value for growers and for the companies that invest in bringing these technologies to the marketplace.

Using genomics and chemistry to screen for secondary metabolites in Bacillus spp biocontrol organisms
Christopher Dunlap,, David Schisler. National Center for Agricultural Utilization Research, USDA-Agricultural Research Service, Peoria, IL 61604, United StatesThe adoption of high-throughput DNA sequencing has greatly expanded our knowledge of the secondary metabolite potential of biocontrol organisms. By combining low cost sequencing and data mining software the ability to characterize the potential of new organisms is greatly enhanced. In the current study, we apply the approach to twoBacillus strains isolated to control Fusarium head blight in wheat. For Bacillus amyloliquefaciens AS 43.3, genomic data mining identified 9 gene clusters with the potential to produce bioactive secondary metabolites. The knowledge of the types of potential metabolites allowed us to develop extraction and mass spectroscopy approaches to confirm the metabolites are produced using traditional chemistry techniques. The results confirmed the presence of all 9 metabolites under liquid culture production. For Bacillus subtilis OH 131, genomic screening identified 5 gene clusters with the potential to produce secondary metabolites. Chemical techniques were able to confirm three of the metabolites, while the other two clusters are inactive due to a fatal point mutation in gene that makes a required precursor molecule for the synthetic clusters. Overall, the study highlights the growing interdisciplinary nature of studying the interactions of biocontrol organisms.

Transcriptional responses to the ingestion of Cry1Ab protoxin and Cry1Ab corn leaves in the gut of Ostrinia nubilalis larvae
Jianxiu Yao1, Chitvan Khajuria2, Lawrent L Buschman3Kun Yan Zhu3, (1) Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, United States, (2) Department of Entomology, University of Nebraska, Lincoln, NE 68583, United States, (3) Department of Entomology, Kansas State University, Manhattan, KS 66506, United StatesWe developed a DNA microarray representing 2,755 gut-specific transcripts from the European corn borer (ECB, Ostrinia nubilalis) larvae. Our microarray analysis putatively identified 174 genes whose expression changed by at least 2 folds after the early fourth-instar larvae of a Cry1Ab-susceptible strain were fed the artificial diet containing Cry1Ab protoxin. Among these, 17 up-regulated genes appeared to be involved in insect defense, signaling, and transport, and 43 down-regulated genes may be involved in binding and other biological processes. A total of 13 genes, putatively encoding 8 different serine proteases, 1 cadherin-like protein, 1 alkaline phosphatase, and 3 aminopeptidases, are potentially involved in Bt toxicity or/and resistance. We further compared the gut transcriptional responses to the ingestion of transgenic Cry1Ab corn (MON810-event) leaves in the early third-instar larvae between a laboratory-selected resistant (R) strain and a susceptible (S) strain. We putatively identified 398 genes from the S strain and 264 genes from the R strain with significantly increased or decreased expression (at least 2.0 folds) levels. When both the S and R larvae were fed transgenic corn leaves, the number of differentially expressed genes and the degree of their expression changes were generally greater in the S larvae than in the R larvae. Among these genes, 17 in the S larvae and 9 in the R larvae were potentially involved in Bt toxicity or/and resistance. Two aminopeptidase genes (contig 4776 and contig 1398) were down-regulated in the S larvae, but up-regulated in the R larvae as compared with the control larvae after they were fed transgenic corn leaves for 6 h. These studies represent the first large-scale surveys of Cry1Ab protoxin and transgenic Cry1Ab corn-induced transcriptional changes in the ECB larval gut and provide a platform for functional investigation of Bt-insect interactions.

Mode of action of Bacillus thuringiensis toxins
Sarjeet S Gill,, Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, CA 92506, United StatesNumerous Bacillus thuringiensis strains are used for control of lepidopteran, dipteran, and coleopteran insects. In most strains insecticidal crystal proteins (Cry) are produced. In addition, these bacteria produce insecticidal proteins during the vegetative phase (Vips); and binary toxins that are structurally unrelated to Cry toxins also have been isolated. Further, all mosquitocidal strains produce cytolytic toxins (Cyt). These Cry, Cyt, Vip, and binary toxins are structurally, and appear to be evolutionarily, unrelated. Nevertheless, these toxins all act on the insect midgut, where they lyse epithelial cells affecting cell function. The Cry and Cyt toxins act in multistage processes. Crystals are ingested by larvae and dissolve in the alkaline midgut environment. The inactive protoxins are cleaved by proteases yielding active toxin fragments, which bind specific membrane receptors on the midgut epithelium. This binding is a key factor in determining Cry toxin selectivity. For Cry toxins, these receptors are cadherin proteins or ABCC transporters, and with alkaline phosphatases and aminopeptidases also contributing to binding. Cadherins, ABCCs and alkaline phosphatases apparently bind domain II of the Cry1A toxins, while aminopeptidases bind a carbohydrate-binding pocket in domain III. This binding leads to the formation of an oligomeric toxin that migrates to and inserts into lipid rafts in the cell membrane, thereby leading to the formation of lytic pores. In contrast, Cyt toxin binding is dependent on unsaturated phospholipids. The bound toxins then aggregate through toxin–toxin interactions leading to formation of lytic pores. Pore formation in both toxins result in paralysis of the insect and eventual larval death. Mutations of cadherin, ABCC and aminopeptidase proteins in a number of insects have resulted in insect resistance. The talk will focus on mechanism(s) of toxin action, resistance mechanisms, and implications for such mechanisms in the field use of Bacillus thuringiensis toxins in insect control programs.

Toxins for transgenic resistance to hemipteran pests
Nanasaheb P. Chougule1, Huarong Li2, Sijun Liu1, Lucas B. Linz1, Kenneth E. Narva2, Thomas Meade2Bryony C. Bonning1, (1) Department of Entomology, Iowa State University, Ames, IA 50011, United States, (2) Dow AgroSciences, Indianapolis, IN 46268, United StatesTransgenic crops expressing Bacillus thuringiensis (Bt) toxins have been widely adopted for management of lepidopteran and coleopteran pest species. In contrast, the sap sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. We demonstrated that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12 amino acid pea aphid gut binding peptide by adding to or replacing amino acids in one of three loops of Cyt2Aa resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.

Assessing the fate of RNA-based agricultural products in the environment

Josh Fischer,, Sam Dubelman, Bonnie Ayden, Fatima Zapata, Josh Uffman, JoAnne Warren, Steve Levine, Dave Carson. Monsanto Company, Saint Louis, MO 63167, United StatesEnvironmental fate data to characterize the extent of biodegradation of dsRNAs in environmental matrices is an essential element of a comprehensive environmental risk assessment of RNA-based agricultural products. The continued use of RNA-based gene suppression for agricultural products necessitates the development of novel analytical methods to determine the biodegradation potential of dsRNAs in the environment. Therefore, we have developed two complimentary techniques to assess the rate of degradation in soil of dsRNA from a biotechnology-derived in planta RNA-based insect-protected corn product. These techniques include a QuantiGene® assay to quantify the amount of dsRNA and an insect bioassay to measure functional toxicity. Results will be presented indicating that soil samples dosed with a dsRNA alone or with a dsRNA-corn tissue mixture demonstrate a loss of the parent dsRNA over time. Furthermore, results from insect bioassay indicate a corresponding time-dependent loss in biological activity. Results from complimentary methods support a lack of persistence of dsRNA in the soil environment and will be broadly useful for the exposure assessment needed to appropriately assess the environmental risk of RNA-based agricultural products.

Functional analysis of four RNAi pathway genes in an economically important corn pest, western corn rootworm (Diabrotica virgifera virgifera)
Chitvan Khajuria1,, Blair Siegfried1, Ken Narva2. (1) Department of Entomology, University of Nebraska, Lincoln, NE 68583, United States, (2) Dow AgroSciences LLC, Indianapolis, IN 46268, United StatesRNA interference (RNAi) is being considered as an important tool to be used in insect pest management. Better understanding of the RNAi pathway will provide information to use this technology effectively for pest management and to inform decisions related to resistant management strategies for RNAi based traits. Four genes (Dicer 2Argonaute 2,Sid 1A and Sid 1C) related to RNAi pathway were identified from transcriptome library generated from the gut of western corn rootworm (WCR). The expression of these genes was knocked down by injecting gene specific dsRNA into adult beetles, and then these adults were fed vATPase A dsRNA which has been demonstrated to cause mortality. The suppression of these genes apparently made the RNAi pathway less effective, thus reducing the mortality due to the second dsRNA treatment with vATPase A dsRNA. This research provides the basis for future studies aimed at improving RNAi technology as an insect resistance trait for WCR.

RNA interference in insect pest management: Assessing potential benefits and risks
Blair D Siegfried1,, Xuguo Zhou2, Chitvan Khajuria1, Jessica Jurzenski1. (1) Department of Entomology, University of Nebraska, Lincoln, NE 68583, United States, (2) Entomology, University of Kentucky, Lexington, KY 40546, United StatesWidely recognized as one of the premier functional genomics research tools, RNA interference (RNAi) has been used extensively in the post-genomics era to assign functions for genes annotated through small (expressed sequencing tags) or large (whole genome) scale sequencing efforts. Recently, the agricultural industry has recognized the potential to utilize RNAi as a mechanism to control the expression of target genes for pest control purposes resulting in a diversity of applications. Efficient delivery mechanisms, RNA stability, and RNA toxicity to the target organism remain as major technical challenges. However, a number of different approaches are being developed to overcome these challenges including transgenic crop plants that express RNAi traits (in planta RNAi). Although RNAi-based insect pest management technologies have yet to be commercialized, they are likely to become an important pest management tool that complements existing control practices including synthetic pesticides and Bt traits. This is especially important for target pest species, such as the western corn rootworm, where Bt traits are being challenged by resistance evolution. However, it is critical that the technology is used in a manner that is both sustainable and environmentally safe. The lack of a formalized/standardized ecological risk assessment (ERA) procedure remains as a major regulatory obstacle to integrate RNAi management approaches into sustainable pest management practices. An essential component of the ERA of RNAi plants involves in vivo RNAi toxicity testing under a defined worst-case scenario of exposure for both potential effects on non-target organisms and for resistance evolution. The studies described here are designed to answer questions directly pertaining to the risk of RNAi to non-target arthropods that are at greatest risk of exposure because of a shared environment and common molecular targets. In addition, studies to address the potential for resistance evolution are also described.

Natural nematicides and analogs
Joel R Coats1,, Angie Knips2, David Soo2, Rong Tsao1, Dong-Sik Park1, Greg Tylka2. (1) Entomology, Iowa State University, Ames, Iowa 50011, United States, (2) Plant Pathology, Iowa State University, Ames, Iowa 50011, United StatesPlants produce numerous defensive chemicals, some of which are active against insect or disease pests. Glucosinolates, cyanohydrins, and monoterpenoids are three classes of botanicals with activity against insects and nematodes. Bioassays were conducted on nematode pests in the laboratory, focusing primarily on the soybean cyst nematode. Laboratory studies determined that several glucosinolate aglycones from rapeseed, crambe, and crucifers had activity against the soybean cyst nematode and stored-grain insect pests, as did several natural cyanohydrins (from cassava, flax, almonds, lima beans, etc.). Some natural nematicides had sufficient volatility to express bioactivity through the vapor phase only. Synthetic analogs of glucosinolate aglycones and cyanohydrins were also screened for their bioactivity.

Mode of action and anthelmintic activity of novel plant-based functional food additives
Brooke W Bissinger1,, Ray M Kaplan2, Bob E Storey2, Ahmad Akashe3, Daniel J Skrypec3, Sheila M Mitchell1. (1) TyraTech, Inc., Morrisville, NC 27560, United States, (2) University of Georgia, Athens, GA, United States, (3) Mondelez International, Glenview, IL, United StatesAscaris lumbricoides is the most prevalent soil-transmitted helminth infection in humans, afflicting approximately 1.5 billion people and primarily burdening children in poor underdeveloped areas of the world. Synthetic anthelmintics are the current method of treatment; however, resistance could potentially develop. Challenges remain in providing inexpensive deworming treatments that do not require a complex and coordinated infrastructure; functional foods may help overcome this. A controlled study was performed in pigs infected with Ascaris suum, an important porcine pathogen closely resembling A. lumbricoides,to determine the efficacy of two microencapsulated essential oil blends, TTN1013 and TTN1014, as functional food compounds. Sixty-four Yorkshire-cross pigs aged 21–24 days were randomly assigned to one of four groups: TTN1013 at a dose rate of 0.5 or 1.0 mg/kg, TTN1014 at 1.0 mg/kg, or 1.0 mg/kg equivalent of empty capsules (placebo). Treatments were administered daily through week 10 inside cream-filled sandwich cookies. Pigs were inoculated with A. suum eggs 5 days/week for four weeks (20 eggs/kg/week) beginning three days post initial treatment. Pigs were necropsied during week 11. All worms were recovered, counted, measured for length and volume and separated according to sex. Fecal egg counts (FEC) were conducted weekly starting at week 6. Data were analyzed using a Poisson regression model accounting for group-dependent over-dispersion. TTN1013 at 1.0 mg/kg yielded a significant reduction in total (77%) and female (76%) worm counts, FEC (69%), and worm volume (63%) compared to the placebo. A dose dependent effect for TTN1013 was observed for these parameters, but differences at 0.5 mg/kg were not significant. TTN1014 did not have a significant effect. All pigs remained clinically normal and showed no adverse reactions or signs of reduced intestinal health. Based on these results, TTN1013 at 1.0 mg/kg shows promise as a daily supplement to reduce Ascaris infection in pigs and possibly humans.

Natural and synthetic isothiocyanates for nematode control
Scott R Yates1,, Dong Wang2. (1) US Salinity Laboratory, USDA-Agricultural Research Service, Riverside, CA 92507, United States, (2) San Joaquin Valley Agricultural Research Center, USDA-Agricultural Research Service, Parlier, CA, United StatesMethyl bromide (MeBr) had been widely used since the 1940s to control soil-borne pests prior to planting crops. Its success as a soil fumigant was largely due to its wide spectrum of control against plant pests and pathogens during many stages of life, the ability to rapidly penetrate soils, and the ease of application. However, in 1991, MeBr was identified as a stratospheric ozone-depleting compound, which has led to its phase-out in the United States. The MeBr phase-out has jeopardized production of high-valued crops, however, research is on-going to find suitable alternative chemicals and approaches to manage plant pests and pathogens. Alternative chemicals include a number of natural and synthetic pesticides, including isothiocyanates. Soil fumigation with alternative synthetic pesticides continues the conventional approach to prepare soil for planting. Biofumigation is an alternate agronomic practice that uses decomposing plant tissues (e.g., Brassica), or seed meal to produce volatile chemicals in soil. Isothiocyanates are the most common chemical produced from Brassica and are related to the active ingredient in commercial products, such as metam sodium and dazomet. Isothiocyanates have been shown to be toxic many soil-borne pests and pathogens. The purpose of this presentation is to provide an overview of some of the problems growers are facing in a post-MeBr world, provide information on the effectiveness of natural and synthetic isothiocyanates in controlling plant pests and pathogens, and to offer possible areas of future research that may assist in protecting the environment and lead to the continued use of this important class of chemicals.

Phytochemically based strategies for nematode control
David J Chitwood,, Nematology Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705, United StatesOne of the most important problems facing global agricultural productivity is the lack of chemically based control strategies for nematodes, a consequence of deregistration of numerous chemical nematicides because of environmental or health-associated problems. Because plant-based products or compounds are often safer than synthetic ones and consequently receive less regulation, researchers are utilizing several strategies for exploiting phytochemicals as nematode control agents. Specific experimental or practicable materials include the incorporation of nematode-antagonistic plant amendments to soil, the application of plant extracts or plant-based formulations, or the direct application of compounds discovered to be nematotoxic. Like any chemical nematicide, the ideal phytochemical or active analog would be inexpensive, effective only against agricultural pests and pathogens, and able to move in soils and persist in a manner that is agriculturally effective yet environmentally safe. Specific compounds and products will be emphasized.

Potent nematicidal activity of redox-active aromatic aldehydes against Meloidogyne incognita
Pierluigi Caboni,, Barbara Liori, Nikoletta Ntalli. Department of Life and Environmental Sciences, University of Cagliari, Cagliari, ItalySustainable agriculture providing with food quality and sufficiency is a major challenge for farmers, agro-industries, researchers, and governments. Synthetic nematicides have long had application in plant protection for the control of the root-knot nematodes (Meloidogyne spp.). Phytonematodes represent one of the most damaging pests causing more than $100 billion of crop losses in fruit and vegetable production annually. In the recent years, the environmental, food safety, and animal welfare issues pose the need for alternative nematode control measures. Plant secondary metabolites, involved in the complex chemical-mediated interactions between a plant and other organisms in its environment, can provide bioactive leading compounds to be studied further as innovative nematode control measures in Integrated Pest Management (IPM) programs. Here, we report the nematicidal activity of selected aromatic aldehydes against the root knot nematode Meloidogyne incognita. The most active aldehyde was phthalaldehyde with an EC50 value of 10.6 ± 5.9 mg/L followed by salicylaldehyde and cinnamic aldehyde with an EC50of 10.8 ± 1.0 and 12.1 ± 5.3 mg/L, respectively. The reactivity of tested aldehydes against a synthetic peptide resembling the nematode cuticle was characterized by means of liquid chromatography mass spectrometry. We report that at the test concentration of 1 mM, the main adduct formation was observed for 3,4-dihydroxybenzaldehyde, 2-methoxybenzaldehyde, and 3,4-dimethoxybenzaldehyde. Considering that 2-methoxybenzaldehyde and 3,4-dimethoxybenzaldehyde were not active against M. incognita in in vitro experiments, we hypothesize a different mechanism of action rather than an effect on the external cuticle modification of nematodes. The results of this investigation reveal that aromatic redox-active aldehydes can be considered as potent nematicides, and further investigation is needed to clarify their mode of action.

G-Protein-Coupled Receptors (GPCRs): A target of plant terpenoids
Aaron D Gross1,, Michael J Kimber2, Kevin B Temeyer3, Robert J Miller3, Andrew Y Li3, Adalberto A Pérez de León3, Joel R Coats1. (1) Entomology, Iowa State University, Ames, Iowa 50011, United States, (2) Biomedical Sciences, Iowa State University, Ames, Iowa 50011, United States, (3) USDA-Agricultural Research Service, Kerrville, Texas 78028, United StatesPlants have evolved beneficial and protection mechanisms including the production of essential oils. Essential oils are the odiferous component of plant extracts, which give plants a variety of unique properties. Essential oils are composed of various terpenoid compounds, particularly monoterpenoids and related aromatic compounds, along with sesquiterpenoids. A variety of terpenoids have been shown to have a toxic effect against insects. It is thought that this toxic action occurs through a neurological mechanism of action. The presented research will focus on arthropod GPCRs which have been and continue to be an under-utilized target for pest control.

Neuropeptides and receptors as targets for bioinsecticidesRonald J Nachman,, Southern Plains Agricultural Research Center, USDA-Agricultural Research Service, College Station, TX 77845, United StatesInsect neuropeptides regulate critical processes and behaviors in insects, though they are unsuitable as tools for arthropod endocrinologists and/or as pest management agents due to poor biostability and/or bioavailability characteristics. Peptidomimetic and non-peptide analogs can overcome these limitations and either over-activate or block critical neuropeptide-regulated functions. Stereochemical and conformational aspects critical for the successful receptor interaction of several classes of insect neuropeptides were exploited to design/discover mimetic analogs with enhanced biostability and selectivity. These classes include the insect kinins, insectatatachykinins (or tachykinin-related peptides), and the pyrokinins. While natural, unmodified peptides of these three classes demonstrate little or no activity, biostable versions elicit potent antifeedant and/or insecticidal properties in insects such as aphids when delivered by an oral route. Some biostable analogs are inactive and do not elicit significant antifeedant or aphicidal effects. In some cases, the aphicidal effects can be blocked by neuropeptide antagonists, providing compelling evidence that the insecticidal activity is specific and mediated via a neuropeptide receptor. The mimetic analogs provide important leads for alternative aphicides. Diapause hormone (DH), an important sub-class of the pyrokinins, has been shown to terminate pupal diapause in heliothine species. Potent biostable analogs have been designed that can prevent the onset of pupal diapause via treatment of the preceding larval stage of a heliothine insect. Conversely, an antagonist, developed by a novel conformation-based strategy, can block the pupal diapause termination activity of DH. The observed activity of both agonists and antagonists is dependent on the presence of a DH core sequence. These mimetic DH analogs provide leads for the generation of agents capable of disrupting the protective state of diapause in economically important lepidopteran pests.

Natural product-based insecticidal structure activity relationship investigations
Charles L Cantrell1,, Abbas Ali3, Julia W Pridgeon2, Junaid U Rehman3, Hiroshi Nakano4. (1) Natural Products Utilization Research Unit, USDA-Agricultural Research Service, University, MS 38677, United States, (2) Aquatic Animal Health Research Unit, USDA-Agricultural Research Service, Auburn, AL 36832, United States, (3) School of Pharmacy, University of Mississippi, National Center for Natural Products Research, University, MS 38677, United States, (4) 2-1-18 Kannondai, NARO Institute of Crop Science, Tsukuba, Ibaraki 305-8518, JapanAn Aedes aegypti larval toxicity bioassay was performed on compounds representing many classes of natural compounds including polyacetylenes, phytosterols, thiophenes, flavonoids, sesquiterpenoids, and triterpenoids. Among the compounds studied, two eudesmanolides, alantolactone and isoalantolactone, as well as many thiophenes showed larvicidal activities against Ae. aegypti and therefore were chosen for further structure-activity relationship investigations. Structural modifications were performed on both alantolactone and isoalantolactone in an effort to understand the functional groups necessary for maintaining and/or increasing its activity and to possibly lead to more effective insect control agents. Structure-activity evaluations of thiophenes found relationships between the number of thiophenes rings and acetoxy groups.

Plant-derived products for control of ticks and biting flies affecting livestock
Andrew Y Li1,, Livio M Costa-Júnior2, Lígia MF Borges3, Adalberto A Pérez de León1. (1) US Livestock Insects Research Laboratory, USDA-Agricultural Research Service, Kerrville, Texas 78028, United States, (2) Center for Agricultural and Environmental Sciences, Federal University of Maranhao, Boa Vista, Chapadinha-MA CEP 65500-000, Brazil, (3) Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiás, Goiânia - GO CEP 74001-970, BrazilLivestock production in many parts of the world, including the Americas, is severely affected by blood-feeding ectoparasites. The cattle tick Rhipicephalus (Boophilus) microplus, the horn fly Haematobia irritans irritans, and the stable fly Stomoxys calcitrans are economically important ectoparasites affecting beef and dairy cattle production systems. Their control has relied on synthetic chemical pesticides. However, the indiscriminate use of synthetic pesticides has led to the development of pesticide resistance. New chemical entities with novel modes of action are needed to manage problems associated with pesticide resistance. This review focuses on essential oils and other plant-derived natural products under evaluation against these target species. Essential oils extracted from several plant species have been tested for activity against different life stages of the cattle tick in different countries. The majority of this work has been done using in vitro assays, and few essential oil preparations have been tested on animals. The major chemical components of some essential oils have been identified, and the activity of individual compounds profiled against tick or biting fly species. Essential oils and related compounds demonstrated various levels of activity against livestock pests. Natural products tend to be evaluated as contact pesticides, and some of essential oils and related natural compounds were shown to be highly effective repellents against biting flies. velopment in several countries for livestock pest control. The commercialization of plant-derived products for use in livestock pest control faces several challenges including an inferior efficacy profile under field conditions, relatively higher production costs, and supply issues to meet market demand. Little is known about the modes of action of plant-derived compounds shown to be toxic to livestock pests. It is expected that recent progress in toxicological and pharmacological studies will help elucidate the modes of action of these promising plant-derived natural products.


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