1a. Objectives (from AD-416):
OBJECTIVE 1: Develop semiochemicals (pheromones, kairomones including plant volatiles, and other attractants and repellents) of significant landscape pests (such as brown marmorated stink bug and exotic scarabaeids) and their natural enemies (e.g., parasitoids and spiders) for monitoring and management. Sub-objective 1.A: Identify and synthesize volatiles and secretions from ornamental landscape pests or their plant hosts that are important in host- or mate-finding behavior, as components for biologically based control strategies. Sub-objective 1.B: Develop effective strategies for using synthesized semiochemicals to reduce plant damage due to ornamental landscape insect pests. OBJECTIVE 2: Develop and genomically characterize effective microbial and arthropod natural enemies as biocontrol agents, including bacteria (e.g., Bacillus thuringiensis [Bt]; Chromobacterium spp.), baculoviruses, and other entomopathogens, against insect pests of landscape plants (such as European and Asian gypsy moth, brown marmorated stink bug, and winter moth). Sub-objective 2.A: Discover additional environmental isolates of C. subtsugae and related species, and characterize their insecticidal properties. Sub-objective 2.B: Make phylogenetic and pathogenic comparisons of soil and phylloplane populations of Bt. Sub-objective 2.C: Evaluate and develop viral pathogens as potential control agents for selected lepidopteran pests in unmanaged urban forest landscapes. Sub-objective 2.D: Compare the floral and extrafloral resources of native and exotic urban landscape plant species for their ability to support natural enemies. Sub-objective 2.E: Use molecular gut-content analysis to identify brown marmorated stink bug egg predators. OBJECTIVE 3: Employ new technologies to characterize genomes and transcriptomes, characterize gene expression patterns, and develop RNAi-based molecular biopesticides for control of insect pests of landscape plants, especially brown marmorated stink bug and gypsy moth. Sub-objective 3.A: Characterize and generate draft reference genome for brown marmorated stink bug (BMSB) and gypsy moth. Sub-objective 3.B. Develop RNAi-based microbial biopesticides targeting brown marmorated stink bug (BMSB) and European and Asian gypsy moth.
1b. Approach (from AD-416):
This highly interdisciplinary project combines concepts and methodologies from analytical and synthetic organic chemistry, insect chemical behavior, insect pest-pathogen genomics, and microbial and insect ecology to develop multiple strategies for managing key insect pests of urban landscapes, orchards, and surrounding forest remnants. Integrated pest management approaches to be developed will include: (1) discovery and deployment of insect- and plant-derived attractants and repellents; (2) conservation of parasitic and predatory insects and spiders through selection of host plants for their ability to support natural enemies and thereby to promote effective biocontrol of pests; (3) pest-specific microbial controls including bacteria (e.g., Bacillus thuringiensis strains and Chromobacterium spp.) and baculoviruses; and, (4) plant genetic resistance using molecular-based gene discovery. Research will target insect pests that cause major damage to woody plants, including key landscape and forest genera, as well as fruit-bearing trees. The combination of semiochemical approaches, biological controls, molecular techniques, and crop resistance will offer a range of non-chemical tactics useful to integrated pest management for major pests of urban landscapes and associated orchard and forest ecosystems.
3. Progress Report:
Objective 1. Develop semiochemicals of significant landscape pests and natural enemies for monitoring and management. The use of the brown marmorated stinkbug (BMSB) aggregation pheromone is an essential element in managing this important agricultural and nuisance pest. The pheromone was identified earlier as a mixture of two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol with 7R configuration, both of which are important for attractiveness. However, manufacture of the individual stereoisomers is very cumbersome and expesive. ARS scientists in Beltsville, Maryland, have determined that some stereoisomers of 10,11-epoxy-1-bisabolen-3-ol with the 7S configuration can also be attractive to BMSB, allowing for the more economical synthesis of racemic mixtures containing both 7R and 7S 10,11-epoxy-1-bisabolen-3-ols for trapping BMSB. ARS scientists in Beltsville, Maryland, have discovered new “green” insecticides and evaluated their toxicities. A U.S. patent for the use of methyl benzoate as an insecticide has been granted, and fourteen additional benzoic acid esters, nine of which are naturally occurring, were found to exhibit varying toxicities to spotted wing drosophila, brown marmorated stinkbug, diamondback moth, tobacco hornworm, gypsy moth and fire ants. An additional U.S. provisional patent application was filed covering the use of these methyl benzoate analogs for insect control. Objective 2. Develop microbial and arthropod natural enemies as biocontrol agents of native and exotic pests of landscape plants. A winter moth baculovirus was isolated and characterized by analysis of its genomic sequence and microscopic analysis of infectious particles. Electron microscopy revealed that purified winter moth baculovirus also contained particles of a cypovirus, indicating that these viruses co-infect and replicate together in winter moth larvae. Analysis of the genome sequence revealed that the winter moth baculovirus is part of an ancient lineage within genus Alphabaculovirus. Inoculation of brown marmorated stink bug nymphs with an iflavirus isolated from adult stink bugs failed to cause any noticeable pathology or mortality. Bioassays of a gypsy moth baculovirus isolate from Harbin, China revealed that it possessed a degree of insecticidal activity against Asian gypsy moth larvae from Harbin that was 7- to 11-fold greater than that of the gypsy moth virus isolate used in the Gypchek biopesticide. Genomic analysis of the Harbin gypsy moth baculovirus indicated that it is most closely related to gypsy moth baculoviruses from Japan and Korea. Chromobacterium isolates from diverse aquatic habitats were screened for insecticidal activity against diamondback moth, seedcorn maggot and red flour beetle. An isolate of a new species identified previously has shown activity against all three of the test species, with activity against diamondback moth significantly greater than C. subtsugae. Chromobacterium genomes were analyzed for the occurrence of polyketide synthases and non-ribosomal peptide synthases which are likely responsible for the biosynthesis of the insecticidal factors. Objective 3. Employ new technologies to characterize genomes and transcriptomes, characterize gene expression patterns, and develop RNAi-based molecular biopesticides for control of insect pests of landscape plants, especially brown marmorated stink bug and gypsy moths. In collaboration with scientists in Canadian institutions, including Natural Resources Canada, Université Laval and McGill University, ARS scientists in Beltsville, Maryland, developed molecular markers from the mitochondrial genome of European and Asian strains of the gypsy moth (Lymantria dispar) that will allow the identification of a likely geographic origin of specimens encountered in international commerce. Research aimed at identifying nuclear encoded genes useful in identifying a gypsy moth specimen’s region of origin is proceeding using the assembled transcriptomes of seven distinct geographic races of the forest pest. ARS scientists in Beltsville, Maryland, made progress in methods development for the delivery of RNA interference (RNAi)-based molecular biopesticides using non-transgenic approaches for the control of invasive insect pests of agriculture, including the brown marmorated stink bug (BMSB), a devastating exotic and invasive landscape pest. RNAi deactivates or silences specific genes to disable a target insect. Availability of methods for sustainable delivery of dsRNA into insects for their control has been an impediment to its use. Regulatory processes involved in using transgenic plants for delivery may be laborious and challenging. Expanding earlier methods we developed using vegetables as a vehicle for oral delivery of dsRNA through feeding (Ghosh et. al., 2017), ARS scientists in Beltsville, Maryland, and Ft. Pierce, Florida, collaboratively demonstrated new methods for oral delivery and persistence of dsRNA to a wide range of insects. These non-transgenic plant delivery methods included foliar sprays, root drenches, trunk injections, and clay granules for sustainable and persistent delivery of dsRNA. These techniques were demonstrated by video to the greater scientific community to enhance development of RNAi-based molecular biopesticides for invasive insect pests. As part of the international i5K working group effort, ARS scientists in Beltsville, Maryland, constructed a draft genome for the brown marmorated stink bug, Halyomorpha halys. The 1.2 Gb brown marmorated stink bug (BMSB) genome sequence, with eleven worldwide university and government research institutions participating, is in its final stages of development: annotation and analysis work by the stink bug genome annotation consortium has been completed and communicated with the ARS project coordinators. A cohesive BMSB genome manuscript is in preparation for journal submission.
1. Discovery of a new class of safer insecticides. Methyl benzoate is a volatile organic compound that exists naturally as a floral fragrance in many plants. Building on their earlier discovery showing that methyl benzoate is toxic to a variety of insects, ARS scientists in Beltsville, Maryland, have examined an additional 14 analogs of methyl benzoate and found that they have insecticidal properties as well. Methyl benzoate and some of its analogs are more toxic to gypsy moth larvae and brown marmorated stinkbug nymphs than commercial insecticides. Studying the relationship between structure and activity of the methyl benzoate analogs may reveal important clues about the insect repelling activity of DEET, which is also a benzyl ester, as well as allowing researchers to improve the efficacy of this new class of insecticides.
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Blackburn, M.B., Farrar, R.R., Sparks, M., Kuhar, D.J., Mitchell, A.D., Gundersen, D.E. 2017. Chromobacterium muskegensis sp. nov., an insecticidal bacterium isolated from Sphagnum bogs. International Journal of Systematic and Evolutionary Microbiology. 67(9):3417-3422.
Feng, Y., Chen, J., Zhang, A. 2018. Commercially available natural benzyl esters and their synthetic analogs exhibit different toxicities against insect pests. Scientific Reports. https://doi.org/10.1038/s41598-018-26242-6.
Ghosh, S.B., Gundersen, D.E., Park, A.L., Hunter, W.B. 2018. Double strand RNA oral delivery methods to induce RNA interference in phloem and plant-sap-feeding insects. Journal of Visualized Experiments. https://doi.org/10.3791/57390.
Greenstone, M.H., Cornelius, M.L., Olsen, R.T., Payton, M.E. 2017. Test of a natural enemy hypothesis on plant provenance: Spider abundance in native and exotic ornamental landscapes. Journal of Entomological Science. 52(4):340-351.
Hock, V., Chouinard, G., Lucas, E., Cormier, D., Leskey, T.C., Zhang, A. 2017. Olfactometer responses of plum curculio Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae) to host plant volatiles, synthetic grandisoic acid, and live conspecifics. Journal of Insect Behavior. https://doi.org/10.1007/s10905-017-9634-0.
Jaffe, B.D., Avanesyan, A., Bal, H.K., Grant, J., Grieshop, M.J., Lee, J.C., Liburd, O.E., Rhodes, E., Rodriguez-Saona, C., Sial, A.A., Feng, Y., Zhang, A., Guedot, C. 2018. Multistate comparison of attractants and the impact of fruit development stage on trapping Drosophila suzukii (Diptera: Drosophilidae) in raspberry and blueberry. Environmental Entomology. https://doi.org/1093/ee/nvy052.
Rice, K.B., Bedoukian, R.H., Hamilton, G.C., Jentsch, P., Khrimian, A., Maclean, P., Morrison III, W.R., Short, B.D., Shrewsbury, P., Weber, D.C., Wiman, N., Leskey, T.C. 2017. Enhanced response of Halyomorpha halys (Hemiptera: Pentatomidae) to its aggregation pheromone with ethyl decatrieonate. Journal of Economic Entomology. 111(1):495-499. https://doi.org/10.1093/jee/tox316.
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