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 aggregation pheromone of the brown marmorated stinkbug is an essential tool for managing this important invasive agricultural and nuisance pest. The pheromone identified earlier consists of two principal components, both of which are important for the attractiveness, however the existing synthetic method only provided satisfactory yields for the major component. To allow an efficient scale-up of pheromone production, ARS scientists in Beltsville, Maryland, developed a new synthetic procedure that provides both components in appreciable yields for bioassays. It was determined that methyl benzoate isolated from apple juice, and naturally-occurring in many other plants, possessed toxicity against various stages of a variety of insect pests, including the invasive brown marmorated stinkbug (BMSB) and spotted wing drosophila (SWD). It was found that methyl benzoate was at least 5 to 20 times more toxic than a conventional pyrethroid (ß-cyfluthrin), sulfur & pyrethrin mixture, and some commercial organic products available on the market against BMSB eggs and SWD larvae and adults. ARS obtained a U.S. Patent (US 9629362 B1) covering this technology. In addition, an attractant blend was developed for spotted wing drosophila (Patent applied for), and a pheromone was identified for the peanut burrower bug. Objective 2. Develop microbial and arthropod natural enemies as biocontrol agents of native and exotic pests of landscape plants. Chromobacterium were cultured from diverse aquatic habitats and screened for insecticidal properties. Genomes of two new insecticidal Chromobacterium isolates displaying toxicity to seedcorn maggot, a significant seedling pest of corn and other crops, were sequenced and fully assembled. Alignment of these genome sequences with those of other species of Chromobacterium revealed that these new strains represent previously unknown species. Two patent applications were filed for the use of two other species of Chromobacterium, identified in prior years, for insect control. Particles of a brown marmorated stinkbug iflavirus were extracted from adult stinkbugs from a laboratory colony of the pest. Reverse transcriptase-polymerase chain reaction experiments confirmed the presence of the iflavirus in the particle preparation. Electron microscopy of the particle preparation revealed the presence of icosahedral virus-like particles occurring in two distinct sizes, 30 nm and 40 nm in diameter. Next-generation sequencing of gypsy moth baculovirus field isolate 3029 revealed the presence of a Bacillus sp. bacterium in the field isolate. This bacterium was not present in clones of 3029 derived from tissue culture, but was present in some of the other field isolates of gypsy moth baculovirus. Cultures of this Bacillus sp. exhibited no pathogenicity against gypsy moth larvae in bioassays. Restriction endonuclease digest analysis of 3029 clones revealed genetic polymorphisms among the different clones, but bioassays of the 3029 clones did not reveal consistent differences in pathogenicity against gypsy moth larvae. Baculovirus bioassays were carried out against larvae of Asian gypsy moth colonies reared in quarantine. Differences were observed in the susceptibilities of larvae from different colonies to different gypsy moth baculovirus isolates. 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. ARS scientists in Beltsville, Maryland, made progress in implementing RNA interference (RNAi) for control of brown marmorated stink bug (BMSB), a devastating exotic and invasive landscape pest. RNAi deactivates or silences specific genes to disable a target organism. Using a newly developed oral delivery method, scientists tested RNAi-mediated silencing of specific genes critical to BMSB survival. Significant depletion in levels of transcripts was observed upon feeding gene-specific dsRNA to BMSB nymphs. Specifically of interest were genes in biosynthetic pathways that may be controlled by other precursors in the pathway; silencing of these genes affected BMSB nymphal-adult development ultimately leading to mortality. Thus, gene-specific dsRNAs inducing RNAi can be deployed in the environment as orally delivered molecular biopesticides against BMSB and other invasive insect pests ARS scientists in Beltsville, Maryland, generated a preliminary draft nuclear genome assembly of the European strain of gypsy moth (EGM) (Lymantria dispar), which was compared with an unpublished assembly of an Asian strain of gypsy moth (AGM). RNA-Seq information for the AGM strain was produced and used by ARS scientists in Beltsville, Maryland, to improve gene structure predictions associated with the AGM genome, which in turn will be used to inform gene predictions of the EGM variant. This ongoing work is being performed in collaboration with scientists in various Canadian institutes, including Natural Resources Canada, Université Laval and McGill University. ARS scientists in Beltsville, Maryland, continued an international i5K working group effort to assemble and annotate the 1.2 Gb brown marmorated stink bug (BMSB) genome sequence, with eleven worldwide university and government research institutions participating. This group has already discovered that, consistent with its generalist dietary habits, BMSB contains the largest number of gustatory receptors that has been observed among sequenced arthropods to date. Aside from managing the overall project, ARS scientists in Beltsville, Maryland, annotated genes from BMSB’s carboxylesterase and cytochrome P450 gene families. In conjunction with previous results associated with the glutathione S-transferase family, these findings were used to facilitate an interspecific comparison of xenobiotic detoxification genes in the BMSB with the closely related hemipteran insect pest, the harlequin bug.
1. New delivery system for giving molecular biopesticides to brown marmorated stink bug (BMSB) and other hemipteran insect(s) through feeding. The brown marmorated stinkbug (BMSB) is an invasive insect native to Asia that has emerged as a very prominent insect pest in the United States. BMSB feeds on many plants and poses a considerable threat to specialty crops (apples, stone and pome fruits, grapes, ornamental plants, vegetables, seed crops) as well staple crops (soybean and corn), and is an aggravating indoor nuisance pest. New molecular genetic materials designed to deactivate or silence specific genes critical to BMSB survival were designed; however, methods for delivering these to BMSB by feeding were needed to use them for insect biocontrol. ARS scientists in Beltsville, Maryland, developed a method for delivering molecular biopesticides derived from these genetic materials to BMSB and other hemipteran or sap-feeding insects through feeding. This method allowed for dispersal of insect-controlling genetic materials to insect pests without need to create genetically modified insects. This method could enable widespread deployment of BMSB molecular genetic biopesticide technologies to control this now ubiquitous invasive pest.
2. Methyl benzoate is toxic to invasive insect pests. ARS scientists in Beltsville, Maryland, discovered that a chemical methyl benzoate, isolated from apple juice and naturally-occurring in many other plants, was toxic to various stages of a variety of insect pests, including the invasive brown marmorated stinkbug (BMSB) and spotted wing drosophila (SWD). Against BMSB eggs and SWD larvae and adults, methyl benzoate was found to be 5 to 20 times more commercial organic products available on the market. Because methyl benzoate is considered environmentally friendly, it has great potential to be used as a safer alternative to synthetic pesticides for sustainable agriculture, reducing threats to human health and the environment caused by over-application of conventional synthetic pesticides.
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