Research Programs and Projects at this Location : USDA ARS

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Research Project: Urban Small Farms and Gardens Pest Management

Location: Invasive Insect Biocontrol & Behavior Laboratory

2018 Annual Report

Objectives
Objective 1: Identify, synthesize, and develop semiochemicals (pheromones, kairomones, plant volatiles, and other attractants and repellants) of significant insect crop pests (such as harlequin bug, bagrada bug, spotted wing drosophila, striped cucumber beetle, kudzu bug) and their natural enemies for use in integrated pest management. Subobjective 1a: Develop attractant volatiles and trap designs to monitor and manage the harlequin bug. Subobjective 1b: Assess known stink bug attractants for value in monitoring and/or managing bagrada bug. Subobjective 1c: Identify plant volatiles that attract squash bugs. Subobjective 1d: Identify plant and/or insect volatiles for detection and attraction of kudzu bug. Subobjective 1e: Improve lures for spotted wing drosophila by identification of fruit-based attractants. Subobjective 1f: Synthesize and test aggregation pheromone in combination with baits and traps for striped cucumber beetle management. Objective 2: Develop arthropod biological controls for managing key vegetable pests such as stink bugs, squash bug, and cucumber beetles, including integration of natural enemies with other tactics such as microbial control, semiochemicals, and cultural pest controls. Subobjective 2a: Evaluate the efficacy of the squash bug natural enemy complex for use in pest management and identify the most effective biological control agents. Subobjective 2b: Assess the value of native egg parasitoids to suppress BMSB. Objective 3: Advance effective microbial controls for key crop pests such as stink bugs, noctuid moths, and other seedling pests, including discovery of molecular and ecological mechanisms for sustained field reproduction and persistence, using diverse Bacillus thuringiensis (Bt) strains, Chromobacterium spp., baculoviruses, and other entomopathogens. Subobjective 3a. Discover additional environmental isolates of C. subtsugae and related species, and characterize their insecticidal properties. Subobjective 3b. Determine ability of Bt strains to persist in common vegetable mulches. Subobjective 3c. Develop effective baculoviruses for key pest diamondback moth. Objective 4: Discover naturally occurring biopesticides (such as botanical compounds and/or RNAis) targeting key vegetable pests such as stink bugs and cucumber beetles. Objective 5: Determine and strengthen the genetic basis of plant defense mechanisms (e.g., pest aversion and resistance) for protection of high-value crops such as cole crops on small farms or gardens against destructive insects, and determine the influence of the microflora of pest insects and/or plants on the plant defense response. Subobjective 5a: Determine the effect of knocking out or over-expressing the infestation responsive TF StZFP2 in its native species, and in Arabidopsis. Subobjective 5b: Determine how infestation inducible Q-type C2H2 TFs affect resistance to pest insects in Arabidopsis and B. oleracea. Subobjective 5c: Determine the influence of plant and insect microflora on the infestation response of Arabidopsis to T. ni.

Approach
The project brings together a research team with diverse expertise for multiple approaches to insect management. The proposed project will focus on control of key insect pests in small farms and urban gardens, both organic and non-organic. Bio-based integrated pest management approaches to be developed will include: (i) discovery and deployment of natural insect attractants and repellents; (ii) conservation and augmentation of beneficial insects including use of their semiochemicals; (iii) pest-specific microbial controls [bacteria (including Bacillus thuringiensis strains and Chromobacterium spp.) and baculoviruses]; and, (iv) crop genetic resistance using molecular-based gene discovery. Research will target insect pests that cause major damage to key crops such as cucurbits and cole crops, although other important crops such as small fruit, beans, and potatoes may receive attention for specific problems. 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 strategies for major crop pests in urban small farms and gardens.

Progress Report
Objective 1: Identify, synthesize, and develop semiochemicals (pheromones, kairomones, plant volatiles, and other attractants and repellants) of significant insect crop pests and their natural enemies for use in integrated pest management. The aggregation pheromone of the harlequin bug (Murgantia histrionica) is a powerful tool for monitoring and managing this important vegetable pest. The pheromone identified earlier consists of two principal components, both of which are important for the attractiveness. To allow an efficient scale-up of pheromone production, ARS scientists in Beltsville, Maryland, developed a new synthetic procedure that provides both components in improved yields for bioassays. Due to the overlap in chemical composition with brown marmorated stink bug pheromone, this new synthesis is valuable to management of both species. Field attraction of bagrada bug (Bagrada hilaris) to 10,11-epoxy-1-bisabolen-3-ols which contain pheromones of the brown marmorated stink bug and harlequin bug, was not reproduced by California cooperators, and therefore an APHIS-permitted colony of this western vegetable pest was obtained for further ecological, behavioral, and semiochemical study starting in July 2017. A study of the ovipositional preferences of two squash bug species, Anasa tristis and Anasa armigera, for different cultivars and species of plants in the family Cucurbitaceae was completed. This study provided evidence that A. tristis had an induced preference for the cultivar it was reared on compared with several other cultivars of the same species. Also, A. tristis had a significant preference in paired choice tests for Cucurbita pepo over cucumber and watermelon, but not C. maxima or C. moschata. This study is the first experimental evidence of the ovipositional preferences of the horned squash bug, A. armigera. Horned squash bugs did not show any preference for cucumber over C. pepo, regardless of which species they were reared on. However, they did show a significant preference for cucumber over watermelon and C. moschata, and they preferred C. maxima to cucumber. Ovipositional preference tests provided the groundwork for further research on the responses of squash bugs to host plant volatiles. Methyl benzoate isolated from apple juice, and naturally-occurring in many other plants, was found to possess toxicity against various stages of a variety of insect pests, including the invasive brown marmorated stinkbug (BMSB) and spotted wing drosophila (SWD). 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). SWD research is supported by the USDA NIFA CPPM grant, “Managing an Invasive Drosophilid Species in Agriculture using Innovative Behavioral Manipulation.” A pheromone was identified for the peanut burrower bug, the first for this family of true bugs (Cydnidae). Research is supported by the USDA NIFA CPPM grant, “Development of Risk Assessment and Monitoring Tools for Peanut Burrower Bug in Southeastern U.S. Peanut Production Systems.” Objective 2: Develop arthropod biological controls for managing key vegetable pests such as stink bugs, squash bug, and cucumber beetles, including integration of natural enemies with other tactics such as microbial control, semiochemicals, and cultural pest controls. A two-year study of parasitism and predation rates on wild and sentinel eggs of the squash bug, Anasa tristis, in squash fields at BARC was completed and the results were published in 2016. Gryon pennsylvanicum accounted for 99% of the egg parasitism. The egg parasitism rate peaked in late July at 72.8%. In addition, laboratory studies evaluating the efficacy of Gryon pennsylvanicum as a biological control agent have been completed. In the laboratory, the average parasitism rate of a single female without prior exposure to eggs was 59.6%, resulting in a squash bug nymphal survival rate of 22.9%. There were significant differences in the parasitism rate based on egg size of squash bug egg masses, with parasitism rates ranging from < 20% for the largest masses and > 90% for the smallest. A two-year field study of sentinel egg masses of the pest brown marmorated stink bug, and the beneficial stink bug, spined soldier bug (SSB, Podisus maculiventris), deployed in 2015-16 to field sites in Maryland, Virginia, DC, Delaware, and West Virginia, has yielded lesser numbers of the adventive Asian parasitoid, Trissolcus japonicus, in the DC region, but new state records for Delaware and West Virginia. This makes the redistribution of the parasitoid within these states allowable for BMSB suppression. However, T. japonicus only attacks BMSB in certain habitats and is not yet abundant. It successfully attacks the beneficial sentinel egg masses of SSB, however, from laboratory rearings these eggs are too small for optimal parasitoid success. Successful emergence of some native egg parasitoids and higher rates of egg mass consumption by native predators was also notable. Further research is supported by USDA NIFA SCRI CAP grant, “Management of BMSB in US Specialty Crops,” through North Carolina State University in cooperation with several other universities and ARS labs. Objective 3: Advance effective microbial controls for key crop pests such as stink bugs, noctuid moths, and other seedling pests, including discovery of molecular and ecological mechanisms for sustained field reproduction and persistence, using diverse Bacillus thuringiensis (Bt) strains, Chromobacterium spp., baculoviruses, and other entomopathogens. Chromobacterium species were isolated from diverse aquatic habitats and characterized by genomic sequencing, biochemical properties, and their insecticidal properties against diamondback moth, seedcorn maggot and red flour beetle. Two previously unknown species toxic to seedcorn maggot were identified in FY17 by comparing their genomic sequences with known Chromobacterium species. These newly described species can be developed into organic insecticides. To date, four new species of insecticidal Chromobacterium have been identified with lepidopteran and/or dipteran toxicity. The phylogenetic relationship of these species with other species Chromobacterium has been analyzed by analysis of their genomic sequences. A manuscript describing the first of these, C. sphagni, has been accepted for publication. The target pests for Bacillus thuringiensis toxins or modified toxins has been broadened by ARS work with University of Florida with support from a USDA NIFA SCRI grant, “Bt toxin-based strategies for management of Diaphorina citri and citrus greening.” Toxins with promise for control of psyllids, including for control of Asia citrus psyllid, the vector of the devastating citrus greening disease, huanglongbing, will be used to develop resistance in citrus. Determination and analysis of the genomes of two baculoviruses from the true armyworm were completed. A novel virus gene that may be involved in protection of viral DNA during infection was identified. Evidence emerged of extensive exchange of genes among the genomes of different true armyworm and Oriental armyworm baculoviruses. Old World bollworm betabaculovirus isolates from the IIBBL collection were evaluated for pathogenicity in bioassays against bollworm larvae. A group of isolates deposited in 1973, 1978, and 1993 killed larvae with symptoms previously reported for this baculovirus. Objective 4: Discover natural biopesticides (such as botanical compounds and/or RNAi’s) targeting key vegetable pests such as stink bugs and cucumber beetles. ARS scientists in Beltsville, Maryland, made progress in developing RNA interference (RNAi) tools for use with harlequin bug. RNAi deactivates or silences specific genes to disable a target organism. The transcriptome, or the total complement of active genes in the harlequin bug, was determined for different life stages and both sexes of the adult stage. Intraspecific comparison of transcripts was made to identifying a wide variety of differentially expressed transcripts and a reliable source to determine genes involved in key physiological processes. Distinct transcripts associated with particular life stages or sexes, or associated with highly specific biological systems, for example semiochemistry-related genes expressed in adult male during pheromone biosynthesis, were identified. These represent candidate harlequin bug-specific gene targets that may be involved in key synthesis of semiochemicals or may serve as candidates for disruption by molecular biopesticide technologies useful in managing the negative impacts of this insect pest. Specific RNAi- inducing dsRNAs for harlequin bug were developed and used to evaluate RNAi efficacy in vivo. Objective 5: Determine and strengthen the genetic basis of plant defense mechanisms (e.g., pest aversion and resistance) for protection of high-value crops such as cole crops on small farms or gardens against destructive insects. In order to determine the role of the potato gene StZFP2 in resistance to insects or pathogens, transgenic potato lines were produced knocking down or over-expressing StZFP2. The StZFP2 gene in potato is turned on during numerous plant stresses such as insect feeding and late blight infection. StZFP2 encodes a transcription factor that regulates the expression of numerous genes. These transgenic lines have been tested for their level of resistance to potato late blight (Phytophthora infestans) and infestation by Manduca sexta. Several of the over-expressing lines show increased resistance to late blight and several of the knock-down lines are significantly less resistant compared to non-transformed.

Accomplishments
1. New species of insecticidal bacteria discovered. Overreliance on a limited number of organic insecticides increases the risk that pests will become resistant to them. Searching for alternatives to existing products, ARS scientists in Beltsville, Maryland, have described a new species of bacteria that produces insecticidal metabolites. The new species, named Chromobacterium sphagni, was discovered growing in Sphagnum bogs in West Virginia and Maine, and is orally toxic to a group of important pests represented by the diamondback moth and gypsy moth. The bacteria produce the toxic factor in artificial culture conditions, and living bacteria are not required to kill moth larvae. Spent cultures of the new species can be processed into an organic insecticide, and will be of interest to manufacturers of organic insecticides.

2. Efficient attractant for spotted wing drosophila (SWD). SWD is an exotic fruit fly from Southeast Asia that was introduced to the temperate regions of North America and Europe in 2008. It attacks a wide variety of fruits and has become a devastating pest of soft-skinned fruit crops. Due to the rapid spread of SWD across the newly-invaded continents, fresh fruit markets have a zero-tolerance policy regarding infestation. Specific and efficient detection tools are needed so that farmers can deliver timely interventions to meet market demands. Since SWD is attracted to damaged and rotting fruits, headspace volatiles from fresh and fermented apple juices were collected and analyzed by advanced scientific instruments to determine the compounds produced and/or enriched during the fermentation process. After performing a series of field tests, ARS scientists in Beltsville, Maryland, identified a five-way blend, which is more efficient and selective for the current standard apple cider vinegar and the commercially available SWD lure. Identification of SWD attractant will help growers accurately detect infestations in orchards, thereby allowing for timely interventions while reducing conventional insecticidal usage to protect our crops, environment, and ecosystem.

3. Economical pheromone for key cucurbit pest. The striped cucumber beetle is a key pest of cucurbit vegetables (squash, cucumbers, and melons) in eastern North America, rapidly colonizing on young plantings, and causing serious feeding damage while transmitting bacterial pathogens. Current control methods are costly. Male beetles produce an aggregation pheromone (attractive to both females and males), that has been identified and synthesized. Until recently, production methods were expensive and did not produce enough pheromone even for field tests. ARS scientists in Beltsville, Maryland, developed a new and less expensive mixed pheromone preparation, and then tested attraction to traps and baits under field conditions in cucurbit vegetable plantings. The results indicate that the new mixed pheromone could enable highly effective, less expensive environmentally-friendly species-specific management of striped cucumber beetle.

U.S. DEPARTMENT OF AGRICULTURE

Invasive Insect Biocontrol & Behavior Laboratory: Beltsville, MD