Research Project: Urban Small Farms and Gardens Pest Management

Location: Invasive Insect Biocontrol & Behavior Laboratory

2019 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 natural enemies. As a result of syntheses initiated by the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL), pheromones are now available in quantity for field deployment of aggregation pheromones for striped cucumber beetle, a key pest of cucurbit crops in North America, and for two species of flea beetles that are key pests of brassica vegetable crops like canola, cabbage, broccoli, kale, collards, and mustard greens. In each case, ARS scientists have discovered that plant compounds are very important in enhancing the attraction and feeding of the beetles, making attract-and-kill techniques potentially more effective. Therefore, the aggregation pheromones will be deployed together and separate from the plant compounds, to determine the most effective pest management approach. Research cooperators are in New York, New Hampshire, Virginia, North Dakota, and California. IIBBL has also been investigating biosynthesis of pheromones and possible pest management applications. RNA samples from male and female striped cucumber beetles collected in the field were sequenced to obtain global male- and female-specific transcriptomes. Data from this analysis are currently being used to assess differential gene expression between male and female adults. Homologs to known pheromone biosynthesis-related genes previously identified in stink bug species have been identified and are being considered as targets of future functional interrogations. Within the stink bug family, ARS researchers at Beltsville, Maryland, as part of the USDA NIFA Foundational grant, “Defining Molecular Mechanisms of Terpene Aggregation Pheromone Biosynthesis in Stink Bugs to Engineer Trap Crops,” have discovered key enzymes and intermediates in the biosynthetic pathways that allow the male insects to make their pheromones. Results include transcriptomic and biochemical discoveries for three pest species (harlequin bug, brown marmorated stink bug, and southern green stink bug), with more species underway. Results offer added targets to disrupt pest reproduction and/or divert pest populations with future incorporation into pheromone-producing trap plants. Objective 2: Develop arthropod biological controls for managing key vegetable pests. ARS scientists in Beltsville, Maryland, and Newark, Delaware, collaborated in identifying volatiles emitted by brown marmorated stink bug (BMSB) that could be used as attractants or repellents for the parasitoid wasp Trissolcus japonicus. Two compounds, tridecane, and (E)-2-decenal, in the BMSB volatiles emission were behaviorally active against this known egg parasitoid. While the former chemical seemed to attract the egg parasitoid, the latter displayed a repellent activity when tested in laboratory bioassay. For brown marmorated stink bug, implementation research is supported by USDA NIFA Specialty Crop Research Initiative (SCRI), Coordinated Agricultural Programs (CAPS), grant, “Management of BMSB in U.S. Specialty Crops,” through North Carolina State University in cooperation with several other universities and ARS labs. A two-year study has been initiated to evaluate parasitism rates and species composition of parasitoids attacking sentinel egg masses of BMSB and two native stink bug species, Euschistus servus and Podisus maculiventris. In the first year of the study, only 4.5% of BMSB eggs were parasitized compared with 31.4% of E. servus eggs and 28.1% of P. maculiventris eggs. Five parasitoid species were identified emerging from BMSB, with Trissolcus brochymenae being the most prevalent. Seven parasitoid species were identified emerging from E. servus and six from P. maculiventris. Results already demonstrate the higher level of parasitism of native species (both herbivore and predator) of stink bugs, showing the necessity of added biological control for the invasive BMSB. The exotic species T. japonicus parasitized a single egg mass of BMSB and a single egg mass of E. servus. The newly discovered microsporidian pathogen of stink bugs, Nosema maddoxi, appears to have caused catastrophic declines of laboratory colonies of brown marmorated stink bug (BMSB), and possibly to have also caused declines in the field, where it infects BMSB and at least two species of native stink bugs. This pathogen is apparently native both to Asia and North America. Its possible value in stink bug management is being investigated under the USDA NIFA SCRI CAP grant, “Management of BMSB in US Specialty Crops,” through North Carolina State University in cooperation with Cornell University and ARS labs. Research continues as to the host range of this pathogen, which may be useful in suppressing other invasive stink bugs such as Southern green stink bug (Nezara viridula) and Bagrada bug (Bagrada hilaris). Research is being conducted to evaluate the efficacy of the egg parasitoid Gryon pennsyvanicum as a biological control agent of two species of squash bugs, Anasa tristis and Anasa armigera and to compare ovipositional behavior of the parasitoid on the two squash bug species. Laboratory bioassays are being conducted to evaluate the effects of increased parasitoid pressure on parasitism rates and successful emergence when a single egg mass is exposed to either one, two, or three parasitoids. Laboratory bioassays are also being conducted to determine if parasitism rates on large egg masses could be increased if parasitoids were continuously provided with nutrition. These studies will provide information on factors influencing parasitism rates on squash bug egg masses and result in better biological control strategies for managing squash bug populations. Objective 3: Advance effective microbial controls for key crop pests, using diverse Bacillus thuringiensis (Bt) strains, Chromobacterium spp., baculoviruses, and other entomopathogens. More progress was made towards identifying a suitable alternative production host for the diamondback moth alphabaculovirus. The amount of diamondback moth virus needed to kill 50% of corn earworm larvae in a bioassay was 1,000-fold higher than that required to kill beet armyworm. Also, this baculovirus was unable to infect and cause mortality of larvae of the fall armyworm. The results suggest that, of the lepidopteran species test, beet armyworm may be the most suitable alternative host for the diamondback moth alphabaculovirus. Objective 4: Discover natural biopesticides (such as botanical compounds and/or RNAi’s) targeting key vegetable pests. ARS scientists in Beltsville, Maryland, continued to evaluate methyl benzoate and its analogs as insecticides, with efficacy against bed bugs, mosquitoes, thrips, nematodes, and some stored product insect pests demonstrated under laboratory conditions. Objective 5: Determine and strengthen the genetic basis of plant defense mechanisms (e.g., pest aversion and resistance) for protection of high value cole crops. The publication of several Brassica whole genome sequences allows mining for genes in cole crops. These encompass several nutritional powerhouses such as broccoli, collards, and kale along with two other similar species Chinese cabbage and canola. To broaden IIBBL investigations of C2H2 transcription factors (TF), we identified 146 C2H2 TF genes in three plant species. The identification and analysis of this gene family helps to focus on genes with similar protein structures to those in the potato (C2H2 TF, StZFP2), which upon over-expression can increase resistance to potato late blight and inhibit growth of the insect pest tobacco hornworm. This advance will allow work directly in these target cole crops. The publication of the biography of Charles Valentine Riley, Founder of Modern Entomology. An ARS entomologist in Beltsville, Maryland, has co-authored this definitive biography, which is the first full account of a fascinating American scientist whose leadership created the modern science of entomology which recognizes both the essential role of insects in natural ecosystems and their challenge to the agricultural food supply that sustains humankind. The book, published by University of Alabama Press, tells the story of how Riley (1843–1895), a young British immigrant to America, came to play a key role in the reorientation of entomology from the collection and arrangement of specimens to a scientific approach to insect evolution, diversity, ecology, and applied management of insect pests. Riley was USDA Entomologist from 1877 until his death in 1895. Among other achievements, he founded the National Insect Collection, vanquished the Grape Phylloxera that ravaged French vineyards through use of pest-resistant rootstocks, initiated the first successful classical biological control project through importation of the Vedalia Beetle from Australia, and discovered the Viceroy-Monarch mimicry as well as the yucca moth system of coevolution of plants and insects. This publication will be of interest to entomologists and other scientists and members of the general public interested in agricultural, demonstrating the value of government-funded science to solve agricultural challenges.

Accomplishments
1. Development of attract & kill strategy for cocoa pod borer pest management. The cocoa pod borer (CPB) is one of the most important insects affecting cocoa production in Southeast Asia. This pest is responsible for more than 50% of cocoa crop losses in the Indo-Malayan archipelago. Until now, management of CPB has heavily relied on pesticide applications, which is neither environmentally sustainable nor economically effective. ARS scientists in Beltsville, Maryland, have developed a more economically productive method to synthesize the sex attractant and developed attract and kill strategy using impure sex attractant as lure and cypermethrin as killing agent. Field tests conducted in Malaysia during 2014–2016 indicated that attract-and-kill strategy is superior to and more feasible than the currently applied conventional synthetic pesticide treatment in CPM management. This will result in reduction of conventional pesticide application in cocoa plantation.

2. Discovery of the harlequin bug pheromone biosynthesis intermediate. ARS researchers at Beltsville, Maryland, in cooperation with Virginia Polytechnic Institute and State University, identified a gene, encoding of which produces an enzyme responsible for the formation of a key intermediate in the biosynthesis if the harlequin bug pheromone. Harlequin bug is a widespread key pest in the southern U.S. that heavily damages cabbage and related crops. Application of the aggregation pheromone for monitoring is an important part in the management of this pest. Earlier ARS researchers at Beltsville identified the two aggregation pheromone components of the harlequin bug and now they demonstrate the structural relationship of the pheromone molecules and the newly discovered biosynthesis intermediate. The knowledge of a total pheromone biosynthesis would allow incorporation of pheromone-producing genes into host plants to pursue new attract-and-kill strategies and develop biopesticides via RNAi-based molecular techniques (gene silencing) for controlling this pest.

Review Publications
Harrison, R.L., Mowery, J.D., Bauchan, G.R., Theilmann, D.A., Erlandson, M.A. 2019. The complete genome sequence of a second alphabaculovirus from the true armyworm, Mythimna unipuncta: Implications for baculovirus phylogeny and host specificity. Virus Genes. 55:104-116.
Harrison, R.L., Rowley, D.L. 2019. The complete genome sequence of an alphabaculovirus from the southern armyworm, Spodoptera eridania. Microbiology Resource Announcements. 8:e01277-18.
Escasa, S.R., Harrison, R.L., Mowery, J.D., Bauchan, G.R., Cory, J.S. 2019. The complete genome sequence of an alphabaculovirus from Spodoptera exempta, an agricultural pest of major economic significance in Africa. PLoS One. 14(2):e0209937.
Feng, Y., Bruton, R.G., Park, A.L., Zhang, A. 2018. Identification of attractive blend for spotted wing drosophila, Drosophila suzukii from apple juice. Journal of Pest Science. https://doi.org/10.1007/s10340-018-1006-9.
Zhang, A., Feng, Y., Larson, N., Feldlaufer, M.F. 2018. Eucalyptol detected by aeration from the eggs of the common bed bug (Hemiptera: cimicidae). Journal of Economic Entomology. https://doi.org/10.1093/jee/toy385.
Lancaster, J., Khrimian, A., Young, S., Lehner, B., Luck, K., Wallingford, A., Ghosh, S.B., Zerbe, P., Marek, P., Sparks, M., Tittiger, C., Kollner, T.G., Weber, D.C., Gundersen, D.E., Kuhar, T., Tholl, D. 2018. De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.1800008115.
Lancaster, J., Lehner, B., Khrimian, A., Muchlinsky, A., Luck, K., Kollner, T., Weber, D.C., Gundersen, D.E., Tholl, D. 2018. An IDS type sesquiterpene synthase produces the pheromone precursor (Z)-a-bisabolene in Nezara viridula. Journal of Chemical Ecology. https://doi.org/10.1007/s10886-018-1019-0.
Morrison III, W.R., Blaauw, B.R., Short, B.D., Nielsen, A.L., Bergh, J.C., Krawczyk, G., Park, Y., Butler, B., Khrimian, A., Leskey, T.C. 2018. Successful management of Halyomorpha halys (Hemiptera: Pentatomidae) in commercial apple orchards with an attract-and-kill strategy. Pest Management Science. 75(1):104-114. https://doi.org/10.1002/ps.5156.
Lawrence, S.D., Novak, N.G. 2018. Over-expression of StZFP2 in Solanum tuberosum L. var. Kennebec (potato) inhibits growth of tobacco hornworm larvae (THW, Manduca sexta L.). Plant Signaling and Behavior. https://doi.org/10.1080/15592324.2018.1489668.
Lawrence, S.D., Novak, N.G. 2018. Comparative analysis of the genetic variability within the Q-type C2H2 zinc-finger transcription factors in the economically important cabbage, canola and Chinese cabbage genomes. Hereditas. 155:29.
Novak, N.G., Perez, F.G., Jones, R.W., Lawrence, S.D. 2019. Detached leaf assays: A simplified approach to study gene expression in potato during infestation by the chewing insect Manduca sexta. Journal of Visualized Experiments. 147:e59153.
Harrison, R.L., Herniou, E.A., Jehle, J.A., Theilmann, D.A., Burand, J.P., Becnel, J.J., Krell, P.J., Van Oers, M.M., Mowery, J.D., Bauchan, G.R. 2018. ICTV virus taxonomy profile: Baculoviridae. Journal of General Virology. 99:1185-1186.
Lawrence, S.D., Novak, N.G., Perez, F.G., Jones, R.W. 2019. Over expression of the Q-type ZFP StZFP2 in potato increases resistance to potato late blight (Phytophthora infestans) infection. Journal of Plant Interactions. 14(1):129-136. https://doi.org/10.1080/17429145.2018.1562109.
Vanhove, W., Zhang, A., Osman, H.I., Vanhoudt, N., Van Damme, P. 2019. Development of an attract-and-kill strategy for Cocoa Pod Borer (Conopomorpha cramerella Snellen) control. Pest Management Science. https://doi.org/10.1080/09670874.2019.1578003.
Sorensen, W., Smith, E.H., Smith, J., Weber, D.C. 2019. Charles Valentine Riley: Founder of modern entomology. Tuscaloosa: University of Alabama Press. 438 p.
Hall, D.G., Borovsky, D., Chauhan, K.R., Shatters, R.G. 2018. An evaluation of mosquito repellents and essential plant oils as deterrents of Asian citrus psyllid. Crop Protection. 108:87-94.