MANAGING BENEFICIAL INSECTS WITH PLANT VOLATILES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1003648
• Project Start Date: Oct 1, 2014
• Project End Date: Apr 3, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Entomology

Non Technical Summary
Objective 1Many crops benefit to varying degrees from insect pollinators, and for some this service is essential. In recent years, however, a severe decline in honey bee populations has raised concerns about pollination shortfalls that will significantly reduce yield. One option to mitigate the loss of these managed pollinators is to increase reliance on ecosystem services provided by wild pollinators including native bees via the protection of fallow areas in close proximity to crop fields. While this technique has shown promise, it may be difficult to implement in the Midwest where agricultural intensification has led to the extensive loss of natural habitat. An alternative strategy not widely considered is to enhance the attractiveness of crop flowers that compete with weeds and other flowering plants for a limited pool of bees. This novel approach is critical if crop flowers lost their pollinator-attracting qualities during domestication, as shown with other beneficial traits such as pest resistance. Because of the historic abundance of honey bees, attractive flowers were likely never selected by crop breeders; however, the current bee decline necessitates a new approach for the sustainable production of fruits and vegetables.Objective 2Despite stagnant growth in many U.S. agricultural sectors, the last decade has witnessed an explosive rise in demand for locally grown produce. This trend is driven by increasing public awareness of the lack of transparency along the food supply chain linking farm-to-fork, including both the source of and production methods used to grow food. Unfortunately, cold weather constrains crop production for much of the year, except in southernmost U.S. states, preventing the vast majority of growers from meeting demand and thereby limiting farm profitability. One of the leading solutions to this dilemma of seasonality in temperate climates is to employ high tunnels, a form of protected agriculture that aids in season extension by creating a more favorable microclimate using trapped solar heat. Because they allow farmers to generate a crop far earlier in the spring and extend production late into the fall compared with open-field cultivation, their popularity among fruit and vegetable growers is on the rise. Starting in 2010, USDA-NRCS initiated a high tunnel funding program that has invested in the construction of >4,500 new high tunnels across the country for enhanced production of high value specialty crops. Due largely to the recent emergence of high tunnels in U.S. agriculture, knowledge on all aspects of their management, including pest control, has lagged considerably. Virtually no data exist on high tunnel pest management, posing logistical challenges for growers to make informed decisions and adapt management practices in this novel environment.Objective 3While we are only just beginning to realize the potential of plant volatiles as a pest control tool, the success of pheromones in commercial agriculture is a testament to what is possible, and can be used as a roadmap for how to effectively employ semiochemicals in crop protection. Despite their fundamental differences compared with pheromones, plant volatiles offer a distinct advantage in their capacity to synergize with existing pest management tactics, namely biological control. This is particularly important in organic systems where insecticide options are limited or nonexistent, and ecological solutions are in desperate need. Volatiles also fit well in the context of broader calls for a more targeted approach to pest control, or "ecological engineering". The proposed research is significant in taking a proactive approach in facilitating the impact of natural enemies on crop pests using novel phytochemical tools.

Animal Health Component

40%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
215	1420	1070	25%
215	1421	1130	25%
215	1460	1070	25%
215	3010	1130	25%

Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
1460 - Tomato; 1420 - Melons; 3010 - Honey bees; 1421 - Cucumber;

Field Of Science
1130 - Entomology and acarology; 1070 - Ecology;

Keywords

plant volatiles

biological control

pest management

beneficial insects

pollinators

natural enemies

Goals / Objectives
The overarching aim of this research project is to effectively manipulate the behavior of beneficial insects (predators, parasitoids, pollinators) in agricultural systems to improve pest management and/or crop pollination. With this aim in mind, the following three specific objectives are proposed:1. Assess the genetic variability in volatile blends emitted from flowers of wild and cultivated watermelon genotypes, and correlate variation in volatile profile with the strength of pollinator attraction.2. Test the potential for induced plant volatiles to retain mass-released predators and parasitoids used for augmenting biocontrol in high tunnel vegetables.3. Determine the volatiles emitted from caterpillar-damaged tomato and evaluate whether these signals prime naïve tomato plants for more rapid and pronounced induced defenses to pest attack.

Project Methods
Objective 1Watermelons and wild Citrullus species will be grown at Meigs Farm/Throckmorton Purdue Agricultural Center (TPAC)near Lafayette, IN. Seeds of 4 elite commercial watermelon varieties (2 triploid seedless, 2 diploid seeded), commonly grown in southwest Indiana, will be planted. Seeds of domesticated watermelon (C. lanatus var. lanatus)and wild citron (C. lanatus var. citroides), as well as three related species (C. ecirrhosus, C. rehmii and C. colocynthis)will be obtained from the Plant Genetic Resources Conservation Unit (Southern Regional Plant Introduction Station, USDA-ARS, Griffin, GA). For each accession and wild species we will choose 3 genotypes from across their geographical origin (Africa, Middle East), resulting in 15 total non-commercial varieties.Volatile organic compounds (hereafter, VOCs)emitted from male and female flowers will be collected and analyzed by GC-MS. We will compare volatile profiles between closed and open flowers, as well as at different times during the day and night, to identify VOCs with emission patterns correlating with pollinator activity. Additional floral traits (e.g. size, pigmentation)will be analyzed via HPLC.All 19 watermelon genetic lines will be cultivated in field plots (n=10 plants/plot)using a randomized complete block design with 5 replicated blocks. Each week two different observers will conduct visual censuses of each plot, recording overall bee activity and number of visitations per flower for 5 minutes per plot during the hours of 7:00 to 11:00 a.m. Mature fruits will be harvested, graded (according to USDA standards), and used to calculate individual fruit weight and yield. Yield will be correlated with bee visitation across genetic lines.Because flowers differ in several other traits aside from VOCs (e.g., size, color, nectar and pollen chemistry), a complementary field experiment will be conducted to more conclusively link bee visitation differences with scent. Floral VOCs from each of the 19 varieties will be collected and the compounds reapplied in a diluted 50% propylene glycol solution. This solution, containing the odor from each genetic-type of flower, will be placed in 'bee bowls', for one week three times during the summer. Pollinator attraction rankings, using both whole-plant and bee bowl surveys will be correlated, with the associated volatile data.Objective 2Field trials will be conducted at TPAC which has 6 FarmTek Premium Round Style high tunnels (14.6 x 7.9 x 3.7 m, LWH), constructed in 2010 with 12-mil ClearSpan™ PolyMax, a polyethylene sheet allowing 85% light transmission. HOBO data loggers will be placed in each tunnel to monitor daily temperatures and ventilate to optimize crop growth. Brandywine tomatoes, an heirloom cultivar, and long English cucumbers, both of which are high value varieties at farmers' markets, will be germinated from seed in the greenhouse and transplanted into tunnels in late-March to early-April. Four rows per tunnel will be planted with plastic mulch beds and weed control fabric between rows. Each crop will be grown vertically using a trellis system to maximize production space. Insecticides will not be applied for the duration of trials, but all remaining aspects of crop production will mirror commercial practices, e.g., disease control, drip fertigation, and routine fruit harvesting.Each year, each of the 6 high tunnels will be randomly assigned to one of the following 3 treatments in a randomized complete block design with 2 replicated blocks:Predator releaseThis treatment will be common to all high tunnels and serve as a non-manipulated control. The 3 predators will be: the predaceous stink bug, P. maculiventris, the pirate bug, O. insidiosus, and the lady beetle, H. convergens. Predators will be purchased from a commercial insectary (Rincon-Vitova Insectaries, Ventura, CA) and released at recommended rates (stink bugs, 5/plant; pirate bugs, 3/plant; lady beetles, 1/ft2). We will conduct spring, summer, and fall releases of each species.Predator release + screeningFine mesh screening will be placed over all high tunnel openings, using a 0.18 mm2 pore size mesh (100% polyolefin, LS EcoNet 4045; Svensson, Inc., Charlotte, NC), to exclude aphids and whiteflies, as well as retaining pirate bugs, the smallest of our released biocontrol agents.Predator release + volatiles and flowering plantsSemiochemicals will be manipulated using methyl salicylate, which is commonly emitted from pest-damaged crops and acts as a broad-spectrum attractant for predators (PredaLure, AgBio, Inc., Westminster, CO). Two synthetic lures will be hung at canopy height, one at each end of the high tunnel. We will integrate cut flowers (zinnias and sunflowers) into high tunnels to provide a supplemental pollen/nectar resource for predators.Predators will be sprayed with rabbit IgG, an immunomark, prior to release. After 48 and 96 hours, predators will be sampled from high tunnel crops using D-vac suction sampling. All individuals of the 3 released predators recaptured will be placed individually in Eppendorf tubes, flash-frozen and later analyzed for presence of the protein marker using ELISA. These data will then be used to calculate % recapture rates per high tunnel in each treatment.Corresponding with each predator release, incipient pest outbreaks will be created on 3 randomly chosen plants per tunnel. For pirate bugs and lady beetles, aphids (Aphis gossypii)will be used on cucumber. These incipient outbreaks will be created the day prior to predator release and densities tracked daily for one week thereafter. A similar bioassay will be performed for predaceous stink bugs, but instead using five 2nd instar hornworm larvae, Manduca sexta, per tomato plant.Objective 3The plant volatile methyl salicylate (MeSA) will be released from slow-release vials in tomato fields. One large tomato field will be planted to Solanum lycopersicon var. MP-1, but with four plots, each randomly assigned to one of the following treatments: (1) No MeSA; (2) No MeSA + insect herbivory; (3) MeSA priming; (4) MeSA priming + insect herbivory.In plots assigned to MeSA priming, a single vial containing 15 mL pure MeSA will be attached to a bamboo stake at the central point of each plot. Those plots assigned to insect herbivory will receive experimentally-imposed damage from hornworm (Manduca sexta) larvae. Forty-eight hrs after MeSA lures are placed in plots, caterpillars will be sleeve-caged onto the plants assigned to insect herbivory for 24 hrs.Volatile organic compounds will be collected from the headspace of undamaged (i.e., systemic) leaves for the initial 6 hrs from the onset of insect leaf damage. Single leaves will be enclosed in 500 mL polyethylene cups and air will be pulled over charcoal adsorbent tubes with a 12 V vacuum pump. Trapped volatiles will be analyzed by GC-MS. Expression of defense genes will be measured by harvesting undamaged tomato leaflets at 1, 3, 6, 12, and 24 hrs after insect herbivory is initiated. Tissue will be flash-frozen in liquid nitrogen and stored at -80°C until analysis. RNA will be isolated from both plants primed with MeSA and non-primed plants at the different time points after herbivore attack and the level of transcripts will be analyzed by quantitative RT-PCR using gene-specific primers. Two 1st instar M. sexta larvae will be added to plants at each of the 12 plot locations. One larvae will be bagged onto a leaf using a mesh sleeve-cage; the other will be placed on an adjacent leaf but not enclosed. Insect survival will be assessed for 7 days, after which we will remove and weigh caged insects and determine final survival of non-caged insects. Background mortality from caged larvae will be subtracted from that of exposed larvae to estimate the contribution of predators to pest suppression. Surviving larvae will be returned to the lab and reared to pupation to evaluate attack from parasitoids.

Progress 10/01/14 to 04/03/17

Outputs
Target Audience:Academic scientists (faculty, postdoctoral researchers, graduate students) and USDA employees Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?In addition to the two publications listed, we contribued the following oral presentations: Carrillo, J., X. Li, M. Garvey, and I. Kaplan. Domestication of tomato has reduced attraction of herbivore natural enemies. Symposium: Crop Domestication Effects on Plant-Insect Interactions: Patterns, Mechanisms, and Future Directions. XXV International Congress of Entomology, Orlando, FL, September 2016 Ingwell, L., R. Foster, and I. Kaplan. Pest management in high Tunnels: Evaluating biological control and screening. Symposium: Agroecology in Specialty Crops: IPM Strategies to Address Pest Management Challenges. Annual Meeting of the North Central Branch of the Entomological Society of America, Cleveland, OH, June 2016 Vidal Gomez, U., and I. Kaplan. Developmental differences in olfactory processing for predaceous insects. XXV International Congress of Entomology, Orlando, FL, September 2016 Garvey, M., C. Creighton, and I. Kaplan. Plant domestication has consequences for direct and indirect plant defense in the Solanaceae. Symposium: From Basic to Applied Science: Tri-Trophic Interactions in Natural and Managed Systems. Annual Meeting of the North Central Branch of the Entomological Society of America, Cleveland, OH, June 2016 Kaplan, I. Manipulating crop volatiles for sustainable insect management in agriculture.Symposium: Everything you wanted to know about volatiles. Volatile Organic Compound (VOC) Workshop, Netherlands Institute of Ecology, Wageningen, The Netherlands, March 2016 Garvey, M., C. Creighton, and I. Kaplan. Ecoimmunology of Manduca sexta: Therapeutic effects of plant chemistry on the immune response as a mediator of tritrophic interactions. XXV International Congress of Entomology, Orlando, FL, September 2016 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The data collection stage for these 3 objectives are now complete and the data are currently in process of being analyzed and written up for publication. A manuscript for Objective 3, based on the research of a M.S. student Elizabeth Rowen,wasjust accepted for publication at Journal of Chemical Ecology. Data for Objective 2 are being analyzed by a postdoctoral scientist, Dr. Laura Ingwell, since the high tunnel experiment was conducted during the summers of 2015 and 2016. This work is currently being written up as a manuscript that will be submitted to the journal Biological Control.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kaplan I. 2017. A cry for help or sexual perfumes? An alternative hypothesis for wasp attraction to the scent of caterpillar?wounded plants. Plant, Cell & Environment, 40: 327-329
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Rowen E, Gutensohn M, Dudareva N, Kaplan I. 2017. Carnivore attractant or plant elicitor? Multi-functional roles of methyl salicylate lures in tomato defense. Journal of Chemical Ecology, in press

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Faculty, graduate student, undergraduate students, and USDA scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We have given the following seminars and presentations: Purdue University, Calumet (Dept. of Biological Sciences), Calumet, IN, October 2016 Wageningen University (Ecology & Evolution), Wageningen, Netherlands, May 2016 University of Neuchâtel (Institute of Biology), Neuchâtel, Switzerland, April 2016 Netherlands Institute of Ecology (NIOO), Wageningen, Netherlands, April 2016 Wageningen University (Dept. of Entomology), Wageningen, Netherlands, March 2016 Max Planck Institute for Chemical Ecology, Jena, Germany, February 2016 Carrillo, J., X. Li, M. Garvey, and I. Kaplan. Domestication of tomato has reduced attraction of herbivore natural enemies. Symposium: Crop Domestication Effects on Plant-Insect Interactions: Patterns, Mechanisms, and Future Directions. XXV International Congress of Entomology, Orlando, FL, September 2016 Ingwell, L., R. Foster, and I. Kaplan. Pest management in high Tunnels: Evaluating biological control and screening. Symposium: Agroecology in Specialty Crops: IPM Strategies to Address Pest Management Challenges. Annual Meeting of the North Central Branch of the Entomological Society of America, Cleveland, OH, June 2016 Garvey, M., C. Creighton, and I. Kaplan. Plant domestication has consequences for direct and indirect plant defense in the Solanaceae. Symposium: From Basic to Applied Science: Tri-Trophic Interactions in Natural and Managed Systems. Annual Meeting of the North Central Branch of the Entomological Society of America, Cleveland, OH, June 2016 Kaplan, I. Manipulating crop volatiles for sustainable insect management in agriculture.Symposium: Everything you wanted to know about volatiles. Volatile Organic Compound (VOC) Workshop, Netherlands Institute of Ecology, Wageningen, The Netherlands, March 2016 Vidal Gomez, U., and I. Kaplan. Developmental differences in olfactory processing for predaceous insects. XXV International Congress of Entomology, Orlando, FL, September 2016 Garvey, M., C. Creighton, and I. Kaplan. Crop domestication in the Solanaceae: Consequences for indirect plant defense. Annual Meeting of the Entomological Society of America, Minneapolis, MN, November 2015 Rowen, E., and I. Kaplan. Predictable ecological and evolutionary drivers of herbivore induced plant volatiles: A meta-analysis. Annual Meeting of the Entomological Society of America, Minneapolis, MN, November 2015 Ingwell, L., R. Foster, and I. Kaplan. Biological control in high tunnel agriculture: An effective pest management tool? Annual Meeting of the Entomological Society of America, Minneapolis, MN, November 2015 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We learned that plant volatiles can be used in retaining certain beneficial predators used in augmentative biocontrol, but the consequences for pest populations requires more investigation. Also, the same volatiles (methyl salicylate) can prime tomatoes for increased resistance to foliar pathogens and insect pests. Objective 1. Nothing to report. Objective 2: We completed analysis for a 2015field experiment that tested the effect of methyl salicylate lures on predator retention in high tunnels at the Throckmorton-Meigs Purdue Agricultural Center. These volatiles were combined with flowering plants to evaluate their combined effect on predator retention and efficacy. Of three predator groups tested (Anthocoridae, Coccinellidae, Chrysopidae), only the species Orius insidiosus responded to the volatile treatments. Significantly more individuals were recaptured with volatiles that in control tunnels or those with screening. However, no impact on aphid population growth was observed. Objective 3: We analyzed the effect of Predalure (the volatile MeSA) on tomatoes in the greenhouse and the field. The greenhouse experiment showed that exposure of tomato plants to MeSA reduces subsequent growth of hornworm caterpillars (Manduca sexta) when placed on leaves. In the field, pathogens were less likely to colonize MeSA-exposed tomatoes and, importantly, this effect did not depend on distance from the lure. This was determined by testing along a distance gradient up to 4 meters. Similarly, directionality did not affect the horizontal diffusion of volatiles to impact tomato physiology. Induced chemical defenses (protease inhibitors, peroxidase, polyphenol oxidase) were strongly induced in response to simulated caterpillar herbivory, but were unaffected by the main effect of MeSA. Polyphenol oxidase showed evidence for priming with stronger responses to herbivory with prior exposure to MeSA.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kaplan I, Carrillo J, Garvey M, Ode PJ. 2016. Indirect plantparasitoid interactions mediated by changes in herbivore physiology. Current Opinion in Insect Science, 14: 112-119
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Rowen E, Kaplan I. 2016. Eco?evolutionary factors drive induced plant volatiles: a meta?analysis. New Phytologist, 210: 284-294

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Other university scientists, USDA scientists, farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?In addition to the reported publications we have presented this research with the following seminar and conference presentations: Kaplan I.Michigan State University (Dept. of Entomology), East Lansing, MI, February 2015 Kaplan I.University of Florida (Dept. of Entomology), Gainesville, FL, January 2015 Kaplan I.University of Illinois (Dept. of Entomology), Urbana-Champaign, IL, October 2014 Rowen, E., M. Gutensohn, N. Dudareva, and I. Kaplan. Tomato (Solanum lycopersicum)volatiles prime defenses against Manduca sexta in the field. Annual Meeting of the Entomological Society of America, Portland, OR, November 2014 Vidal Gómez, U., and I. Kaplan. Associative learning of Podisus maculiventris (Hemiptera:Pentatomidae) to herbivore-induced plant volatiles. Annual Meeting of the Entomological Society of America, Portland, OR, November 2014 Garvey, M., C. Creighton, and I. Kaplan. Medicinal effects of plant chemistry on the immunesystem of Manduca sexta (Lepidoptera: Sphingidae). Annual Meeting of the Entomological Society of America, Portland, OR, November 2014 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Completed a 2nd year of common garden comparing the volatiles and attraction of insects to 22 lines of Citrillus (watermelons) to determine differences in floral compounds. Statistically correlated release rates of individual volatile compounds with attraction of native bees and managed honey bees. Also, performed 2nd year of high tunnel predator releases in conjunction with methy salicylate (MeSA) volatile lures to reduce emigration of biocontrol agents. Predators were protein marked and sampled with a D-vac suction device to evaluate dispersal. Also, completed tomato project to assess priming of tomato for defense response to caterpillar herbivory. This work is being written up for publication before the end of the year.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Gutensohn M, Nguyen TTH, McMahon RD, Kaplan I, Pichersky E, Dudareva N. 2014. Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate. Metabolic Engineering, 24: 107-116
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Thaler JS, Olsen EL, Kaplan I. 2015. Jasmonate-induced plant defenses and prey availability impact the preference and performance of an omnivorous stink bug, Podisus maculiventris. Arthropod-Plant Interactions, 9: 141-148
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Kaplan I, Lewis D. 2014. What happens when crops are turned on? Simulating constitutive volatiles for tri-trophic pest suppression across an agricultural landscape. Pest Management Science, 71: 139-150