INVESTIGATING THE INDIRECT ROLE BENEFICIAL NEMATODES PLAY IN PLANT PROTECTION AGAINST HERBIVORES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022810
• Grant No.: 2017-67012-31498
• Cumulative Award Amt.: $42,198.10
• Proposal No.: 2018-05094
• Project Start Date: May 1, 2020
• Project End Date: Apr 30, 2021
• Grant Year: 2020
• Program Code: [A7201]- AFRI Post Doctoral Fellowships

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Entomology

Non Technical Summary
Chemical cues play important roles in many ecological interactions, especially among plants and animals. Many previous studies in this field have focused on odor cues that influence insect behavior, such as floral scents that attract pollinators. A relatively recent and exciting discovery is that plants also perceive and respond to chemical cues, often detecting cues associated with herbivores as a warning to prepare for future attack. Finding novel ways to harness these natural cues and behavioral responses for enhancing crop protection represents a new frontier in agroecology. Concerns about insecticide resistance and losses of beneficial arthropods, have contributed to a growing interest in developing alternative strategies for pest management. Biological control by beneficial nematodes (EPNs) is one strategy that offers a promising alternative for controlling soil-dwelling insect pests. EPNs are important natural enemies that attack and kill many root-feeding herbivores. However, there has been reluctance among many growers to reduce insecticide use and adopt EPN biological control. A major goal of this project is to uncover additional benefits of EPNs for plant protection and to increase growers' incentives for implementing this alternative pest management strategy.My preliminary findings have revealed that EPNs emit odors that can negatively influence insect herbivores. In this project, I will investigate the influence of EPN odors on plants and insect herbivores, as both cases would have important consequences for protecting plants against herbivore pests. For example, if plants detect EPN odors as a warning cue that insect herbivores are present and likely to attack, they can enhance their anti-herbivore defenses to reduce future damage. If the insect herbivores themselves perceive and avoid odors from their EPN natural enemies, they will likely avoid nearby plant roots where EPN are present, resulting in a further reduction of herbivore damage. I also plan to characterize the odor blend from EPNs and identify the compounds involved in these interactions. Through this project, I hope to provide additional knowledge about EPN biological control, which will contribute to sustainable pest management decisions and inform fundamental concepts about ecological interactions. Overall, this work will result in several short- and long-term benefits to growers and the environment that include: a) More information about alternative protection of plants b) Reduced insecticide use, and c) Increased efficacy of available biological controls.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3130	1070	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3130 - Nematodes;

Field Of Science
1070 - Ecology;

Keywords

belowground volatiles

chemical eavesdropping

entomopathogenic nematodes

plant defense

Goals / Objectives
The long-term goal of this project is to elucidate indirect benefits of entomopathogenic nematodes (EPNs) for plant protectionagainst herbivores, thereby increasing growers' incentives for implementing EPN biological control as an alternative pestmanagementstrategy. To accomplish this goal, I plan to investigate the influence of EPNs on plants and insect herbivores, as both cases would have important consequences for protecting plants against herbivore pests. The key objectives of this proposal are: 1) Quantify the influence of EPNs on plant defenses and pest performance, 2) Determine the behavioral responses of above and belowground herbivores to EPN cues.

Project Methods
Objective 1: Investigate whether EPN cues influence plant defenses and pest performance. I will compare defense traits of plants exposed to EPN odors and unexposed controls and measure herbivore performance, quantify levels of herbivore damage, and analyze a suite of plant chemical defenses. Squash plants will be grown in an insect-free greenhouse from commercially available seeds. Four-week old squash plants will be used in these experiments. Laboratory colonies of striped cucumber beetle (SCB) will be maintained on their host plants. The EPN species Steinernema carpocapsae and Heterorhabditis bacteriophora will be reared from waxworm larvae (G. mellonella). I will expose roots of plants to live EPNs, EPN volatiles, and appropriate controls. For exposure to live EPNs, I will introduce either EPN-infested cadavers (of either pest species or G. mellonella), or infective juveniles (IJs) in water, to the soil of each potted plant. Control plants will receive either freeze-killed larvae or water. I will repeat these experiments using S. carpocapsae and H. bacteriophora. I will expose plants to EPN volatiles, by aerating EPNs in clean glass chambers, forcing filtered air through the EPN headspace (at 100 ml/min) and through a glass connection onto the plant roots. Mesh filters will prevent EPNs from physically contacting the plants. All exposure treatments will begin 24-hours before herbivores are applied to plants and will continue throughout the experiments. To assess the influence of exposing plants to EPN odors on herbivore performance, I will measure larval growth and development times. For striped cucumber beetle larvae feeding on squash, I will allow neonate larvae to feed on fresh cuttings from exposed or unexposed squash roots. I will add five neonates to a diet cup containing fresh root cuttings, replacing root cuttings every few days. I will weigh the larvae on the fifth and tenth days, in addition to counting the number of days required to reach the third instar. I will also repeat this experiment with SCB larvae feeding on intact squash roots with or without exposure to EPN odors. For this experiment, I will apply ten SCB neonates to each potted plant. After ten days, I will harvest plants to recover and weigh the larvae. To measure the effects of EPN exposure on herbivore damage, I will conduct similar experiments to the herbivore-performance studies. For SCB larvae feeding on squash roots, I will allow five, third instar larvae to feed on fresh root cuttings from exposed or unexposed plants for 48 hours. I will record the mass of root tissue before and after feeding damage, adjusting for desiccation of the root cuttings. To investigate the influence of EPN exposure on plant chemical defenses, I will analyze a suite of anti-herbivore defenses in exposed and unexposed squash plants, before and after herbivore damage. After exposing plants to EPN odors and the appropriate controls, I will collect undamaged leaf and root tissue for chemical and molecular analyses. Then, I will allow SCB larvae to feed on squash roots, and I will collect tissue samples from leaves and roots at 4 h, 24 h, and 48 h after feeding is initiated. This will allow me to quantify the induction and accumulation of plant defenses over time. I will quantify levels of the defense phytohormones jasmonic acid (JA) and salicylic acid (SA), in squash roots and leaves. These hormones are induced by herbivore and pathogen damage, and mediate a variety of downstream defenses. Previous studies have found that plants exposed to chemical cues associated with herbivores often induce higher quantities of jasmonic acid in response to damage, making it a good biochemical indicator of enhanced plant defenses. I will also measure levels of the defense compounds cucurbitacins in roots and leaves of exposed and control plants before and after SCB damage. Cucurbitacins are bitter and typically toxic compounds that deter feeding by most herbivores but serve as a feeding stimulant for SCB and other ?diabroticites. Objective 2: Examine the behavioral responses of herbivores to EPN odors. I will conduct behavioral experiments examining responses of larval and adult herbivores to EPN-emitted volatiles. To measure larval preference for plants with or without EPN odors, I will conduct experiments using a 6-arm belowground olfactometer, following established methods. In all experiments, I will use 2-3 week old squash plants and SCB neonate larvae. For the first experiment, two pots will contain squash plants, each with 5 EPN-infested cadavers, 2 will contain only squash plants, and 2 will be empty sand controls. All connecting arms will be fitted with a fine mesh screen to prevent EPN from leaving the chambers or physically contacting SCB. After allowing 24 hours for odor diffusion to occur, I will introduce 20 SCB neonates into the central chamber and allow them to choose among the 6 arms. After 48 hours, any larvae that did not move into one of the arms will be counted as non-responders. After 48 hours, I will count the proportion of larvae that chose each treatment. I will repeat this experiment using IJs for both S. carpocapsae and H. bacteriophora and both SCB and G. mellonella cadavers. To determine whether ovipositing female beetles discriminate between plants with or without EPN odors, I will examine female behavior and quantify the number of eggs laid on each treatment. Female SCB typically lay their eggs in the top layer of soil surrounding host plants. I will place 1 mated female SCB inside a cage with one EPN-exposed and 1 unexposed host plant. The EPN-exposed plant will have EPN-infected cadavers or IJs added to the soil prior to the experiment (S. carpocapsae or H. bacteriophora). After 24 hours, I will filter the soil to retrieve and count the SCB eggs in each treatment. The results from the experiments in objective 1 are being analyzed using one-way ANOVA and nested ANOVA, data from the behavioral experiments in objective 2 are being analyzed using a Chi-squared test, and all analyses are being performed with R statistical software. Compounds are quantified/identified through GC/MS or HPLC by comparing spectral data with commercially available standards and spectra in MS libraries, and/or confirming with retention times of authentic standardsEfforts and Evaluation: To ensure the successful completion of this project, I have developed a project timeline. I split each objective into experimental units and created a 1-year plan and time budget for completing these goals. Results will be presented at scientific meetings and manuscripts will be submitted for publication in peer-reviewed journals. Relevant findings will be shared at extension and outreach events.

Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The target audience for this project included scientists, students, growers, and members of the broader public. Our efforts to reach these groups included the following: We published findings from this project in 3 peer-reviewed scientific journals and shared the work through 25 presentations at professional scientific meetings and seminars. This allowed us to communicate the research with the scientific community. I mentored 3 graduate and 3 undergraduate students for this project, including 3 students from underrepresented backgrounds. I also taught a graduate level course in chemical ecology and an undergraduate course in insect ecology, where we discussed this research. My lab also participated in outreach events like the Texas A&M Darwin Day, where we shared our research with the broader public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training Activities I mentored one graduate student and 3 undergraduate students as part of this project. The students were trained in a variety of chemical ecology techniques, including using gas chromatography-mass spectrometry (GCMS) to analyze plant, insect, and nematode metabolites, high-performance liquid chromatography (HPLC) to quantify plant metabolites, and using olfactometers for above- and below-ground behavioral experiments. I also trained these students in experimental design, data analysis, scientific writing, and oral presentation skills. Professional development I participated in the ADVANCE Faculty Success Fellows Program and the Texas A&M College of Agriculture and Life Sciences Tenure-Track Faculty Development Program focused on mentoring and supporting early career faculty as they develop their research and teaching programs and navigate the promotion and tenure process. The students in my research group completed an online scientific writing course and presented their work at several scientific conferences and symposia. How have the results been disseminated to communities of interest?The results from this project were published in peer-reviewed scientific journals, presented at scientific conferences, and shared with the general public through outreach events. We published findings from this project in 3 peer-reviewed scientific journals and shared the work through 25 presentations at professional scientific meetings and seminars. My lab also participated in the Texas A&M Darwin Day event in 2019 and 2020, where we shared our research with the broader public. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Statement Chemical cues play important roles in many ecological interactions, including among plants and animals. Some examples of these interactions include pollinator attraction to floral scents and plants that enhance their defenses in response to chemical cues associated with herbivores. Finding novel ways to harness these natural cues and behavioral responses for enhancing crop protection represents a new frontier in agroecology. Concerns about insecticide resistance and losses of beneficial arthropods, have contributed to a growing interest in developing alternative strategies for pest management. Biological control with beneficial entomopathogenic nematodes (EPNs) is one strategy that offers a promising alternative for controlling belowground insect pests. EPNs are important natural enemies that attack and kill herbivores in the soil. However, there has been reluctance among many growers to reduce insecticide use and adopt EPN biological control. A major goal of this project was to uncover additional benefits of EPNs for plant protection and increase growers' incentives for implementing this alternative pest management strategy. The findings from this project have important implications for stakeholders and the scientific community. Through this project, we found that chemical cues from EPNs can increase plant resistance against insect herbivores and are directly repellent to herbivores. These results suggest farmers and gardeners can experience multiple benefits of using EPNs for biological control, as EPNs both directly kill insect pests and provide further pest protection by deterring herbivores and boosting plant defenses. Furthermore, this project revealed that chemical cues from a belowground natural enemy (EPNs) influence plant and insect responses, indicating their important role in shaping ecological interactions, which was previously unknown. Objective 1: Quantify the influence of EPNs on plant defenses and pest performance 1) Major activities completed / experiments conducted: We quantified herbivore performance on plants exposed to EPNs or EPN cues and quantified plant defense traits (e.g., defense phytohormones) following plant exposure to live EPN IJs or EPN chemical cues. Experiments were conducted with cucumber plants (Cucumis sativus), striped cucumber beetles (Acalymma vittatum), and the EPN species Heterorhabditis bacteriophora. 2) Data collected: We collected performance data (mass gain, mass tissue removed, survival) for striped cucumber beetle larvae feeding on roots of cucumber plants exposed to EPNs or EPN chemical cues (or unexposed control plants). We also quantified levels of plant defense hormones (jasmonic acid and salicylic acid) in root and leaf tissue of cucumber plants exposed to EPNs, EPN chemical cues, or unexposed control plants. 3) Summary statistics and discussion of results: We found no difference in larval performance on EPN-exposed or control plants for any of the metrics analyzed. We did find evidence for elevated defenses in root and leaf tissue in plants exposed to EPNs compared to unexposed control plants. 4) Key outcomes or other accomplishments realized: The results from this objective provide evidence that plants can detect and respond to beneficial nematodes. These findings will ultimately contribute to promoting increased adoption of biological control using EPNs and a decrease in reliance on synthetic insecticides. Objective 2: Determine the behavioral responses of above and belowground herbivores to EPN cues. 1) Major activities completed / experiments conducted: We quantified herbivore preference for plants with or without EPN cues in olfactometer and Petri dish experiments. Experiments were conducted with cucumber plants (Cucumis sativus), striped cucumber beetles (Acalymma vittatum), and the EPN species Steinernema carpocapsae and Heterorhabditis bacteriophora. 2) Data collected: We collected preference data (% choice) for striped cucumber beetle larvae for roots of cucumber plants with EPN-infected insect cadavers or with freeze-killed control cadavers. We also collected oviposition preference data for female cucumber beetles for plants with or without EPN-infected cadavers. 3) Summary statistics and discussion of results: We found that cucumber beetle larvae avoid chemical cues from Heterorhabditis bacteriophora-infected cadavers, but do not respond to chemical cues from Steinernema carpocapsae. Female beetles did not appear to respond to EPN chemical cues. 4) Key outcomes or other accomplishments realized: The results from this objective provide evidence that chemical cues from beneficial nematodes can repel belowground herbivores. These findings will ultimately contribute to promoting increased adoption of biological control using EPNs and assisting with selection of EPN species to achieve the maximum benefits for biological control.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Helms, A.M., Ray, S., Matulis, N.L., Kuzemchak, M.C., Grisales, W., Tooker, J.F., Ali, J.G. Chemical cues linked to risk: Cues from belowground natural enemies enhance plant defences and influence herbivore behaviour and performance. Functional Ecology. 33, 798-808 (2019). DOI: 10.1111/1365-2435.13297
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Grunseich, J.M., Thompson, M.N., Aguirre, N.M., Helms A.M. The Role of Plant-Associated Microbes in Mediating Host-Plant Selection by Insect Herbivores. Plants. 9, 6 (2020). https://doi.org/10.3390/plants9010006
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Grunseich, J.M., Thompson, M.N., Hay, A.A., Gorman, Z., Kolomiets, M.V., Eubanks, M.D., Helms A.M. Risky roots and careful herbivores: Sustained herbivory by a root-feeding herbivore attenuates indirect plant defences. Functional Ecology. 34,17791789 (2020). https://doi. org/10.1111/1365-2435.13627
• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Grunseich, J.M., Aguirre, N.M., Thompson, M.N., Ali, J.G., Helms A.M. Olfactory cues from entomopathogenic nematodes vary across species with different hunting strategies, triggering different behavioral responses in prey and competitors (Under Review)
• Type: Theses/Dissertations Status: Accepted Year Published: 2021 Citation: Grunseich, J.M. OLFACTORY CUES MEDIATE MULTITROPHIC INTERACTIONS AMONG CUCUMBER PLANTS, CUCUMBER BEETLE LARVAE AND ENTOMOPATHOGENIC NEMATODES. M.S. Thesis. Texas A&M University Department of Entomology.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Helms, A.M., Grunseich, J.M., Hay, A.A.,Thompson, M.T., Aguirre, N.M., Ali, Jared G. Challenges and opportunities for enhancing biological control of cucumber beetles. Invited oral presentation in IOBC-NRS symposium Early career professionals promoting biological control in a changing world. Entomological Society of America Annual Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Helms, A.M., Grunseich, J.M., Hay, A.A.,Thompson, M.T., Manuel, E., Aguirre, N.M.The smells of dinner, death, and danger: How organisms navigate multitrophic interactions in a chemical world. Invited seminar. University of California Davis Department of Entomology.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Helms, A.M., Grunseich, J.M., Hay, A.A.,Thompson, M.T., Manuel, E., Aguirre, N.M.The smells of dinner, death, and danger: How organisms navigate multitrophic interactions in a chemical world. Invited seminar. Purdue University Department of Entomology.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Helms, A.M., Grunseich, J.M., Hay, A.A.,Thompson, M.T., Manuel, E., Aguirre, N.M.The smells of dinner, death, and danger: How organisms navigate multitrophic interactions in a chemical world. Invited seminar. Texas A&M University Department of Entomology.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Helms, A.M., Grunseich, J.M., Hay, A.A.,Thompson, M.T. Entomopathogenic nematodes and their bacterial symbionts influence plant defenses and herbivore performance. Invited oral presentation. Entomological Society of America Annual Meeting. St. Louis, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Helms, A.M., Grunseich, J.M., Hay, A.A.,Thompson, M.T. Chemical cues linked to risk: plants and insect herbivores respond to chemical cues from entomopathogenic nematodes. Invited oral presentation in symposium Signaling and perception in plant-herbivore interactions. Asia-Pacific Association of Chemical Ecologists (APACE) Hangzhou, China.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Helms, A.M., Grunseich, J.M., Hay, A.A.,Thompson, M.T., Manuel, E., Aguirre, N.M.The smells of dinner, death, and danger: How organisms navigate multitrophic interactions in a chemical world. Invited seminar. University of Nevada Reno Ecology, Evolution, and Conservation Biology Program.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Helms, A.M., Grunseich, J.M., Hay, A.A., De Moraes, C.M., Mescher, M.C., Tooker, J.F. Ray, S., Matulis, N.L., Kuzemchak, M.C., Grisales, W., and Ali, J.G. Chemical cues linked to risk: eavesdropping by plants and insect herbivores. Invited seminar. Agrilife Research Center, Texas A&M University. Weslaco, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Helms, A.M., Grunseich, J.M., Hay, A.A., De Moraes, C.M., Mescher, M.C., Tooker, J.F. Ray, S., Matulis, N.L., Kuzemchak, M.C., Grisales, W., and Ali, J.G. Chemical cues linked to risk: eavesdropping by plants and insect herbivores. Invited seminar. Natural Science Division Pepperdine University, Malibu, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Helms, A.M., Ray, S., Matulis, N.L., Kuzemchak, M.C., Grisales, W., Ali, J.G. Chemical cues from beneficial nematodes enhance plant protection against herbivores. Gordon Research Conference Plant-Herbivore Interactions. Ventura, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Helms, A.M., Grunseich, J.M., Hay, A.A., De Moraes, C.M., Mescher, M.C., and Tooker, J.F. Ray, S., Matulis, N.L.*, Kuzemchak, M.C., Grisales, W., Ali, J.G. Chemical cues linked to risk: eavesdropping by plants and insect herbivores. Invited seminar. Plant Pathology Department Texas A&M University, College Station, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Grunseich, J.M., Thompson, M.N., Hay, A.A., Helms, A.M. Temporal changes in olfactory cues from plant roots influence foraging by entomopathogenic nematodes. Oral Presentation. North-Central/Southwestern Branch Entomological Society of America Joint Virtual Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Grunseich, J.M., Helms, A.M. A specialist herbivore uses chemical cues from host plant roots to avoid competition and elevated predation risk. Poster presentation. Entomological Society of America Meeting. St. Louis, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2019 Grunseich, J.M., Helms, A.M. The effects of belowground chemical cues from entomopathogenic nematodes and conspecifics on host plant selection of diabroticite beetle larvae. Poster presentation. Southwestern Branch Entomological Society of America Meeting Tulsa, USA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Grunseich, J.G., Helms, A.M. A root-feeding herbivore suppresses indirect plant defenses to avoid elevated predation risk. Oral Presentation. Texas A&M Entomology Graduate Student Forum.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Grunseich, J.G., Helms, A.M. A root-feeding herbivore attenuates indirect plant defenses to avoid elevated predation risk. Oral Presentation. Entomological Society of America Virtual Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020 Grunseich, J.G., Helms, A.M. A root-feeding herbivore suppresses indirect plant defenses to avoid elevated predation risk. Poster presentation. Texas Plant Protection Association Annual Virtual Meeting.