Source: MONTANA STATE UNIVERSITY submitted to
INSECTICIDE RESISTANT ALFALFA WEEVILS IN THE WESTERN US: QUANTIFYING THE SCOPE OF RESISTANCE AND IMPLEMENTING A PLAN TO MANAGE THE THREAT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1020627
Grant No.
2019-70005-30286
Cumulative Award Amt.
$499,988.00
Proposal No.
2019-02811
Multistate No.
(N/A)
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2022
Grant Year
2020
Program Code
[AFRP]- Alfalfa and Forage Program
Project Director
Wanner, K.
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Office of Sponsored Programs
Non Technical Summary
Alfalfa weevil, the key economic pest of alfalfa throughout the continental US, has developed resistance to the pyrethroid group of insecticides (Warrior®, Mustang® Maxx and others) used to control it, making its economic damage more severe in these areas and foretelling broader management problems. Several production areas in the Western US and Canada have reported failures of insecticides to control alfalfa weevil, and we have corroborated producer reports of pyrethroid-resistant weevils with laboratory data. In two affected areas at minimum, producers have abandoned alfalfa as a crop due to an inability to control insecticide-resistant weevils. This threatens not only the economics of the $20 billion alfalfa forage industry, but also the $67 billion and $38 billion beef and milk commodities that depend in part on alfalfa forage production.A rapid research and Extension response is needed to mitigate economic loses by prolonging the useful life of insecticides through resistance management. Research will quantify the degree and geographic scope of insecticide resistance using dose-response experiments and monitoring bioassays. Science-based results will be incorporated into Extension media that provides resistance management recommendations to stakeholders within the framework of integrated pest management (IPM).A multi-state team of research scientists and Extension specialists representing Arizona, California, Colorado, Montana and Oregon will develop and deliver resistance management recommendations in the Western US.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21116401130100%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
Alfalfa weevil is the predominant economic pest of forage alfalfa across the continental US. When left unmanaged it can cause substantial damage to this crop with thin economic margins. Pest managers turn to synthetic pyrethroid insecticide (i.e. Warrior ®, Mustang® Maxx and others) as the first choice for control because of their low cost and efficacy. However, pyrethroid resistant alfalfa weevils, now documented in the western US are causing yield loss and increased management costs.A multistate team of research scientists and Extension specialists representing Arizona, California, Colorado, Montana and Oregon will quantify the scope of insecticide resistance and begin to mitigate its economic impact in the western US with Extension.Specific Objectives:1) Quantify and demonstrate alfalfa weevil resistance to lambda-cyhalothrin and zeta- cypermethrin (Warrior II® and Mustang® Maxx, MoA group 3A) using laboratory dose- response assays.2a) Monitor pyrethroid resistance in alfalfa weevil populations across the western US to map its geographic spread using a vial assay mail-out kit.2b) In California, correlate pesticide use history, local landscape, and cropping practices with patterns of insecticide resistance levels to predict risk of insecticide resistance.3) Measure baseline susceptibility of alfalfa weevil to indoxacarb (Steward®, MoA group 22A) across the western US region. 4) Deliver resistance management recommendations through multistate Extension.
Project Methods
Methods overview:The lethal concentration of an insecticide that results in 50% and 90% mortality (LC50 and LC90) are often calculated by probit analysis of mortality recorded at different doses of the active ingredient. LC50 values obtained using a standardized protocol is the most accurate method to compare resistance levels in different pest populations and provides a baseline to track future changes in resistance levels, with statistical significance. First, alfalfa weevil resistance levels to pyrethroid insecticides will be quantified with dose-response experiments. Next,the focus shifts to monitoring changes in resistance across the alfalfa production landscape.Single dose (discriminating dose) and two- dose approaches are commonly used to monitor insecticide resistance after it has beendemonstrated quantitatively in a pest population. Finally, baseline LC50 values and resistance monitoring data form the foundation of Extension recommendations to manage resistance, and provides the ability to evaluate the effectiveness of the efforts to reduce the magnitude of resistance and its spread.1. Laboratory dose-response assays to quantify resistance to lambda-cyhalothrin (Warrior II®) and zeta-cypermethrin (Mustang® Maxx)Dose-response experiments accurately quantify the degree of insecticide resistance, provide a baseline to track its development over time, and allow differences to be assigned statistical significance.Dose-response experiments will focus on alfalfa weevil populations with suspected resistance based on observations of poor or failed control after pesticide application in commercial fields.2a. Monitoring pyrethroid resistance in alfalfa weevil populations in the western US. A two-dose discriminating bioassaywill be used to monitorinsecticide resistance levels in the western US. One dose that results in 90% mortality of susceptible populations and a dose 5 times higher than this dose (using data from Objective 1), along with an untreated control, will be used to distinguish insecticide- resistant and susceptible alfalfa weevil populations.Resistance testing kits will be mailed to participants in thewestern US alfalfa production areas using arbitrary and targeted approaches.2b In California pesticide use history, local landscape, and cropping practices with be correlated with patterns of insecticide resistance levels to predict risk of insecticide resistance.In this research objective, we will perform detailed sampling in the Scott and Palo Verde Valleysof CA. We will use the dose-response assay from Objective 1 to determine LC50 values for populations within each of these regions. Over the course of two years, we will test 15-20 populations per region. The California Pesticide Information Portal project (CalPIP, calpip.cdpr.ca.gov) provides access to the extensive pesticide use database maintained by the Department of Pesticide Regulation dating back to 1990. Using this database, historical pyrethroid use is reported as acres treated and pounds of active ingredient at a square mile resolution (County/Meridian/Township/Range/Section, COMTRS). We will relate measured resistance values in a field to the local pesticide use history.3. Measure baseline susceptibility of alfalfa weevil to indoxacarb (Steward®, MOA group 22A) across the western US region.We will measure baseline susceptibility of alfalfa weevil populations using dose-response assays as described in Objective 1 with modification. Indoxacarb acts mainly through ingestion. Bioassays described in Objective 1 will be modified to use alfalfa shoots dipped in insecticideor by incorporating insecticide into diet. Alfalfa fields with a history of indoxacarb use or of no use will be selected to establishbaseline weevil susceptibility to this newer active ingredient and to quantify any resistance if present.4.Deliver resistance management recommendations through multistate Extension.Research results from the first three objectives will be integrated into Extension programing to provide multistate recommendations to manage insecticide resistance in forage and seed alfalfa production systems, including:Science-based recommendations for western US forage and seed alfalfa producers; developing multistate recommendations for resistance management; creating common use publically available extension media; and reaching stakeholders through an establishednetwork of research and Extension specialists in the western US that are strongly positioned to disseminate the results and recommendations of this research.

Progress 09/01/19 to 10/22/21

Outputs
Target Audience:Efforts to dissemminate information about the objectives of this project to quantify the degree and extent of insecticide resistance in alfalfa weevils in the western region, and efforts to mitigate their economic impact, reached a range of stakeholders related to forage alfalfa production. Stakeholders include industry, government and academic agriculuturalists, educators and researchers along with producers and farm and range managers. The first target audience reached were producers, ranchers and dairymen that directly grow alfalfa for on-farm use or for sale. Second, agricultural businesses such as the agrochemical industry, seed companies and commercial pesticide applicators were made aware of the issue of insecticide resistant alfalfa weevils and the efforts to research them. Third, scientists and agricultural educators were reached by our efforts, including entomologists, forage specialists, agronomists and county agents working in the field of alfalfa and forages. The target audiences have been reached using a variety of media including a website, business cards, publications in trade magazines and pressentations at meetings. The western US represents the primary target region but the national audience is relevant. Changes/Problems:Travel and research restrictions related to the 2020 covid virus pandemic created challenges in completing some project tasks. This is a multi-state project and the degree of these challenges varied with state-specific guidelines. Adjustments were made (such as online outreach and extension efforts) and overall significant progress was achieved. What opportunities for training and professional development has the project provided?Mentoring: Two graduate students were recruited and and have been trained during the two years of this project. Erika Rodbell, PhD program, Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT and Madison Hendrick, MS Entomology Program, Entomology Department, University of California Davis, Davis CA. Both students have attened regional producer-focused alfalfa meetings and presented their results, as well attending regional and national scientific meetings where they presented their results, contributing to their professional developement. Both students have been engaged in outreach and extension activities as well writting scientfic publicatons to disseminate their results. In addtion, each summer two undergraduate research assistants are employed to assist with laboratory and field entomology/agricutlure research. How have the results been disseminated to communities of interest?Efforts to dissemminate information about the objectives of this project to quantify the degree and extent of insecticide resistance in alfalfa weevils in the western region, and efforts to mitigate their economic impact, reached a range of stakeholders related to forage alfalfa production. The first target audience reached were producers, ranchers and dairymen that directly grow alfalfa for on-farm use or for sale. This audience was reached via a variety of Extension venues, including on-farm visits, in person and online Extension presentations, attendacne and presentations at regional alfalfa producer meetings and research center farm field days. Second, agricultural businesses such as the agrochemical industry, seed companies and commercial pesticide applicators were made aware of the issue of insecticide resistant alfalfa weevils and the efforts to research them. Third, scientists and agricultural educators were reached by our efforts, including entomologists, forage specialists, agronomists and county agents working in the field of alfalfa and forages. The target audiences have been reached using a variety of media including a website, business cards, publications in trade magazines and pressentations at meetings. The western US represents the primary target region but the national audience is relevant. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the two year period of this research and extension project the magnitude and geographic extent of pyreethroid resistant alfalfa weevils was determined in six western region states. Alfalfa weevil populations that were highly resistant, moderatley resistant and suceptibile to lambda cyhalothrin (one of the most commonly used pyrethroid active ingredients for alfala weevil management) were identified in each state where experiments were conducted. This data demonstrates that the genetic basis for pyrethroid resistance is well established in alfalfa weevil populations across the western US. The presence of suceptible and moderatley resistant populaitons, in addition to highly resistant pockets, demonstrates the potenial to mitigate the effects of insecticide resistance. A second major outcome of this research was the demonstration of cross-resistance between different pyrethroid active ingredients. Several different active ingredients within the pyrethroid class (mode of action group 3A, MoA3a) are registered in many different commerical formulations and trade names for alfalfa weevil cotrol. Our demonstation of cross resistnace between the different active ingredients provides evidence that all MoA3A products contribute to the development of pyrethroid resistance and when resistance is present, none of these products will be effective. Our disemination of these science-based results through extension and scientific manuscripts has begun to change management practicies used by producers and agricultural managers. In areas of high resistance alfalfa producers have begun to chage the MoA insecticide used to management alfalfa weevils and to employ the practices of integreated pest management (IPM). The use of economic thresholds to guide decisions to apply insecticide and the incorporation of cultural practices such as early harvest serve to reduce the pressue to select resistant insects and prolong the usefullness of the MoA3A pyrethroid class of insecticide. Objectives 1 & 2 A glass vial bioassay was developed to quantify the effective concentration of pyrethroid that is lethal to 50% of the insects in a lab assay (LC50 value). Alfalfa weevils were collected from field sites in six different western region states to assay the effectiveness of three pyrethroid active ingredients, lambda cyhalothrin, zeta cypermethrin and permethrin. Alfalfa weevil larvae from six western region states were assayed with lambda-cyhalothrin, and when possible zeta-cypermethrin and permethrin, using concentration response experiments. Larvae from most sites, when placed in treated vials for 24 hours, responded to increasing concentrations of lambda-cyhalothrin with increasing mortality that fit a sigmoidal relationship when plotted against the log [concentration]. However, this was not the case for larvae from highly resistant sites, where a clear concentration dependent and sigmoidal response of mortality was not observed.The LC50 values are listed in Tables 1-3. Montana Results Table 1. Lethal concentrations of three active ingredients that result in 50% mortality (LC50, µg/cm2) for alfalfa weevil larvae sampled from five Montana counties. Multiple sample collection dates within the same county are indicated by subscript letters that follow the field site number. Chi-square and p-values are provided as a measure of goodness of fit with the Probit analysis (the fit is not rejected if p > 0.05). Active ingredient County Field Site Date T-ratio of the slope LC50 µg/cm2 ??2df p-value Number of concentrations Lambda-cyhalothrin Big Horn 1d 4.12 >3.3 9.78 5 0.08 8 2 c 2.51 >1.0 3.09 2 0.21 5 2 e 4.08 >3.3 7.62 4 0.11 7 Broadwater 1 5.1 0.26 7.39 5 0.19 8 2 8.22 0.26 4.79 5 0.44 8 3 7.82 0.77 2.68 2 0.26 5 Madison 1 2 8.33 7.18 0.02 0.06 2.47 5 6.41 5 0.78 0.27 8 8 Powder River 1 4.58 0.03 2.73 2 0.26 5 2 7.14 0.1 5.51 4 0.24 7 3 7.83 0.09 6.58 4 0.16 7 Richland 1 6.58 0.09 5.13 5 0.40 8 Zeta-cypermethrin Big Horn 1 5.18 >3.3 9.02 5 0.11 8 2 5.57 >3.3 3.12 5 0.68 8 Active ingredient County Field Site Date T-ratio of the slope LC50 µg/cm2 ??2df p-value Number of concentrations Permethrin Big Horn 1 a 2.82 4.74 0.00411 0.95 4 1 b 4.96 1.18 0.78 2 0.68 5 1 d 7.9 1.54 3.22 5 0.67 8 2 e 5.56 7.05 3.9 4 0.42 7 Broadwater 1 6.12 1.28 5.19 3 0.16 6 Madison 1 7.5 0.17 5.45 3 0.14 6 Powder River 1 5.81 0.55 8.06 5 0.15 8 2 7.36 0.1 6.58 4 0.24 7 3 5.85 0.19 5.45 3 0.14 6 a- 5/25/2020 b- 5/30/2020 c- 6/1/2020 d- 6/4/2020 e- 6/6/2020 California Results Table 2. Pyrethroid LC50 (proportion of label rate) values from populations sampled in California. An LC50 of 1.0 = the highest label rate; 0.10 = 1/10th of the highest label rate; and 10.0 = ten times the highest label rate. Green = susceptible; yellow = moderate resistance; and red = high resistance. LC50 (LC50 values from five Montana Counties are reported in Table 3. County Site Active Ingredient LC50 (fl oz/ac) Year Level Riverside 1 lambda-cyhalothrin 0.40 2020 2 (organic) lambda-cyhalothrin 0.10 2021 3 (organic) lambda-cyhalothrin 0.09 2021 Merced 1 lambda-cyhalothrin 0.51 2020 2 lambda-cyhalothrin 0.71 2021 3 lambda-cyhalothrin 0.06 2021 4 lambda-cyhalothrin 0.09 2021 5 lambda-cyhalothrin 0.13 2021 Kern 1 lambda-cyhalothrin 0.01 2020 2 lambda-cyhalothrin 0.01 2020 3 lambda-cyhalothrin 0.09 2021 Kings 1 lambda-cyhalothrin 0.14 2020 2 lambda-cyhalothrin 0.13 2020 3 lambda-cyhalothrin 0.01 2020 4 lambda-cyhalothrin 0.05 2020 Tulare 1 lambda-cyhalothrin 3.46 2020 2 lambda-cyhalothrin 2.22 2020 Yolo 1 lambda-cyhalothrin 0.01 2020 2 lambda-cyhalothrin 0.51 2020 3 lambda-cyhalothrin 0.32 2021 4 lambda-cyhalothrin 5.63 2021 5 lambda-cyhalothrin 0.17 2020 6 lambda-cyhalothrin 0.09 2020 7 lambda-cyhalothrin 0.13 2021 8 lambda-cyhalothrin 0.75 2021 *equivalent technical grade lambda-cyhaltohrin to Warrior II label rate (high label rate for alfalfa weevil = 1.92 fl oz/acre) Western Region Results Table 3. Alfalfa weevil larval resistance level to lambda-cyhalothrin in the western region of the United States by state. State Co. Site year LC50 µg/cm2 T-ratio slope χ2df P value # of Conc. Resistance Level AZ La Paz 12021 1.55 6.09 8.395 0.14 8 H 22021 0.65 8.27 7.95 0.16 8 M 32021 2.78 9 5.045 0.41 8 H 42021 0.51 6.24 0.0011 0.97 4 M Mohave 12020 0.01 3.45 17.55 0.004 8 S Pinal 12021 0.15 8.52 6.084 0.19 7 S Yuma 12020 0.18 5.77 2.051 0.15 4 S 22021 0.12 6.64 3.34 0.51 7 S 32021 1.35 8.27 7.95 0.16 8 H OR Umatilla 12021 0.06 5.48 5.894 0.21 7 S 22021 0.17 5.88 7.94 0.10 7 S 32021 0.01 3.38 3.455 0.63 8 S WA Klickitat 12020 >3.3 3.76 6.565 0.26 8 H Yakima 12021 >10.0 3.76 6.565 0.65 8 H 22021 0.21 9.27 8.565 0.13 8 S WY Converse 12021 1.84 6.54 8.924 0.06 7 H 12021 6.92 4.2 9.65 0.08 8 H Platte 12021 0.52 7.97 3.924 0.41 7 M 12021 0.24 6.99 6.74 0.22 7 S Sheridan 12020 2.23 5.05 11.095 0.05 8 H Objectives 3 & 4 Multi state extension to deliver resistnace management reccomendations are listed in the "Products" section of this report. Outreach included attending and presenting resistance results and reccomendations at regional alfalfa conferences and workshops as well as state state level Extension venues.The effectiveness of Steward, to obtain baseline levels of its efficacy, was assessed in Montana and California using vial assays, in Montana using whole plant dip bioassays and in field trials conducted in Montana. The best protocol for standardized testing of Steward continues to be developed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rodbell, E. A., and K. W. Wanner. 2021. First report of alfalfa weevil (Coleoptera: Curculionidae) resistance to lambda-cyhalothrin in Montana. J. Econ. Entomol. https://doi.org/10.1093/jee/toab152.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rethwisch, M.D., Peairs, F., Pierce, J., Mostafa, A., Price, S., Ramirez, R., Rondon, S., Schell, S., Vardiman, J., Walsh, D.B., Wanner, K., and Wenninger, E. 2019. Resistance in Alfalfa Weevil and Related Implications in Other Alfalfa Insect Pests. In: Proceedings, 2019 Western Alfalfa and Forage Symposium, Reno, NV, Nov.19  21, 2019. UC Cooperative Extension, Plant Sciences Department, University of California, Davis, CA 95616. (http://alfalfa.ucdavis.edu)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Kevin W. Wanner, Ian M. Grettenberger, Erika Rodbell and Madison Hendrick. ALFALFA WEEVIL MANAGEMENT & RESISTANCE ISSUES. In: Proceedings, 2021 Western Alfalfa and Forage Symposium, Reno, NV, Nov.16  18, 2021. UC Cooperative Extension, Plant Sciences Department, University of California, Davis, CA 95616. (See http://alfalfa.ucdavis.edu for this and other alfalfa symposium proceedings.)
  • Type: Journal Articles Status: Other Year Published: 2022 Citation: E.A. Rodbell1, M. Hendrick, I. Grettenberger and K.W. Wanner. 2021. Alfalfa weevil (Coleoptera: Curculionidae) resistance to lambda-cyhalothrin in the western region of the United States. In Preparation.
  • Type: Websites Status: Published Year Published: 2020 Citation: RAW: Resistant Alfalfa Weevil Project. https://www.montana.edu/resistantalfalfaweevil/
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Rodbell and Wanner. Entomological Society of America- Pacific Branch meeting 2021 PhD paper competition, Remote. Pyrethroid resistant alfalfa weevils in Montana.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Hendrick and Grettenberger. ESA Pacific Branch Meeting, Virtual  Apr. 2021. Insecticide resistance monitoring in California populations of alfalfa weevil (Hypera postica).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Hendrick and Grettenberger. ESA National Meeting, Virtual  Dec. 2020. Insecticide resistance in California populations of alfalfa weevil (Hypera postica).
  • Type: Other Status: Published Year Published: 2021 Citation: Rodbell and Wanner. Extension presentation, Pyrethroid resistant alfalfa weevils in Montana and Wyoming. Wyoming Pest Seminar 2021, Remote, Western Region attendees.WYOMING PEST VIRTUAL SEMINAR ALFALFA WEEVILS, MARCH 2, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Rodbell and Wanner, Managing Weevil & Aphids in Alfalfa. CROPLAN Alfalfa Training Event Feb 4th, 2021, Remote.
  • Type: Other Status: Published Year Published: 2020 Citation: Grettenberger, Extension talk, Alfalfa Weevil Insecticide Resistance. SJC and Delta Field Crops Meeting  Jan. 2020
  • Type: Other Status: Published Year Published: 2019 Citation: Grettenberger, Extension talk, Alfalfa Weevils: Management & the Spectre of Resistance. Pest Management Seminar  Siskiyou Co., Yreka, CA  Dec. 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Wanner, Management of insecticide resistance in alfalfa, 2020 Alfalfa Integrated Pest Management Virtual Workshop,December 3-4, 2020,University of California Cooperative Extension, UC Davis, UC Agriculture & Natural Resources
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Wanner. Insecticide Resistant Alfalfa Weevils, 2020 Western Alfalfa Seed Growers Association, January 26-28, 2020, Reno, NV.
  • Type: Other Status: Published Year Published: 2020 Citation: Wanner, virtual Extension Talk, Alfalfa weevil management and insecticide resistance. Southcentral Ag Forum, Billings, MT Dec. 1, 2020.
  • Type: Other Status: Published Year Published: 2019 Citation: Wanner, Poster: RAW - Resistant Alfalfa Weevil Project. 2019 Western Alfalfa and Forage Symposium, Nov.19  21, 2019. UC Cooperative Extension, Plant Sciences Department, University of California, Davis, CA 95616. (http://alfalfa.ucdavis.edu)
  • Type: Other Status: Published Year Published: 2020 Citation: Grettenberger, Extension talk, Alfalfa Insect Pest IPM Update: Aphids, Weevils, and Worms, Kearney REC Alfalfa and Forage Virtual Field Day  Sept. 2020
  • Type: Other Status: Published Year Published: 2020 Citation: Grettenberger, Extension talk, Pest management strategies with less chlorpyrifos, Golden State Dairy Management Conference  Mar. 2020
  • Type: Other Status: Published Year Published: 2020 Citation: Grettenberger, Extension talk, Insecticide resistance of alfalfa weevil: Current status and how we can mitigate the problem. California Plant and Soil Conference, Fresno, CA  Feb. 2020
  • Type: Other Status: Published Year Published: 2019 Citation: Grettenberger, Extension talk, Alfalfa Weevil Management and Insecticide Resistance, Kearney REC Alfalfa and Forage Field Day, Parlier, CA  Sept. 2019
  • Type: Other Status: Published Year Published: 2021 Citation: Grettenberger, Extension talk, Insecticide resistance in California populations of alfalfa weevil (Hypera postica. California Plant and Soil Conference, Virtual  Feb. 2021.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Efforts to dissemminate information about the objectives of this project to quantify the degree and extent of insecticide resistance in alfalfa weevils in the western region, and efforts to mitigate their economic impact, reached a range of stakeholders related to forage alfalfa production. The first target audience reached were producers, ranchers and dairymen that directly grow alfalfa for on-farm use or for sale. Second, agricultural businesses such as the agrochemical industry, seed companies and commercial pesticide applicators were made aware of the issue of insecticide resistant alfalfa weevils and the efforts to research them.Third, scientists and agricultural educators were reached by our efforts, including entomologists,forage specialists, agronomists and county agents working in the field of alfalfa and forages. The target audiences have been reached using a variety of media including a website, business cards, publications in trade magazines and pressentations at meetings. The western US represents the primary target region but the national audience is relevant. Changes/Problems:Travel and researchrestrictions related to the 2020 covid virus pandemic created challenges in completing some research tasks. Adjustments were made and significant progress was achieved and the research is on track for completion in the second year. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Summary: During the first nine moths significant progress was made towards the grant goals and objectives. A website and other media were developed to advertise the project and disperse the results to stakeholders, including presentations at producer meetings. A bioassay to test pyrethroid insecticide resistance was developed and validated. Alfalfa weevil from alfalfa fields in several states/counties of the western US were assayed are both pyrethroid susceptible and resistant populations identified. Testing continues during the last quarter of the first grant year. Progress09/01/2019 - 06/01/2020 Stage 1.Recruit and train graduate students to support research activities (Fall 2019). Two graduate students have been recruited and supported by this grant beginning Sept. 2019. i. Erika Rodbell, PhD program, Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT. Erika graduated from Iowa State University, Ames, IA ii.Madison Hendrick, MS Entomology Program, Entomology Department, University of California Davis, Davis CA. Stage 2.Develop outreach media to advertise the project to stakeholders and to provide a platform to deliver results and extension programing (Fall 2019). An internet website with supporting content was developed to advertise the project and to serve as a platform to deliver result and outreach material. Outreach media included a poster that presented at alfalfa commodity meetings attended by the PI and the graduate students. Business cards were also developed and printed to advertise the project as well as articles in trade magazines. i. Resistant Alfalfa Weevil Project website, https://www.montana.edu/resistantalfalfaweevil/ Figure 1. Opening page of the Resistant Alfalfa Weevil Project webpage with the "RAW" logo developed by the Wanner and Grettenberger. Figure 2. Description of the Resistant Alfalfa Weevil Project "RAW" Figure 3. Link for stakeholders to request insecticide testing kits. ii. Business Cards for the Resistant Alfalfa Weevil Project Figure 4. Business card designed to advertise the resistant alfalfa weevil project. Front side: RAW logo.Back side: Link to the RAW website and contact information for PI Wanner and co-PI Grettenberger. iii. Figure 5. Poster summarizing the Resistant Alfalfa Weevil Project iv. Alfalfa producer meetings attended and trade magazine articles published. Western Alfalfa & Forage Symposium, November 19-21, 2019, Grand Sierra Resort, Reno, NV Attended by: Kevin Wanner (PI) Erika Rodbell (Graduate Student) Madison Hendrick(Graduate Student) Poster Presented: RAW - Resistant Alfalfa Weevil Project WesternAlfalfaSeedGrowers Association 2020 Golden Nugget inLasVegas, NV on January 26-28, 2020. Attended by: Kevin Wanner (PI) Talk Presented: 20 minutes, "Insecticide Resistant Alfalfa Weevils" AgNet West (Trade Magazine) article Ian Grettenberger, "New Resistant Alfalfa Weevil Project Underway",AgNet West FEBRUARY 17, 2020, http://agnetwest.com/new-resistant-alfalfa-weevil-project-underway/ Stage 3.Develop and validate the bioassay that will be used to quantify pyrethroid resistance using larvae from the southern desert alfalfa production areas of California and Arizona (January - March 2020). During February 2020 co-PI Grettenberger and graduate studentHendrick began sampling and testing alfalfa weevils from southern California. PI Wanner traveled to southern Arizona and southern California duringFebruary 2020. The purpose of this early sampling was to validate the bioassay methods. Methods Developed: Pure, technical-grade active ingredients (lambda-cyhalothrin and zeta-cypermethrin) were obtained free of charge from the manufacturers (Syngenta and FMC Corp.).Pure permethrin was purchased from Sigma-Aldrich (now Millipore Sigma). Technical grade insecticide is weighed onto a glass microscope slide and dissolved into 95% acetone to make a working stock solution (taking into account % purity of the active ingredient). The working stock solution is used to make a series of 3x serial dilutions in 95% acetone. A 1.0 ml aliquot of insecticide dissolved into 95% acetone is dispensed into a glass vial measuring 8.3 cm in height with an inner surface area of 57.3 cm2. The vials are rotated on hot dog rollers (with the heat turned off) to allow the acetone to evaporate and to leave an even coating of active ingredient on the inner surface of the vial (Figure 6). The vials are then infested with 10 3rdor 4thinstar alfalfa weevil larvae in to each vial and each dose is replicated by 5 vials for a total of 50 larvae tested for each dose. ?Results: Infested larvae are maintained at 70°C for 24 hours prior to evaluation. After 24 hours larvae are placed on a hot plate heated to 100°C. Under these conditions normal healthy begin crawling immediately. Paralyzed or dead larvae are unable to crawl. The number of live and dead/paralyzed larvae are scored. During February and March Grettenberger tested larvae from California. Wanner and graduate student Rodbell travelled to Arizona to test larvae before the field season in Montana began. Date of weevil collections: 02/18/2020-02/27/2020 Locations: Arizona; Bullhead City; Parker; Roll, California: Blythe. Preliminary results indicate that pyrethroid resistance in the Palo Verde valley of California has increased during the last three years and now is more widespread than previously thought.Figures 8-10 illustrate dose response results from alfalfa weevil populations susceptible and resistant to lambda cyhalothrin (Warrior II).Figures 11 and 12 demonstrate susceptible and resistant alfalfa weevil populations from two counties in California. Figure 8. Response of alfalfa weevil larvae to increasing concentrations of lambda cyhalothrin. Sampled from an alfalfa field near Mohave Arizona. Mortality increases at lower doses and plateaus above 90% mortality for the last three doses. LD50 = 0.01 ug ai per cm2. Figure 9. Response of alfalfa weevil larvae to increasing concentrations of lambda cyhalothrin. Sampled from an alfalfa field near Blythe California. Mortality increases slowly at lower doses and never exceeds 80%. LD50 = 0.3 ug ai per cm2. Figure 10. Overlapping response curves for susceptible larvae (top curve, Mohave AZ) and resistant larvae (bottom curve, Blythe CA). The LD50 for the resistant larvae is 30 times higher than the LD50 for susceptible larvae. Figure 11.Zonnefeld Farms in Kings County California, susceptible population, mortality plateaus above 90% at the 4 highest concentrations. Figure 12. Riverside CountyCalifornia, resistant population, mortality does not exceed 60% at the highest concentrations tested. Stage 4.Scale up insecticide testing in the western US Region (April - July 2020). Insecticide resistance testing during April and May was slowed by restrictions associated with the covid outbreak. Nevertheless we were able to proceed with testing under restricted operating rules. Alfalfa fields in southern and central California have now been assayed, fields in northern California are currently being tested. California counties completed: Riverside (3 fields); Kern (2 fields); Kings (4 fields); Madera (2 fields); Merced (4 fields); Yolo (7 fields); Siskiyou (5 fields); and Lassen (1 field).Collaborators have sent larvae from Washington, Oregon and Kansas to Montana State University for bioassay and we are currently awaiting larvae from a collaborator in Wyoming. Alfalfa weevil larvae are now peaking as 2ndand 3rdinstars in the warmer regions of Montana and testing is now in full operation. Testing in Montana will continue through to the end of June and early July in the northern areas of the state.

Publications

  • Type: Websites Status: Published Year Published: 2019 Citation: Resistant Alfalfa Weevil Project website, https://www.montana.edu/resistantalfalfaweevil/
  • Type: Other Status: Published Year Published: 2020 Citation: Ian Grettenberger, New Resistant Alfalfa Weevil Project Underway, AgNet West FEBRUARY 17, 2020, http://agnetwest.com/new-resistant-alfalfa-weevil-project-underway/
  • Type: Other Status: Published Year Published: 2019 Citation: Wanner, K.W. and Rodbell, E. Poster Presented: RAW - Resistant Alfalfa Weevil Project. Western Alfalfa & Forage Symposium, November 19-21, 2019, Grand Sierra Resort, Reno, NV
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Wanner, K.W. Insecticide Resistant Alfalfa Weevils, Presentation at the Western Alfalfa Seed Growers Association 2020 Meeting in Las Vegas, NV on January 26-28, 2020.