Source: KANSAS STATE UNIV submitted to
DEVELOPING A RAPID RESPONSE PROTOCOL FOR PHOSPHINE RESISTANCE MANAGEMENT AT FOOD FACILITIES BY LEVERAGING TOOLS AT OR CLOSE TO MARKET
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031038
Grant No.
2023-70006-40604
Cumulative Award Amt.
$324,979.00
Proposal No.
2023-02981
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
Aug 31, 2026
Grant Year
2023
Program Code
[ARDP]- Applied Research and Development Program
Project Director
Zhu, K.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
(N/A)
Non Technical Summary
This is a multi-state, multi-institution collaborative applied research (single-function) project for Applied Research and Development Program (ARDP). The project aims to improve phosphine resistance management in insect pest populations in food facilities. Stakeholders often inquire for help after resistance has already become a problem, and there are no comprehensive guidelines for steps to take with phosphine resistant populations in the US. It is clear that stakeholders require advance warning that resistance is becoming a problem, and they need recommended, empirically-driven guidance to support resistance management programs. We aim to develop this information for stakeholders by: 1) optimizing an early, warning phosphine resistance screening tool based on recessive allele frequency; 2) conducting a survey of key Great Plains food facilities in corn-, rice-, and wheat-producing states for phosphine resistance; 3) validating and implementing a diversified suite of integrated pest management tactics in bulk storage and processing facilities to manage phosphine resistance; and 4) engaging stakeholders with a suite of interaction strategies to communicate the importance of screening for resistance, what to do if resistance is found, and associated costs. Our project includes personnel representing Kansas, Nebraska, and Arkansas, and will thus help protect the US's role as the breadbasket of the world at a time when the world faces a global food crisis, supporting USDA Strategic Goal 2 and 4, and helping to reduce health risks and environmental effects from pests and management strategies.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21631101130100%
Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
3110 - Insects;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
Most agricultural research focuses on crops prior to harvest, despite the enormous economic value associated with the postharvest agricultural supply chain. One of the key sources of postharvest losses are caused by stored product insects, which globally result in $100 billion in losses each year. The main way to combat these insect pests in bulk storage of grain and related facilities is through calendar-based fumigations (typically) or fumigations timed with grain movement (e.g., deliveries, shipments, movement between silos). As such, one of the major challenges to fumigation is the increasing worldwide surge in resistance to phosphine in a range of important pest species. However, determining the best management approach is complicated by the fact that testing for phosphine tolerance or resistance is not commonly practiced at food facilities, thus stakeholders typically do not know the level of resistance in the populations with which they are dealing. Further, there are no set recommendations for resistance management tactics for a specific level of phosphine resistance at food facilities.Our overall goal of this project is to increase resilience in the postharvest supply chain at food facilities by training personnel of food facilities to implement new screening procedures for phosphine resistance, and where necessary, validating and recommending a suite of commercially available integrated pest management (IPM) tactics to manage phosphine resistance. Our specific objectives of this project are to: 1) optimize a rapid test for detecting and monitoring phosphine resistance on the Great Plains and compare the test to a commercial standard test, 2) apply the most effective rapid phosphine tolerance/resistance test to food facilities on Great Plains and screen for resistance among multiple stored product insects, 3) deploy a suite of tactics for phosphine resistance management to reduce effects of resistance in food facilities, and 4) transfer knowledge to and engage stakeholders in implementing effective solutions to overcome phosphine resistance.
Project Methods
Objective 1. Develop quick, sensitive, and reliable techniques for detecting and monitoring phosphine resistanceWe will optimize bioassay techniques to determine the phosphine ET50 (median effective time) values in the laboratory phosphine-susceptible and resistant strains of RFB by using the PTT kit. The test insects within the plastic syringe will be observed at different exposure times. The phosphine ET50 values will then be calculated using Probit analysis for two populations of phosphine-resistant (weak and strong) and two phosphine-susceptible strains of RFB.We will use a molecular technique ARMS-qPCR to detect the P45S-related mutation. The presence and absence of the P45S mutation will be determined based on the cycle threshold (Ct) value. To confirm the presence or absence of the mutation in the ARMS-qPCR products, we will sub-clone and sequence the ARMS-qPCR-generated DNA products (30 samples showing the mutation and other 30 samples showing no mutation).To establish a relationship between the level of phosphine resistance and the frequency of resistance mutation, we will first artificially create laboratory colonies of RFB with different levels of phosphine resistance. We will then determine the phosphine ET50 value for each of the mixed colonies using the PTT kit, and compare their ET50 values against the ET50 value of the susceptible strain to obtain resistance levels. We will then determine the frequencies of the P45S mutation in these mixed colonies using ARMS-qPCR.Objective 2. Apply quick, sensitive and reliable techniques for detecting and subsequently monitoring phosphine resistance in insect populations of food facilities on the Great Plains We will analyze phosphine resistance mutation in RFB samples at food facilities using ARMS-qPCR and compare the results with those determined using PTT kit over time. We will collect 60-100 RFB from each of at least 12 food facilities in different locations in each of three states (KS, NE, and AR). The RFB samples will be collected at three time points (April, July, and October) from these 36 locations in each of two years to understand how phosphine resistance may fluctuate over time. The level of phosphine resistance will be determined for each RFB sample by linear regression analyses based on the relationship between the frequency of the phosphine resistance mutation and the level of phosphine resistance as established in Objective 1.Objective 3. Deploy suite of phosphine resistance management programs to reduce effects of phosphine resistance on food facilitiesWe will evaluate three different strategies for phosphine resistance management. First, we will examine if increasing concentration or period of fumigation can temporarily recover phosphine efficacy. This experiment will be performed using a 3-metric tonne (3-MT) grain bin. After each fumigation, the cohorts of insects will be checked for condition of individuals (alive, affected, or dead, as above).We will then deploy phosphine resistance management programs to reduce effects of phosphine resistance on bulk storage food facilities. We will test a combination of proposed tactics for bulk storage (e.g., crack-and-crevice around the perimeter, top dressing with grain protectant on commodity, insecticide netting sealing entryways, and fumigation with sulfuryl fluoride) with three alternate scenarios, including: a) sulfuryl fluoride fumigation only, b) phosphine fumigation only (stakeholder standard), and c) no management tactics (untreated control) with at least one facility each in AR, KS, and NE. The number of progeny, insect-damaged kernels (IDK), and the weight of damaged and undamaged grain will be recorded.Finally, we deploy suite phosphine resistance management programs on processing facilities and warehouses. We will begin with testing a combination of proposed tactics for processing and warehouses with three alternate scenarios, including: a) aerosols only, b) phosphine fumigation only (stakeholder standard), and c) no management tactics (untreated control). Deltamethrin will be used to treat crack-and-crevice. Cylinderized pyrethrin, piperonyl butoxide, pyriproxyfen, and other ingredients, with a carbon dioxide carrier, will be used to treat the mesocosms in a large indoor warehouse. The number of progeny, insect-damaged kernels (IDK), and the weight of damaged and undamaged grain will be recorded. Objective 4. Dynamically transfer knowledge to and engage stakeholders in implementing effective solutions to overcome phosphine resistance We will use a mixed strategy approach to reach the greatest number of stakeholders. We will first administer a survey at the start of the project to 100 stored product facility managers and industry personnel in the Great Plains. At the end, the same survey will be administered again to track changes in perception and willingness to adopt other IPM tactics.We will make all the results from this project available on our project website to stored product stakeholders nationally. The online repository will host: 1) images of stored product damage, life stages, and habitats, 2) latest fact sheets and bulletins from land grants around the US on stored products and IPM, 3) latest published primary literature by our team with brief lay-synopses, 4) links to other postharvest resources around the web, 5) topical videos on stored product IPM, and 6) media coverage of this and related projects.We will also take advantage of cooperative extension in AR, KS, and NE, focusing on rice, wheat, and corn, respectively. Results will be delivered through: 1) electronic newsletters; 2) one-on-one consultations with collaborating stored product facilities and mills; 3) winter and industry meetings; 4) regional extension meetings in AR, KS, and NE; 5) short videos detailing advances from the research and its implications for management via YouTube; 6) summary factsheets detailing the most important findings and what they mean for food facilities; and 7) on-site demonstrations of the new technologies, and how they can help manage insects.Finally, stored product stakeholders will be invited to a full-day program with interactive workshops and demonstrations at the USDA-ARS Center for Grain and Animal Health Research in KS that will feature the state-of-the-art in diversified IPM tactics and their efficacy for resistance management. This will bring together stakeholders in AR, KS, MO, NE, and OK. The program will feature tours of the CGAHR, demonstrations of new technologies, tours of local food facilities, and presentations by experts across the region.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The target audiences for this project include participants in the post-harvest food supply chain, including millers, pest control operators, food facility managers, as well as domestic and international researchers, extension professionals, and the public. Partially, this list includes women, people of color, and people from rural and relatively disadvantaged areas. Science-based knowledge was delivered through 4H programs to youth in multiple states, and workshops targeted middle school girls to increase participation in science. Changes/Problems:We initially hired a graduate student in fall who backed out of coming at the last minute. Unfortunately, this set the timeline back a bit. However, we now have a PhD student who started this past August and is working at full speed on the project. Combined with shortages at Detia Degesch for the PPT Kit, we have been a bit delayed this first year, but should be able to make up time in the coming years. What opportunities for training and professional development has the project provided?This project has allowed for the training of a Ph.D. student, 2 post-doctoral researchers, and a lab technician in various aspects of molecular techniques and insect bioassays with phosphine. How have the results been disseminated to communities of interest?The findings, such as the efficacy of long-lasting insecticide-incorporated netting on laboratory phosphine-susceptible and -resistant strains of the red flour beetle, have been shared with relevant scientific communities through various platforms. What do you plan to do during the next reporting period to accomplish the goals?We will continue to develop bioassay techniques for determining the phosphine resistance level in the laboratory phosphine-resistant strains of the red flour beetle, develop molecular techniques to determine the frequency of the phosphine resistance mutation in the laboratory resistant strain, and establish a relationship between the level of phosphine resistance and the frequency of the resistance mutation.

Impacts
What was accomplished under these goals? Overall, we have completed 30% of Obj. 1, 25% of Obj. 2, 0% of Obj. 3, and 15% of Obj. 4. We also summarize the work that has been done for each objective separately below: Objective 1. We have completed 30% of Obj. 1. We have established a resistant strain from Enid, OK and a susceptible laboratory strain of red flour beetle from the 1970s. In order to compare the baseline resistance ratio, we have exposed both strains with n = 3 replicates to 3000 ppm of phosphine for 30 min then checked condition at 1, 2, 4, 6, 24, 48, and 72 h later. We found almost all of the susceptible strain was affected or dead immediately after exposure and up to 3 d later. By contrast, all of the resistant strain were still moving after exposure. There was over 4-fold more individuals still moving in the resistant strain after exposure to phosphine. However, to ensure that resistance to phosphine is fixed in the population, we will be exposing 400 individuals of the resistant strain to 3000 ppm for 30 min, and will take all highly mobile individuals (unaffected) and start subcultures from these individuals only. We have also used mRNA of the Dld gene to sequence its cDNA to verify the point mutations in the resistant strains. This will allow us to proceed in developing the ARMS-qPCR. Objective 2. We have completed 25% of Obj. 1. We have started using the Detia Degesch phosphine tolerance test (PTT) kit to evaluate resistance of red flour beetle populations from Kansas, Nebraska, and Arkansas food facilities. We have obtained multiple populations through the course of the season and fumigated with phosphine using the PTT kit. Colonies of these will be held for performing ARMS-qPCR. We have been working with Detia Degesch to obtain a sufficient number of PTT kits. Ecolab in Arkansas and industry collaborators in Nebraska have been cooperating with research personnel to perform the fumigations in each state. Objective 4. We are developing questions for the initial survey of stakeholders this winter. In addition, we have been speaking to researchers, extension personnel, and industry about the importance of testing for phosphine resistance and updates from the project.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Abshire, J., R. Harman, A. Bruce, and S. Gillette, and J.M. Maille, S. Ranabhat, D. Brabec, E.D. Scully, K.Y. Zhu, A.R. Gerken, W.R. Morrison III. 2024. Flight capacity and behavior of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) in response to kairomonal and pheromonal stimuli. Environmental Entomology, 53: 567576.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Scully, E.D., Q. Ming, W.R. Morrison III, D.S. Scheff, A.R. Gerken, K.Y. Zhu, J.F. Campbell. Molecular mechanisms for insecticide recovery. Proceedings of the IOBC-WPRS Working Group on Integrated Stored Product Protection at Novi Sad (Serbia), Sep 1620, 2024. IOBC-WPRS Bulletin 173: 132134.
  • Type: Other Status: Published Year Published: 2024 Citation: Presentations Invited: Aug '24 Morrison III, W.R., J. Duran, S. Ranabhat, T.W. Phillips, G.V. Bingham, T. McKay, K.Y. Zhu. Novel behaviorally-based tactics to combat phosphine resistance among stored product insects at food facilities. XXVII International Congress of Entomology, Kyoto, Japan. Aug '24 Scully, E.D., Q.L. Ming, J.F. Campbell, K.Y. Zhu, W.R. Morrison III. Mechanisms of recovery after exposure to contact insecticides in the presence of food. XXVII International Congress of Entomology, Kyoto, Japan. Jul 24 Morrison, III W.R. Developing behaviorally-based pest management programs for stored product insects. 39th Annual Meeting of the International Society of Chemical Ecology, Prague, Czech Republic. Jul '24 Morrison, III W.R, H.E. Quellhorst, C.G. Athanassiou, R. Harman, M.A. Ponce, A.R. Gerken, K.Y. Zhu. Using behavior and microbial ecology to help improve surveillance and management programs for stored product insects in a warming world. XX International Plant Protection Congress, Athens, Greece. Feb '24 Morrison III, W.R. and S. Ranabhat. Informing management by trapping stored product insects at food facilities and in the landscape with pheromones. 2024 NSPCA Urban Pest Management Conference, Lincoln, NE. Nov 23 Morrison, III W.R., and S. Ranabhat. Latest in IPM. Midwest Grain Seminar, Doniphan, NE. Nov 23 Bingham, G.V., G. Chipabika, L. Rault, W.R. Morrison III, A.R. Gerken, D.S. Scheff, S. Ranabhat, W. Mittmann, and A. Mortensen. Use of specialized packaging, alternative tools, and digital technologies to protect global food facilities against cosmopolitan pests; maize weevil (Sitophilus zeamais), red flour beetle (Tribolium castaneum), lesser grain borer (Rhyzopertha dominica) and larger grain borer (Prostephanus truncatus). 71st Annual Meeting of the Entomological Society of America, National Harbor, MD. Contributed Poster: Mar 24 Abshire, J., R. Harman, S. Gillette, J.M. Maille, S. Ranabhat, E.D. Scully, K.Y. Zhu, A.R. Gerken, W.R. Morrison III. Food cues and sex pheromone affect flight capacity and behavior of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) in the laboratory. 79th Annual Joint Meeting of the North-Central Branch of the Entomological Society of America, Ft. Collins, CO. Nov 23 Quellhorst, H.E., K.Y. Zhu, and W.R. Morrison III. Improving management of the larger grain borer, Prostephanus truncatus, and the maize weevil, Sitophilus zeamais. 71st Annual Meeting of the Entomological Society of America, National Harbor, MD.