Progress report for OW22-370
Clover seed weevil (CSW), Tychius picirostris Fabricius (Coleoptera: Curculionidae), is a key insect pest in white clover seed production in Oregon. Historically, clover seed growers have relied heavily on broad-spectrum pyrethroid insecticides for CSW management. Since 2017, growers and crop consultants have observed poor efficacy with pyrethroid insecticides, causing serious concern for the white clover seed industry. Moreover, reduced effectiveness of pyrethroid insecticide treatments in a 2021 Oregon State University (OSU) field trial warrants further CSW pyrethroid resistance status evaluation. Available insecticides are limited, leaving few options for growers to rotate between different modes of action (MoA). We aim to i) determine the resistance status to pyrethroids and other MoA using laboratory bioassays, ii) evaluate new MoA and different insecticide timing under field conditions, and iii) develop a scouting method and treatment decision-making guidelines based on the larvae density per seed head. Our education efforts will focus on i) enhancing grower knowledge of CSW biology and optimizing control timing; ii) facilitating training and workshops for calibrating pesticide application equipment and scouting techniques specific to CSW, and iii) gathering input on growers' perceptions of alternative methods of the integrated management plan (IPM). Data from this research will be disseminated during OSU Extension events throughout the project duration. The expected outcomes will include one factsheet, one extension publication, and at least one peer-reviewed journal article. The sustainability goal will be achieved by 1) reducing unnecessary chemical applications and 2) increasing economic return by lowering inputs and improving seed yield.
Our research objectives include:
- Identify the level of pyrethroid resistance development and toxicology of other insecticide modes of action using laboratory bioassays.
- Conduct field efficacy tests using new modes of action and different insecticide timings specifically targeted to control clover seed weevil (CSW) larvae.
- Develop a grower-friendly scouting method and treatment decision-making guidelines based on CSW larvae density per seed head.
The educational objectives of this project will focus on 1) improving current knowledge of clover seed growers and crop advisors on CSW biology and behavior to better time insecticide applications, 2) facilitating the incorporation of insecticide resistance management strategies in clover seed production systems through training and workshops, and 3) gather input from growers and local crop advisors about their perceptions of alternative methods included in the integrated management plan (e.g., host plant resistance, cultural control, and biocontrol).
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1) Laboratory bioassays to determine the extent of resistance development to pyrethroid insecticides and toxicity of anthranilic diamides
Tychius picirostris (CSW) collections
Field collections of adult CSW were used for laboratory bioassays. Adult and larval populations were collected in western Oregon by sweep net sampling and Berlese funnels in commercial white clover seed fields at the pre-bloom and maximum bloom growth stages. Collected CSW specimens were held in temperature-regulated growth chambers for at least 24 hours before laboratory screening.
Surface contact toxicity assays
Glass-vial assays. Log-probit assays were conducted to determine the susceptibility of CSW populations to pyrethroid insecticides using a glass-vial bioassay procedure (Miller et al. 2010). Five replicates for eight concentrations of technical grade bifenthrin (97% purity) and chlorantraniliprole (99% purity) were dissolved in acetone solvent. These technical grade materials were tested to determine potential cross-resistance between chemistries. Preliminary assays determined an appropriate geometric series of concentrations for each chemistry to achieve a response range from 0 to 100 percent mortality. Each concentration solution was transferred to glass vials (0.5 mL) and rolled on a hotdog roller at room temperature until the solvent evaporated to ensure uniform distribution on the vial's interior surface. Control vials were treated with acetone only. Five CSW adults were placed in each vial with screw-on caps loosely placed to allow sufficient oxygen exchange. Dead or nearly dead (moribund) adults were evaluated based on the inability of adults to right themselves and walk. Vials were inspected for CSW survival at 24 h. Mortality was corrected using Abbott's formula (Abbott 1925). Tests were discarded if control mortality was above 20%. Data were analyzed using the Proc Probit procedure in SAS software to generate the lethal concentration to kill 50% of the population (LC50), 95% fiducial limits, and other descriptive statistics.
Fig 1: Glass vial assay
Synergism assays. To evaluate potential mechanisms of insecticide resistance development to pyrethroid insecticides, we also screened three phase-1 detoxification enzymes, including a mixed-function oxidase inhibitor (piperonyl butoxide; PBO), esterase inhibitor (S,S,S-tributyl phosphotrithioate; DEF), and a glutathione S-transferase (GST) inhibitor (dimethyl maleate; DEM). For synergism assays, the glass-vial procedure above were used with the same geometric series of concentrations for bifenthrin addition to 5 µL of each synergist added to each test vial, including the untreated control. Insects were evaluated as described above. In addition to the descriptive statistics analyzed for glass-vial assays, we also calculated synergistic ratios (SR50) for synergism assays by dividing the LC50 without synergists by the LC50 when synergists were added.
Topical assays. To test the toxicity of anthranilic diamides as a potential replacement or rotation option for pyrethroid insecticides, we developed a topical assay using formulated product of chlorantraniliprole, malathion and bifenthrin. For topical assays, a Potter Spray Tower (Burkard Manufacturing Co. Ltd., England) was used to deliver pesticide solutions as a fine mist, forming a uniform coating on insects without visible droplets. Formulated product were diluted in water, and at least eight concentrations were used in a geometric progression to determine LC50 values. Ten adult insects were immobilized with a flash chilling treatment and placed in a 140 mm plastic petri dish. Each petri dish was placed on the Potter Spray Tower stage and sprayed with 1.5 mL of the appropriate insecticide solution or water control using an air pressure of 47 kPa (6.8 psi) and a spray distance of 22 cm. Three replicates per treatment concentration were made. Mortality was determined as described above and corrected using Abbott's formula (Abbott 1925). These chemistries were screened in field efficacy trials described in Objective 2.
Fi 2: Potter spray tower
2) Field efficacy tests using standard and new modes of action
A replicated field experiment to test the efficacy of insecticides against CSW was conducted in June 2021 in a commercial white clover production field near Tangent, Oregon. These data informed field trial design for 2022. In 2021, materials were segregated into one of four categories (see Figure 3):
- Initial knockdown activity on adults three days after treatment, but minimal impact on larval counts (Brigade, Malathion – both grower standards).
- Knockdown activity on adults and impacts on larval counts (Avaunt).
- No knockdown activity on adults but impacts on larval counts (Harvanta, Exirel).
- No activity on adults or on larval counts (Besiege, Warrior II).
Figure 3. Average adult clover seed weevils per straight line sweep at 3 days after treatment (DAT) and 14 days after treatment (left). Average larvae per Berlese funnel sample 14 days after treatment (right).
For 2022 experiment, a replicated plot was identified in a commercial white clover field to determine the effect of different insecticides on white clover seed yield potential. Plot size were approximately 29 X 300 ft. Insecticide treatments were arranged in a randomized complete block design with three replications. Insecticide treatments included:
- Untreated control(no insecticide)
- Brigade 6.4 floz/acre @ 20%brown down (current industry standard)
- Harvanta 16.4 floz/acre @ larval detection
- Harvanta 16.4 floz/acre @ larval detection + Brigade 6.4 floz/acre @ 20% brown down
- Exirel 20.0 fl oz/acre @ larval detection
- Exirel 20.0 fl oz/acre @ larval detection + Brigade 6.4 fl oz/acre @ 20% brown down
- Steward 6.0floz/ acre @ larval detection
- Steward 6.0floz/acre @ larval detection + Brigade 6.4floz/acre @ 20%browndown
Larval and adult clover seed weevil populations were monitored via Berlese funnels and straight-line sweeps, respectively, throughout the duration of the bloom and seed fill periods. To determine seed yield, plots were swathed and combined using grower’s equipment, and weighed using a Parkan GW200A weigh wagon. Sub-samples of the harvested seed were collected to determine thousand seed weight, percent cleanout, and total clean seed yield.
Fig 4: Field efficacy trial
3) Development of scouting methods based on the larval density per seed head (DIY Berlese funnels efficiency). Insect identification and determination of population density are crucial for any successful integrated pest management plan. Since the 2021 efficacy trials (see data, Objective 2) indicated that several promising control materials reduced larval counts, but not adult counts, it is critical that growers and crop advisors scout fields at the correct time and make informed management decisions based on larval, not adult, densities. We intended to test the efficacy of larval sampling techniques (prototypes of DIY Berlese funnel, Figure 3) that are user-friendly and easy to construct.
Sampling Methods Comparison. We compared the efficacy of the shake sampling method (Hoff et al. 2002) and Berlese extract methods (various prototypes developed in this study) to detect larvae from white clover florets. Three CSW infested commercial field sites were selected in May 2022. Each field were divided into four quadrants, and sweep net sampling was performed in each quadrant to determine adult CSW numbers per week from May to July. Developing clover seed heads from three to five randomly selected 1 m2 area were collected in each quadrant and bagged. Plant material were divided into subsamples (100 flower/seed heads) and extracted using the shake technique or a Berlese funnel for 36 h (until the foliage is well dried). Larvae was recovered in 70% ethanol.
For analysis, the counts were adjusted to a per flower head basis for samples from the shake-bucket and Berlese funnel. Larvae counts recovered using both techniques were compared to adults recovered using a sweep-net on a weekly basis. Using a repeated-measures analysis of variance (ANOVA) for each year, we analyzed the proportion of larvae per flower head per sampling date and associations with adult weevil counts per straight line sweep.
Fig 4: DIY Berlese funnel Fig 5: Standard Berlese funnel
We evaluated the user-friendliness of tested sampling methods for growers and crop advisors via surveys distributed during grower workshops and other OSU Extension events. The producer partners worked with their grower network to seek suggestions for design improvements and to facilitate adoption.
A series of dose-response bioassays were conducted in the laboratory using technical grade and formulated bifenthrin for four Oregon CSW populations and one Canadian CSW population collected from commercial white clover seed production fields. Elevated resistance levels to bifenthrin were detected in CSW populations collected from Oregon (LC50 = 3.70-21.94) when compared to a susceptible Canadian population (LC50 = 0.02-0.04). This study is the first documentation of CSW insecticide resistance development. Based on this research finding, we recommend stopping further reliance on pyrethroid-based insecticides in the CSW management program. Alternative control strategies including refining sampling techniques and targeting the larval stage to manage this pest in the Oregon clover seed production system are being explored. The growers and crop consultant advisors who attended the educational events during year 1 were advised to incorporate a newly registered product representing a new mode(s) of action targeted toward the egg and larval stage of CSW. Information on the life cycle and biology of this insect pest was provided at all educational events. Two oral presentations included Year 1 project findings of insecticide resistance screening in the laboratory and field insecticide efficacy trial results:
Year 1 data indicating the efficiency of two types of berlese extraction techniques and field efficacy results of newer chemistries were presented to growers and other industry stakeholders during two extension events and grower meetings (Nutrien Grower Meeting, Nov 30, 2022, Oregon Clover Growers Annual Meeting, Feb 1, 2023, and Coffee Hour, OSU Extension Service, March 17, 2023).
One peer-reviewed technical publication was submitted with 2022 project findings to the OSU Seed Production Report.
Education and Outreach
1)Improving current knowledge of clover seed growers and crop advisors on CSW biology and behavior to better time insecticide applications. For year 1, we tailored our informal consultations, and oral presentations during grower and extension events to enhance grower/ other industry partners' knowledge about the insect life cycle and develop a management plan to incorporate new mode(s) of action. Elevated resistance to bifenthrin was identified and we reported the research findings via grower meeting talks and popular press articles for industry-wide dissemination.
2) Facilitating the incorporation of insecticide resistance management strategies in clover seed production systems through training and
workshops. Year 1 data indicating the efficiency of two types of berlese extraction techniques and field efficacy results of newer chemistries were presented to growers and other industry stakeholders during two extension events and grower meetings (Nutrien Grower Meeting, Nov 30, 2022, Oregon Clover Growers Annual Meeting, Feb 1, 2023, and Coffee Hour, OSU Extension Service, March 17, 2023). One peer-reviewed technical publication was submitted with 2022 project findings to the OSU Seed Production Report (Tiwari_SPRR_23)
3) Gather input from growers and local crop advisors about their perceptions of alternative methods included in the integrated management plan (e.g.,
host plant resistance, cultural control, and biocontrol). One online questionnaire was developed and distributed to collect baseline data about growers' current practices and perceptions of alternative methods of the integrated management plan (IPM). Data from this survey was disseminated during a coffee hour presentation.
All our extension events and grower meetings were well attended (>50 audiences representing growers, crop advisors, and other stakeholders at all events). Project evaluations were done during the Oregon clover growers annual meeting on Feb 1, 2023. The results are attached and a positive feedback was received from the audience who attended this event.
WSARE project evaluation year 1
Baseline data from the industry on spray records for CSW management and insecticide use patterns revealed that over-reliance on bifenthrin-based products, low spray volumes, and the poor timing of insecticide application to be an industry-wide issue that may have caused poor control in the first place leading to resistance problem. We discussed these survey results with our audience during a coffee hour presenation and recommendation were made to incoporate insecticide resistance management strageies including targetting weak links in insect life cycle (selective chemistries for larval control), and rotating mode (s) of action in CSW management plans.
Education and Outreach Outcomes
Our research and educational activities effectively engaged the growers and other stakeholders from the white clover seed industry. The insecticide trial was performed at two grower cooperators' field sites. Eight different grower cooperators were involved in recruiting fields for CSW collections and insecticide resistance screening. All producers indicated that they would continuously participate in this study and will likely use some aspects of this project. Overall positive feedback was received during the project evaluation survey (n=23)
Adoption of new chemistries, Using diverse mode(s) of action, Networking, and peer-to-peer teaching among producers,