Progress report for LNE20-413R
Project Information
The ultimate goal of this project is to create a sink in local cucurbit pest populations by luring striped cucumber beetle (SCB), Acalymma vittatum F. (Coleoptera: Chrysomelidae) to baited traps, where they are captured and/or killed. In 2020, we made progress in developing a vittatalactone lure, or the “attract” component of our attract & kill system. In 2021 and 2022, we made progress in developing our experimental bait, or the “kill” component that includes a feeding stimulant (cucurbitacin) and a poison (e.g. spinosad). We also continued our large-scale experiment to evaluate the season-long impacts of manipulating beetle movement during springtime invasions. We have not yet collected enough data to confirm the efficacy of this approach at the field scale, and will replicate some of experiments in 2023. However, we have confirmed that the components of our system effectively attract striped cucumber beetle (SCB), spotted cucumber beetle (SpCB), Diabrotica undecimpunctata Mannerheim (Coleoptera: Chrysomelidae) and adult & nymph squash bug (SB), Anasa tristis De Geer (Hemiptera: Coreidae), and squash vine borer Melittia cucurbitae Harris (Lepidoptera: Sessiidae).
The ultimate goal of this project is to develop an effective and adoptable attract-and-kill (A&K) approach for managing striped cucumber beetle (SCB), using attractive odors, selective feeding stimulants, and the judicious use of bee-safe insecticides.
Problem, Novel Approach and Justification:
Striped cucumber beetle (SCB) is a serious pest of cucumber, melon, squashes, and pumpkin. While effective chemical controls are available in conventional systems, concern for pollinators visiting these pollinator-dependent crops has driven demand for non-chemical or bee-safe alternatives. Behavioral controls can replace chemical controls, or be integrated with other tools and tactics to achieve control with a dramatic reduction in insecticide use and unwanted non-target effects.
Attract-and-kill (A&K) approaches lure pest insects to a source of poison, which is often combined with an arrestant or feeding stimulant. Here we seek to develop and field test an A&K approach using SCB aggregation pheromone (vittatalactone) to lure beetles to a bait station containing a feeding stimulant (cucurbitacin) laced with a gut poison (spinosad).
Background & Rationale
Striped cucumber beetle is a serious pest of cucumber, melon, squashes, and pumpkin. While effective chemical controls are available in conventional systems, concern for pollinators visiting these pollinator-dependent crops has driven demand for non-chemical or bee-safe alternatives. Behavioral controls can replace chemical controls, or be integrated with other tools and tactics to achieve control with a dramatic reduction in insecticide use and unwanted non-target effects.
Attract-and-kill (A&K) approaches lure pest insects to a source of poison, which is often combined with an arrestant or feeding stimulant. Here we seek to develop and field test an A&K approach using SCB aggregation pheromone (vittatalactone) to lure beetles to a bait station containing a feeding stimulant (cucurbitacin) laced with a gut poison (spinosad).
While adult SCB will often consume nectar and pollen from a broader range of host plants, members of the Cucurbitaceae family are required for their successful reproduction. Adult beetles overwinter under leaf litter in wild habitats and emerge in the spring to feed and find mates. Eventually they move into cultivated cucurbit crops, often in response to crop flowering. Male striped cucumber beetles emit an aggregation pheromone, vitattalactone, that aids in attracting both male and female conspecifics to hosts as well. Females then lay their eggs at the base of their host plant, and the subsequent larvae burrow down in to the soil to feed on roots until the pupate and emerge in a new generation of adults. In the northeastern United States, SCB will typically complete two generations but longer seasons can allow for a third generation from time to time.
Male striped cucumber beetles emit an aggregation pheromone, vittatalactone (2-methyl-3-(2,4,6,8 tetramethyloctyl) oxetan-3-one), when feeding on a favored host. Chauhan and Paraselli (2017) devised and patented a synthesis that produces eight stereoisomers of vittatalactone with the ring configuration (2R,3R), including the one active isomer. Mixed vittatalactone (eight stereoisomers of (2R,3R)-vittatalactone) has proved highly attractive to both sexes of SCB. This semistereospecific synthesis (i.e. that produce an isomeric mix at three chiral centers, of the five in the molecule) enables less expensive production of the SCB aggregation pheromone in quantities useful for field testing and application.
Mixed vittatalactone lures were found to attractive to SCB, SpCB, and (surprisingly) squash bug adults and nymphs in preliminary testing during the fall of 2019 (Brzozowski et al. 2022). While replication is necessary to confirm this finding, our cheaper, “new and un-improved’ lure may be an important component for management of multiple key pests of cucurbit crops.
Research
Research Plan:
Experiments will address questions A&K deployment regarding trap development and development of attract & kill components (Fig. 1). Initial project plans include experiments conducted at research farms in NH & MD and commercial farms in NH, VT, and ME. We have also coordinated our efforts with on-going cucumber beetle research outside of our region, and will include pertinent details regarding unpublished results from our collaborations with researchers at South Dakota State University, Virginia Tech University, Cornell University, and Trécé Inc.
Dose-Response Trials.
Our lure contains a man-made version of cucumber beetle aggregation pheromone (vitattalactone), which evaporates from our trap to create an attractive odor plume. The potency of our lure could be affected by the concentration of this compound, which is affected by the initial dose applied to our lures. If the dose is too low, the beetles won’t be able to detect it. If the dose is too high, the beetles might not find it attractive.
In order to understand how the dose affects beetle response, we deployed clear sticky traps baited with various concentrations of vitattalactone in farmland with a history of cucumber beetle infestation (Beltsville, MD). We observed how many beetles were trapped weekly. We also changed traps, refreshed lures, and re-randomized treatment locations to replicate the experiment in the spring and fall of 2020.
Trap Design Trials.
Our lure is a powerful attractant for striped cucumber beetle, spotted cucumber beetle, and squash bug but we must identify a reliable method of capturing or killing the pest insects lured to our man-made aggregation.
In order to identify the best trap, we deployed various trap types in farmland with a history of cucumber beetle infestation (Beltsville, MD). Traps were baited with 1 mg vitattalactone, unless they were un-baited as a control. We observed how many beetles were trapped weekly. We also changed traps, refreshed lures, and re-randomized treatment locations to replicate the experiment.
Season-long Lure Trials.
We deployed 8 sticky traps at each of two commercial farm sites in Newmarket & Rollinsford, NH through the 2020 growing season. Half of these traps were baited with 1 mg vittatalactone lures and the other half were unbaited as control. We changed traps, refreshed lures, and re-randomized treatment locations to replicate the experiment through the growing season. We also observed several crops for beetle densities through this same period. At least 20 plants of each crop type were observed each week for the number of striped cucumber beetles, the number of spotted cucumber beetles, the number of squash bug egg masses, nymphs, and adults, signs of injury, and signs of bacterial wilt. We observed the undersides of leaves for at least 30 seconds per plant, if not all leaves. We supplied weekly reports of pest observations to growers, who treated all plants according to their wishes.
Early-Season Intervention Trials.
- At three farms (VT, NH, ME), we deployed vitattalactone-baited boll weevil traps in the perimeter of one side of a plot of winter squash to see if early season trapping would remove enough pest insects from the system to result in different pest densities on plants through the season. Traps were 10 m apart and the “treatment” end of each field was at least 50 meters from the “control” end. Weekly visits were made to each site to scout each side of each field for striped cucumber beetles, spotted cucumber beetles, and squash bug egg masses, nymphs, and adults. For each field, we observed the number of beetles on 20 plants on the perimeter and 20 plants in the interior of each plot. The undersides of the leaves were examined on each plant for at least 30 seconds. We supplied weekly reports of pest observations to growers, who treated both plots according to their wishes, and growers supplied us with their spray records at the end of the season.
- We repeated a similar experiment at five farms (VT, NH, ME), where we deployed bait stations in the perimeter of one side of a plot of winter squash; traps were 10 m apart and the “treatment” end of each field was at least 50 meters from the “control” end. Bait stations (Fig. 2) were deployed within a week of planting/transplanting and maintained for 4-5 week, in order to poison insects as they migrated into crop space from overwintering sites. This invasion period typically occurs in June or July and is typically well timed with flower initiation in the crop. We reapplied the bait (Spinosad + bitter watermelon juice) every week and replaced lures (1 mg vittatalactone) every other week .
- We repeated a similar experiment at four farms (VT, NH), where we deployed 4 bait stations in the perimeter of one side of a plot of winter squash; traps were 10 m apart and the “treatment” end of each field was at least 50 meters from the “control” end. Bait stations (Fig. 2) were deployed within a week of planting/transplanting and maintained for 4-5 week, in order to poison insects as they migrated into crop space from overwintering sites. This invasion period typically occurs in June or July and is typically well timed with flower initiation in the crop. We reapplied the bait (Spinosad + bitter watermelon juice) every week and replaced lures (1 mg vittatalactone) every other week. Caged Bait Station Assays. Additionally, collected additional field data to compare potential spillover effects of our trap designs. We also conduced caged bioassays to compare the efficacy of the four trap designs, which will need to be replicated in 2023.
We made weekly visits to each site to count the number of pest insects captured or killed by bait stations, and to scout treatment and control plots for striped cucumber beetles, spotted cucumber beetles, and squash bug egg masses, nymphs, and adults. For each plot, we observed the number of beetles on 20 plants, by examining the undersides of all leaves on each plant, or for at least 30 seconds. We supplied weekly reports of pest observations to growers, who treated both plots according to their wishes, and supplied us with their spray records at the end of the season.
Roller Bait Station Experiment
The experimental bait station included in 2021 field experiments includes several components. In order to understand the importance of each component, we attempted to “deconstruct” a bait station with three bait components, all accompanied by an organic-approved insecticide restricted to the bait itself. We evaluated how many pest insects were captured/poisoned by bait stations with or without vittatalactone lure (standard 1mg), and sweet watermelon juice with or without added bitter component (cucurbitacin-E-glycoside; 0.05% v/v).
Cucurbitacin Content of Melon Juice
In order to make the bait we used in field trials, we crushed and juiced bitter Hawkesbury watermelons grown in UNH greenhouses from Jan-June 2021 or at USDA-ARS research farm in Beltsville Maryland summer 2020. Juice was frozen at -20°C until use, when it was thawed at room temperature.
In addition to producing the raw bitter melon juice for our experiments, we have partnered with Trécé Inc. and researchers at South Dakota State University and Virginia Tech University to develop an insecticide synergist containing water-soluble cucurbitacin. This feeding stimulant (tradename CideTrak) is derived from the fruit of bitter Hawkesbury watermelon. However, currently available genetic stocks are highly variable in terms of their cucurbitacin content. We conducted a preliminary experiment that determined a wide range in cucurbitacin content in the fruit juice we were relying on for our field experiments (0-1.5 mg/L). While some melons accumulated very high concentrations, we could not detect our compound of interest in ~25% of the lines we tested. For all subsequent field experiments in 2021, we used only the fruits of those high producing lines.
Furthermore, we conducted another preliminary experiment to evaluate the role of genetics and fruit maturity on the cucurbitacin content of fruit grown from the two high accumulators and one low accumulator seed line. Cucurbitacin synthesis occurs in the leaf tissue but, in order to avoid autotoxicity, the plant will divert these compounds and accumulate them in some other storage structure. For bitter Hawkesbury watermelon, cucurbitacins are accumulated in the fruit. We therefore wanted to investigate the potential that all seed lines were given the appropriate about of time to accumulate cucurbitacins in their fruit by harvesting at maturity (45±3 d after pollination), past maturity (59±3 d), and well past maturity (73±3 d). We found that cucurbitacin content in fruit from both “high lines” was not different, and the range was similar to that of their parents. However, fruit from the “low line” accumulated significantly less cucurbitacin than that from the two high lines. Fruit maturity did not have a significant effect on cucurbitacin (Fig. 1). We plan to replicate this experiment with additional lines to confirm this trend. (cucurbitacin analysis carried out by Fathi Halewiesh & Trevor Ostlund at SDU)
Bait Efficacy in Foliar Applications
While our project originally aimed to develop A&K bait stations, there is a potential benefit in deploying bait sprays (foliar application of insecticide mixed with cucurbitacin). Efficacy trials were conducted to evaluate the potential benefit of including bitter watermelon juice or a commercial cucurbitacin product (CideTrak) in cucumber (Painter, VA & Durham, NH), summer squash (Painter, VA), and melons (Whitethorne, VA). Treatments were randomly assigned to 20’ plots in an RCB design with 4 replicates. Experiments conducted in Virginia are reproduced with permission from Kuhar et al. (2022; https://www.pubs.ext.vt.edu/content/pubs_ext_vt_edu/en/author/k/kuhar-thomas_p.resource.html). In VA, all foliar treatments were applied with a 3-nozzle boom equipped with D3 spray tips and powered by a CO₂ backpack sprayer at 40psi delivering 30 gpa. In NH, treatments were applied using a hand-pump applicator with one nozzle, delivered using approximately 100 gpa.
In 2022, we conducted a similar experiment in order to evaluate the potential benefit of adding a feeding stimulant to foliar applications. In this experiment, we compared the number of striped cucumber beetles, spotted cucumber beetles, and squash bug egg masses, nymphs, and adults in plots treated with foliar applications of Entrust, Assail, or Lambda Cy versus the same rates plus the high rate of CideTrak L applied as a dribble next to the plants (presumably lower risk to non-target insects). We repeated this experiment in zucchini, cucumber, and melon crops. We also destructively sampled our zucchini experiment in order to assess squash vine borer damage.
- Dose-Response Trials.
We concluded the following (Figs. 4 & 5):
- An initial dose of 1 mg vitattalactone is appropriate for further testing.
- We found a positive linear dose-response for striped cucumber beetle, spotted cucumber beetle, and squash bug, with no signs of repellency in high concentrations.
- This dose-response is strongest in the early season, before host plants are present.
Trap Design Trials.
We concluded the following (Fig. 6):
- We found that sticky traps, boll weevil traps, and yellow gallon jug traps captured more striped cucumber beetles than unbaited traps, and captured more beetles than other baited designs.
- Given concerns regarding non-target trapping of beneficial hymenopterans, we opted not to use yellow sticky cards or yellow gallon jug traps in further testing, and continued our testing with clear sticky cards and boll weevil traps.
Season-long Lure Trials.
We concluded the following (Fig. 7 & 8):
- When there are not actively growing crops, significantly more beetles were captured by baited traps than un-baited controls.
- Stimuli from crop plants likely outcompete our man-made attractants at this trap density.
Early-Season Intervention Trials.
This experiment has not yet been replicated in a sufficient number of instances to make conclusions on whether or not our experimental approach creates a significant sink in cucurbit pest populations. While we captured economically threatening number of beetles at all three sites during the early part of the season, the pest pressure at our ME site was too low to include in analysis for 2020 & 2021 seasons.
We have concluded the following from data collected in 2020 (Fig. 9) and 2021 (Fig. 10):
- Although we have not collected enough data on whether or not this approach contributes to pest management of this crop, we feel like we’ve collected enough information to conclude that creating man-made aggregations will not make pest pressure worse than no action. This assertion will make recruiting new collaborating farmers easier as the project continues.
- The boll weevil trap fell short of expectations in terms of successful trap capture rate. In several instances, we found beetles on traps or near traps rather than inside traps. Future Early-Season Intervention Trials will be continued with attract-and-kill bait stations.
- While A&K bait stations (paper tube bait stations) were relatively effective at killing beetles observed within 0.5 m of each stake, this approach was likely not effective at poisoning squash bug as the majority of SB adults we observed on bait stations were walking and standing (Fig. 11).
2022 Caged Bait Station Assays. We conduced caged bioassays in order to estimate efficiency of our traps and bait stations, and found that boll weevil traps captured significantly fewer SCB than our control clear sticky trap but there was no difference in efficiency between our bait stations and the control (Fig. 12).
Roller Bait Station Experiment
We evaluated how many pest insects were captured/poisoned by bait stations with or without vittatalactone lure (standard 1mg), and sweet watermelon juice with or without added bitter component (cucurbitacin-E-glycoside; 0.05% v/v).
- Both SCB and SpCB were captured by the traps.
- Striped CB captures were most strongly influenced by presence of their own pheromone, which resulted in a 14-fold increase in captures. Melon juice and cucurbitacin also increased captures, but only 4-fold each (Table 1).
- In contrast, spotted CB responded most strongly to the presence of cucurbitacin, but also to vittatalactone and melon juice (Table 2).
The roller bait traps rely on a combination of all three factors: pheromone lure (presumably for long-distance volatile attraction), melon juice (presumably augmenting the volatiles, and possibly also encouraging bait consumption), and cucurbitacin (a known feeding stimulant and arrestant with no volatility). Both species rapidly succumbed to the 0.4% Entrust, which is a fraction of the field rate for a foliar spray application. This material is registered in all cucurbits but does not have either cucumber beetle on the label. However, when ingested, as in this bait, it is very effective.
This preliminary trial shows not surprisingly that vittatalactone is the most important factor in capture of striped cucumber beetles, since it is their aggregation pheromone. Since 2020 we have known that SpCb and SB are attracted to vittatalactone (Brzozowski et al. 2022), presumably eavesdropping on the signal from the other species, so the 6-fold attraction of SCB to this pheromone is not surprising in that context. Spotted cucumber beetles are even more influenced as to trap captures by the presence of the cucurbitacin, at an almost 13-fold rate.
Table 1: Proportions of striped cucumber beetle captured by roller bait traps, October 2021, Beltsville, MD. Total captured n=60.
|
striped cuke btl |
PLUS |
MINUS |
MULTIPLE |
uncorrected P< |
proportion |
lower95 conf.int. |
upper95 conf.int. |
|
PHERO |
56 |
4 |
14.0 |
0.0001 |
0.933 |
0.838 |
0.982 |
|
MELON |
48 |
12 |
4.0 |
0.0001 |
0.800 |
0.677 |
0.892 |
|
CUX |
48 |
12 |
4.0 |
0.0001 |
0.800 |
0.677 |
0.892 |
|
TOTAL = 60 |
Table 2: Proportions of spotted cucumber beetle captured by roller bait traps, October 2021, Beltsville, MD. Total captured n=824.
|
spotted cuke btl |
PLUS |
MINUS |
MULTIPLE |
uncorrected P< |
proportion |
lower95 |
upper95 |
|
PHERO |
707 |
117 |
6.0 |
0.0001 |
0.858 |
0.832 |
0.881 |
|
MELON |
491 |
333 |
1.5 |
0.0001 |
0.596 |
0.562 |
0.630 |
|
CUX |
764 |
60 |
12.7 |
0.0001 |
0.927 |
0.907 |
0.944 |
|
TOTAL = 824 |
Bait Efficacy in Foliar Applications
Efficacy trials were conducted to evaluate the potential benefit of including bitter watermelon juice or a commercial cucurbitacin product (CideTrak) in cucumber (Painter, VA & Durham, NH), summer squash (Painter, VA), and melons (Whitethorne, VA).
Pest pressure was very low at experimental cucumber plots established in Durham, NH, so treated plant leaves were used in an excised-leaf bioassay to determine the influence of aging on SCB feeding behavior and mortality. Plots were treated in the evening, within an hour of sunset, on July 22nd and August 6th. For each treatment date, leaves were collected from plots the next morning (1 DAT) and again 4 DAT, and returned to the lab for bioassay. One or two SCB (field collected from insecticide-free host plants) were isolated with treated leaves in a 90 mm Petri dish for 48 hours. Each treatment was replicated in 4 bioassays. The number of living, dead, and moribund beetles were observed and recorded. Living beetles were standing and walking. Dead beetles did not move when probed. Leaves were imaged for later assessment of leaf area consumed.
- We found no differences in the amount of leaf tissue consumed in this assay.
- While we observed differences between treatments (ANOVA; F5,29 = 9.98, p = 0.0123), we did not find evidence that the addition of the feeding stimulant improved efficacy as materials were aged in the field (Fig. 13).
- The higher rate of mortality in control treatments versus bitter melon treatments may be an artifact of high rates of parasitism (tachinid fly) observed in bioassay subjects.
-
Figure 13: Mean (±SEM) % mortality of SCB in excised cucumber leaf bioassays, using leaf tissue 1 and 4 days after treatment in the field. Bars with the same letter were not different according to Tukey’s HSD means test (α = 0.05). In 2022, we compared control of foliar applications to the same material combined with CideTrak, but dribbled the same rate/volume per plot next to the plant rather than applied to the crop (zucchini, melon, cucumber; Durham, NH). Again, pest pressure was very low at experimental plots established in Durham, NH. However, we found that SCB adults were likely more attracted to plots with CideTrak additions, which may have made infestations worse than controls (Fig. 14). When we destructively sampled zucchini plots to observe squash vine borer infestations, we found that all of our treatments had fewer squash vine borer larvae than control plots (Fig. 15). This was a surprising and encouraging finding, and to our knowledge, the first record of this feeding stimulant having activity on this species.
All experiments need replication before making recommendations.