Trap cropping for management of Harlequin bug in cole crops

2010 Annual Report for GS09-081

Project Type: Graduate Student
Funds awarded in 2009: $9,523.00
Projected End Date: 12/31/2010
Grant Recipient: Virginia Tech
Region: Southern
State: Virginia
Graduate Student:
Major Professor:
Dr. Thomas Kuhar
Virginia Tech

Trap cropping for management of Harlequin bug in cole crops


Harlequin bug (HB), Murgantia histrionica (Hahn) (Hemiptera: Pentatomidae), is a pest of cole crops (Brassicaceae). This study seeks to identify plant species/varieties that are preferred by this pest species in order to develop a trap crop system for its management, to attract pest feeding away from the protected crop to a nearby preferred “trap crop.” This method of management has potential in both conventional integrated pest management as well as organic vegetable production. This study also attempts to identify olfactory cues used by HB in host plant selection.

Objectives/Performance Targets

  1. 1. Survey the pest status and incidence of HB on various cole crops in Virginia. 2. Identify host plant preference for adult feeding and oviposition of HB. 3. Determine the role of volatile organic compounds (VOCs) of in attracting HB to its host plant(s). 4. Test a trap crop approach to efficiently manage harlequin bug.


1. Survey the pest status and incidence of HB on various cole crops in Virginia.

A survey was conducted in 2009-2010 of vegetable farmers representing 17 counties/cities in Virginia. A questionnaire was distributed through extension agents and at grower meetings. Email, telephone and in person interviews were conducted at farmer’s markets, grower meetings and at local farms. Although none of the farmers surveyed were federally certified “Organic,” many follow the “organic philosophy” and either did not use any chemical insecticides or used only those products approved for use for certified organic growers. A very broad range of brassicaceous crops were reported to be grown by those farmers surveyed and those crops are grown mainly in the spring and fall months. Crops listed by farmers included arugula, bok choi, broccoli, Brussels sprouts, cabbage, cauliflower, collards, daikon, kale, kohlrabi, horseradish, mustard greens, rapini, radishes and turnips. Two growers in the central part of the Commonwealth reported a summer crop of collard greens. Anecdotally, suggestions for potential trap crop varieties included komatsuna, cleome and horseradish. Several growers reported leaving mustard in the field to overwinter and used that stand for vegetable greens the following spring as well as early season nectar sources. Several growers in the southeastern region of Virginia reported cover cropping with overwintering oilseed radish, with the purpose of breaking through hard pans as well as adding organic matter to the soil. Of the 42 growers surveyed, all but five were familiar with HB and reported its presence in the field on a yearly basis; however, only two identified harlequin bug as a key pest that required chemical management. Three growers reported using a trap crop to divert HB.

2. Identify host plant preference for adult feeding and oviposition of HB.

Knowing the varieties of plants preferred by HB adults will aid in selecting the best trap crop for any given cash crop. To determine what species of plants are preferred by HB in caged choice tests in June 2009 and September 2010. Field-collected HB adults were offered plants of five Brassicaceous species (mustard ‘Southern Giant Curled,’ rapeseed ‘Athena,’ collard ‘Champion,’ rapini ‘Ruvo’ and arugula) and one non-brassica species (snap bean). Five plants of each variety were planted (randomized by row) in each of 4 walk-in mesh field cages (3 x 3 x 2m) at the Eastern Shore Agricultural Research and Extension Center (ESAREC) in Painter, VA. Thirty to fifty HB adults were introduced to each cage at five evenly spaced release points when plants were 10 weeks old. Observations of HB adult location and presence of eggs masses were made at 48, 72 and 96 hours after introduction of insects. Data were analyzed using ANOVA and Tukey’s HSD in JMP (SAS Institute, Cary, NC).

Harlequin bug adults showed a clear preference for mustard over other plants. In five of the eight field cage experiments, more HB adults were observed on mustard than all other plant species (Table 1) and virtually no bugs were observed on the non-host plant, bean.

Very little oviposition occurred within each experimental time period; however, the number of egg masses observed corresponded to the number of HB adults present on plants. A new experiment for determining HB oviposition preference is underway. Copulating couples will be field collected and isolated to choice and no choice tests to determine if females prefer to oviposit on one plant species and if time to oviposit differs between test plants.

3. Determine the role of volatile organic compounds (VOCs) of in attracting HB to its host plant(s).

This objective aims to investigate why these plants are more attractive and whether or not volatile organic compounds (VOCs) from plant surfaces are used by HB in its host plant searching and/or selection.

a. Olfactometer bioassays to evaluate insect response to whole plant VOCs.
The goal of this study is to determine if olfactory cues from plants play a role in host plant selection. More specifically, our objectives are to determine whether or not HB are attracted to VOCs from all plants (green leaf volatiles), VOCs from brassicaceous plants in particular, as well as whether or not plant VOCs are enough to attract HB, or if the adult male and his aggregation pheromone must be present.

Plants used as stimulus in this experiment were grown in quart-sized containers under greenhouse mistbeds: 3-5 week old bean plants (Phaseolus vulgaris) with at least two trifoliate leaves, 7-10 week old mustard plants (Brassica juncea ‘Southern Giant’) with at least 4 true leaves or 7-10 week old collard plants (B. oleracea ‘Champion’) with at least 4 true leaves. Plants were taken directly from the greenhouse before assay and two leaves were removed; cut ends were immediately wrapped in a wet paper towel.

Harlequin bugs used as stimulus were virgin males, 3-7 days past eclosion, reared on collard leaves. For each bout, stimulus males were isolated to a diet of the accompanying vegetable leaves (i.e. collard, mustard, bean, no food source) for at least 6 hours prior to bioassay and moved to olfactometer flasks along with plant material prepared as described above and allowed to acclimate for 30 minutes before bioassay.

Participant HB were field collected (June-September 2010) from collard and mustard (Painter, VA). Each participant was isolated to its own Petri dish and kept in the dark at 25oC for 24 hours before assay, which occurred in a darkened room. Daily bouts were conducted, each consisting of 20 males and 20 females and each experiment consisted of 5 bouts in plant VOC experiments and 4 bouts in pheromone experiments. Insects were each introduced to the base of the “flying-T” olfactometer where the participant climbed up the post towards the sole light source above and encountered two plumes of odor, each originating from flasks containing the stimulus being tested. If the participant climbed 1 cm up an arm of the apparatus after displaying behaviors such as pausing, turning and antennating, a choice was recorded.

A test of binomial distribution was performed for each pair of stimuli using JMP (SAS Institute, Cary, NC) assuming a null hypothesis of 50:50 chance of choosing one arm over another.

Harlequin bug males responded to odors from mustard and bean, but not to odors from collard when given a choice between plant and clean air. Harlequin bug females responded to odors from collard, but not mustard or bean. Both males and female participants responded to odors produced by virgin males feeding on mustard and those feeding on collard, but did not respond to odors produced by virgin males feeding on bean or virgin males in an empty jar. Results from these experiments suggest that HB respond to olfactory cues; however, this system is complex and involves plant volatiles, male-produced pheromone and gender differences in responses. These pairings of stimuli will be repeated using virgin participants reared in lab conditions so that physiological stage/mating status is known.

b. Identifying VOCs from whole plants

In a preliminary study gas chromatography linked to mass spectrometry (GC-MS) was used to identify the volatile organic compound profile emitted from stimulus plants. Collard leaves were trimmed to fit into VOC collection jars (one leaf per jar, roughly 16 cm in length) and the cut end was wrapped in wet paper towel and aluminum foil. Baby arugula leaves (about 6 leaves per jar, each roughly 5 cm in length) were trimmed and cut ends put into small beakers of distilled water. Closed-loop headspace VOC collection was conducted using Super-Q absorbent filters (ARS, Florida) of intact leaves for 16 then 4 hours, after which leaves were torn in half and another 4 hour collection was taken. Another collection was taken from collard leaves using closed-loop activated charcoal filters for 24 hours. Each blank collection was conducted on empty jars that were cleaned with ethanol or acetone. Super-Q filters were eluted with 200 microliters of dichloromethane and cleaned with dichlomethane and allowed to dry over night prior to a new collection. Charcoal filters were eluted with microliters of dichloromethane and cleaned with a series of organic solvents (methanol:dichloromethane, 3:1, dichloromethane, acetone) and left to dry over night before they were used for a new collection.

Several compounds were consistently identified, which could be distinguished from background contaminants in each set of collections. These compounds included the green leafy volatile hexenyl acetate and several terpenoids (e.g. sabinene, ?-myrcene). For each species, mechanical damage of leaves resulted in higher emissions of hexenyl acetate than those observed from intact leaves.

Additional experiments are underway to identify VOC profiles from stimulus plants used in Olfactometer assays.

4. Test a trap crop approach to efficiently manage HB.

Harlequin bug populations and damage in collard plots (Brassica oleracea ‘Vates’) were compared to those in plots of collard with border rows of mustard (Brassica juncea ‘Southern Giant Curled’) that was left alone (trap only), treated with a systemic insecticide (Platinum 75SG; attract and kill) or vacuumed weekly (attract and manually remove; n = 6). Plants were direct seeded at the Virginia Tech ESAREC in Painter, VA. Treatments were applied when the naturally occurring harlequin bug population was first observed in plots.

Drought conditions caused the loss of the majority of plants in this experiment before collard plants reached maturity so it was not possible to collect yield data in replicate but a clear preference for mustard over collard was demonstrated on the field level as the number of HB adults per ten plants were observed over time in both the collard plots and mustard border rows (Table 2). Based on cumulative mean number of bugs from July 9th through August 3rd, 91% of bugs found in the experimental plots occurred on mustard borders rather than collards. Yield data was collected from one plot in each of the treatments as this was what remained when collard plants matured. Twenty leaves were randomly selected from plants on the edge of plots and twenty plants from the center of plots and observed for HB feeding damage (Table 3).

This experiment will be conducted in a similar fashion in the spring and fall of 2010 at three locations, Kentland Farm (Blacksburg, VA), Hampton Roads AREC (Virginia Beach, VA) and the Eastern Shore AREC (Painter, VA).

Impacts and Contributions/Outcomes

This study will aid growers in proper selection and maintenance of a trap crop to manage harlequin bug on cole crops. In 2011 a minimum of 10 vegetable growers in Virginia are planning to plant a mustard trap crop on their farms. We will assess HB populations on the mustard and on cash crops on these farms and report the results to growers.


Anna Wallingford

[email protected]
Graduate Research Assistant
Virginia Tech
Department of Entomology
216 Price Hall
Blacksburg, VA 2406-0319
Office Phone: 5402309902