Harlequin bug is a pest of cole crops (Brassica oleracea), such as broccoli, cabbage, collards, cauliflower, Brussels sprouts, etc. There is potential to control this pest by using a trap crop, a preferred host plant planted near a protected cash crop, to draw insect feeding from the cash crop and to concentrate insecticide application to that trap crop rather than the cash crop. The research presented herein identifies plant species that are preferred by the harlequin bug over collards, a B. oleracea cash crop, identifies the role of plant odors in host plant selection, and evaluates a trap crop control strategy using border rows of mustard in the field.
Harlequin bug (HB) (Murgantia histrionica) is a pest of cole crops (Brassicaceae) and, while it has been reported to feed on plants of other families, it does so only in the absence of other brassicals (McPherson and McPherson 2000). Both adults and nymphs are piercing-sucking feeders on leaves and stems. Feeding causes blotching of leaf tissue, which reduces the marketability of crops sold as greens, such as collards and turnips. As feeding continues wilting and browning of leaves may occur eventually leading to the death of the plant (White and Brannon 1933). There are several broadspectrum insecticides (mostly carbamates, pyrethroids, or neonicotinoids) that provide effective control (Kuhar and Doughty 2009). However, there has been a shift toward the use of narrow-spectrum, reduced-risk insecticides in cole crops primarily for control of other pests such as lepidopteran larvae, or aphids. Unfortunatley, the majority of these newer chemicals have little to no toxicity to stink bugs such as HB.
There is potential to implement a trap cropping system as an alternative to broad spectrum foliar insecticide applications to manage HB. Insect feeding is diverted to a preferred host plant or “trap crop” planted near the protected cash crop (Hokkanen 1991). This could result in an elimination of chemical sprays targeted to this pest, or in a dramatic reduction in insecticide, as any necessary sprays would be applied to the trap crop only. In some cases there exists a “dead-end” trap crop, which is more attractive than the cash crop, but that the insect cannot complete development because that host lacks essential nutrition or due to toxicity (Shelton and Nault 2004). Toxicity can be applied to an attractive trap crop through genetic modification or by systemic insecticide. A “dead-end” trap crop also eases the fear of attracting more of the pest to the general area and acting as a source of herbivores rather than a sink.
Ludwig and Kok (1998) found that a perimeter border row of mustard (Brassica juncea) was successful at slowing the movement of HB into broccoli plots in low populations. A perimeter planting ensures that the trap crop is the first thing encountered by invading insects, keeping them in the edge. However, border rows may be a better fit into existing planting schemes over a complete perimeter border around the cash crop. Male HB produce a semiochemical, murgantiol, that attracts both male and female HB (Zahn et al. 2008). Production of this aggregation pheromone indicates that long-distance olfactory cues play a role in attraction of HB to a host plant; however, long-distance attraction of HB may rely on a combination of plant and insect odors.
Trap crops have been used for the control of other brassica specialists (Shelton and Badenes 2006), and long-distance orientation to crucifer compounds has been demonstrated (Pivnick et al. 1992, Bartlett 1996, Smart et al. 1997, Rojas 1999). Glucosinolates, a family of chemical toxins (e.g. sinigrin, glucobrassicin, allyl isothiocynate) produced by brassica plants for herbivore defense, have been shown to stimulate feeding and oviposition in several brassica specialist herbivores (David and Gardiner 1966, Feeny et al. 1970, Nault and Styer 1972, Stadler 1978, Renwick and Radke 1990, Renwick et al. 1992, Huang et al. 1995).
This project seeks to identify host plant species preferred by HB, to better understand the role of plant and insect cues involved in long distance host plant selection, and to evaluate the efficacy of a border row trap crop of mustard at the field level.
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a. Survey the pest status and incidence of HB on various cole crops in Virginia.
b. Identify HB host plant preference and performance.
b1. Field-cage choice tests using whole plants
b2. Lab-cage choice tests using potted plants
b2. Lab feeding performance tests using potted plants
c. Determine the role of olfactory cues from host plants in long-distance attraction of HB.
d. Evaluate a trap crop strategy using mustard border rows for management of HB on the field level.
Surveys were conducted in 2009 and 2010 of vegetable farmers representing 17 counties/cities in Virginia to evaluate the pest status of HB. A questionnaire was distributed through extension agents and 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 chemical insecticides or used only those products approved for use for certified organic growers.
A series of experiments were conducted to evaluate HB host plant preference for habitation, feeding, oviposition, and developmenton five Brassicaceous species and one non-brassica species, bean (Table 1). All experiments used plants that were roughly 10 weeks old, with the exception of bean, which were roughly 6 weeks old. Any plants developing reproductive structures were eliminated from experiments.
b1. Field-cage choice tests using whole plants.
Field-cage choice tests were conducted in June 2009 and September 2010 to evaluate host plant preference for habitation and feeding. Five plants of each species (Table 1) were planted (randomized by row) in each of 4 walk-in mesh field cages (3 x 3 x 2 m) at the Virginia Tech Eastern Shore Agricultural Research and Extension Center (ESAREC) in Painter, VA. Field collected HB adults (30-50) were introduced to each cage when plants were 10 weeks old. Observations of HB adult location were made at 24 and 48 hours after introduction of insects. Data were non-normal and did not respond to transformation so a Kruskal-Wallis test was conducted using JMP (SAS Institute, Cary, N.C.) to determine significant difference between number of HB found on each plant variety. Mean separation was conducted by nonparametric multiple comparison based on rank sums (Zar 1984).
b2. Lab-cage choice tests using potted plants.
Caged choice tests were conducted in the greenhouse in May 2011 to evaluate oviposition preference. Three mated HB pairs were introduced to each of 6 cages (12x12x18”) containing individual potted plants of each species (Table 1), 10 weeks old and potted in pint containers. Each plant was observed for egg masses at 12, 24, and 48 hours after introduction. Total number of observed egg masses were pooled for each experiment and data were log transformed to normalize variances. Analysis of variance was conducted using JMP (SAS Institutue, Cary, NC) to test for effect of treatment (plant species) on number of egg masses observed on each plant species (n=2). Tukey’s HSD was used to separate means.
b3. Lab feeding performance tests using potted plants.
Second instar HB were isolated to one plant species until they reached adulthood and the rate of HB development was observed for each host plant species (Table 1). Fifty 2nd instars were isolated to each of 6 containers (25x30x45 cm) containing potted plants, 8-12 weeks old, and plants were replaced twice weekly. Plants were observed every 2-3 days for number of insects in 2nd, 3rd, 4th and 5th instars and only those individuals on the plants were counted, while those wandering the containers were ignored. Observations continued until more than half of the remaining insects on any particular species reached adulthood.
Olfactometer behavioral assays were conducted to evaluate response of male and female HB to plant and insect odors. Participant insects were field-collected from collard (Painter, Virginia Beach and Henrico, VA). Each participant was isolated to a Petri dish (8 cm diameter), starved and held without light for 24 hours before each assay, which occurred in a darkened room. Daily bouts were conducted consisting of 20 males and 20 females, and each experiment was repeated 4 times.
An open Y-track olfactometer (“Flying T”) apparatus modified after Visser and Piron (1998) and described in detail by Dickens (2000) was used for laboratory choice experiments. Hydrocarbon-free air, supplied at the rate of 1 L/min, was humidified by passing it through distilled water prior to delivery of the plant volatiles to the apparatus.
Each participant climbed up the post towards the sole light source above and encountered two plumes of air, passing through one of two flasks containing the stimulus material(s) (Table 2). Bugs were considered to have made a choice after traveling 1 cm up either arm of the “Flying T”. Plant stimuli was 1 trifoliate of a 6 week old bean plant (Phaseolus vulgaris) or two leaves of a 10 week old mustard plant (Brassica juncea ‘Southern Giant’); plants were taken directly from the greenhouse before assay. Insects used as stimuli were 14 day old virgin males, field collected as 5th instars and isolated by gender to feed on collard leaves before assay. Five virgin males were used as insect stimulus and allowed to acclimate to flasks for 20 minutes prior to assay.
Data were analyzed by test of bionomial proportions assuming a null hypothesis of 50:50 chance (Zar 1984).
Mustard (Brassica juncea ‘Southern Giant Curled’) was used as a trap crop to divert feeding from collard (Brassica oleracea ‘Champion’). Plots were direct seeded at the Virginia Tech Hampton Roads AREC in Virginia Beach and at the Virginia Tech Kentland Research Farm (Montgomery Co.) in April 2011 and May 2011, respectively. Plots were replicated 4 times in a randomized block design and consisted of 6 rows of collard 20’ long to evaluate three “treatments:” 1) no trap crop, 2) mustard border rows and 3) mustard border rows were drenched with a systemic insecticide thiamethoxam + chlorantraniliprole (Durivo; Syngenta Inc., Greensboro, NC) at 0.275 lb ai/A to target HB as well as lepidopteran pests. Insecticide applications for treatment 3 were applied when naturally-occurring HB were first observed in plots. Twice weekly the number of adults, egg masses and nymphs were recorded from observations of 10 collard plants and 10 mustard plants. When collard leaves reached marketable size, 20 leaves were randomly selected from each plot and observed for HB feeding scars. Observations continued for at least two weeks after collards reached marketable size.
Because data were non-normal and did not respond to transformation, a Kruskal-Wallis test was conducted using JMP (SAS Institute, Cary, NC) to determine significant difference between percent marketable leaves and number of insects observed in each treatment for each observation date. Mean separation was conducted by nonparametric multiple comparison based on rank sums (Zar 1984).
A student’s t-test was conducted using JMP (SAS Institute, Cary, NC)to determine significant difference between the number of HB adults observed in collard vs. untreated mustard (treatment 2).
A broad range of brassicaceous crops were reported to be grown by those 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 species included komatsuna, cleome and horseradish.
Several growers reported leaving crops such as collard or mustard in the field to overwinter and used 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. Those locations with overwintered brassica crops would be the most likely to see a sizeable springtime HB population where HB are usually not observed in managed fields until mid-summer.
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 current use of a trap crop to divert HB.
b1. Field-cage choice tests using whole plants
In both experiments, the number of HB observed on mustard was significantly higher than the “cash crop” collard or bean (Figure 1 and Figure 2). The number of HB observed on rapeseed, rapini and arugula varied and was periodically equivalent to the number observed on mustard, but at no time any different from collard or bean (Figure 1 and Figure 2). Cooler temperatures during the 2010 experiment allowed for better growth of arugula plants and this is the likely explanation for the difference in attractiveness of this species between 2009 and 2010 experiments.
b2. Lab-cage choice tests using potted plants
The number of HB egg masses observed on rapeseed plants was significantly greater than the number found on arugula, bean, mustard or rapini, while the number of egg masses found on collard was greater than the number found on bean and mustard (Figure 4; p = 0.0016). This is a curious finding considering that the relative incidenceof the insects on plants in this experiment mirrored that of the field-cage study in that HB were most often found on mustard (data not shown), but oviposition preference appears to be opposite to that of habitation and feeding in this grouping of plant species. Thus, these results suggest that while HB adults may prefer to aggregate and feed on mustard, they appear to choose other plant species such as rape seed or collards to deposit their eggs. The reasons for this are unknown. It is possible that females are avoiding overcrowding for their offspring. Alternatively, HB females may prefer a certain leaf texture for oviposition, and that, as long as the plant is a brassica, a female HB prefers a smoother surface on which to oviposit This was the case with our oviposition experiment, as the mustard assayed was a curly variety compared to the smooth, waxy surface of the rapeseed or collard leaves.
Regardless of the reason, a concern raised here is that adult HB attracted to a mustard trap crop may move into crops such as collards to oviposit. However, this could be avoided if the bugs were killed on the trap plants during initial aggregation and feeding.
b3. Lab feeding performance tests using potted plants.
HB did not develop on bean; although a small portion of the test population survived for about 10 days, none molted to 3rd instar. Nymphs that were fed mustard or rapini had the highest survival rate and the shortest development time (34-38 days for 50% to reach adulthood). Only about half of the rapeseed-fed or collard-fed individuals survived the duration of the experiment and development time was longer (40-42 days for 50% to reach adulthood). Only a small percentage of nymphs that were fed arugula survived and those individuals required more than 45 days to reach adulthood.
The strong performance of nymphs on mustard and rapini clarify the evolutionary purpose of preferring these species for habitation and feeding. However, the reduced performance of HB nymphs on collards and rapini did not agree with the oviposition preference on these plants.
Male HB responded to both bean and mustard odors over air (Figure 4; p < 0.0001, p = 0.0046), and when given the choice between the two stimuli, male harlequin bugs preferred the odors from mustard over bean (p = 0.0016). Female HB showed no significant preference for any plant stimulus.
Neither male nor female HB responded to odors from HB males that were not on plants; however, both female and male participants responded to odors from mustard with male HB over mustard odors alone (Figure 5; p = 0.043, p = 0.013, respectively). Additionally, male participants chose bean alone over bean with HB males (Figure 5; p = 0.0068).
These data suggest that HB respond to a complex of olfactory cues from both plants and conspecific males. Both stimuli may be essential for host plant selection. These data support a model in which males find the host plant with the help of plant cues and, if he finds an appropriate host plant, he produces an aggregation pheromone which brings additional individuals.
Collard plots with mustard border rows had less HB damage than plots without mustard border rows, but the addition of a systemic insecticide made no difference in the amount of damage observed (Figure 6 and Figure 7). Data presented from both locations were collected after the 3-4 week window of protection provided by the systemic neonicotinoid insecticide. To achieve a “dead-end” trap crop, insecticide would have to be applied more than once over the duration of crop growth, depending on the timing of harlequin infestation.
Table 3. Number of days until half of HB nymphs reached each lifestage and the percent of the total (50) on that date for 3rd, 4th, 5th and adult intars. None of the nymphs reared on bean alone reached 3rd instar.
Educational & Outreach Activities
The concepts and data presented in this report were presented several venues including vegetable grower meetings and extension field days, and were published in conference proceedings and a peer-reviewed journal. More publications and presentations are in preparation.
Titles to date:
Wallingford, A., T. Kuhar and P. Schultz. 2009. Host plant preference of Harlequin bug, Murgantia histrionica (Hahn) (Hemiptera: Pentatomidae). Poster presentation ESA annual meeting, Indianapolis, ID December, 2009.
Wallingford, A., T. Kuhar and P. Schultz. 2010. Host plant preference of Harlequin bug, Murgantia histrionica (Hahn) (Hemiptera: Pentatomidae). 10-minute talk EB-ESA meeting, Annapolis, MD March, 2010.
Wallingford, A., T. Kuhar and P. Schultz. 2010. Investigating the role of olfaction in host plant selection of harlequin bug, Murgantia histrionica (Hemiptera: Pentatomidae). Annual Meeting of the Entomological Society of America, San Diego, CA, Dec. 11-16, 2010.
Wallingford, A.K. and T.P. Kuhar. 2010. Managing insects in vegetables using IPM. Mid-Atlantic Crop Management School, November 17, 2010 Ocean City, MD
Wallingford, A.K., P. Schultz, and T.P. Kuhar. 2010. Harlequin bug host plant preference and potential for trap cropping in brassica crops. 2010 Eastern Shore Annual Research Field Day, Painter, VA, July 14, 2010.
Wallingford, A.K., T.P. Kuhar, P.B. Schultz and J.H. Freeman. 2011. Harlequin bug biology and pest management in brassicaceous crops. Journal of Integrated Pest Management 2: H1-4.
Wallingford, A., T. Kuhar and P. Schultz. 2011. Investigating the role of olfaction in the harlequin bug (Murgantia histrionica), insect pest of cole crops. Virginia Academy of Science Annual Meeting, Richmond, VA, May 25, 2011.
This research has led to a better understanding of the chemical ecology and host plant selection of HB, which may help us to more effectively manage this pest. In the field, border rows of mustard were shown to provide effective control of HB in collards and to keep feeding damage below 25%, which would be acceptable in many circumstances. The mustard trap crop is likely to be effective in controlling HB in other cash crops of the same species (Brassica oleracea), such as broccoli, cabbage, Brussels sprouts and cauliflower, particularly considering the higher threshold of feeding injury that can be sustained by crops marketed for plant parts other than their leaves.
This cultural management practice is effective and also reduces or eliminates the need for broad-spectrum insecticides in cole crops, thereby saving natural enemy complexes important to the control of key pests of cole crops.
Several farmers surveyed were enthusiastic about adopting trap cropping in the future. Many farmers surveyed had come across this concept by accident and were currently “trap cropping” in some form, although they had not heard of this term previously. Demonstration plots were established at 8 commercial organic and conventional farms. One participating farmer reported a plan to adopt a mustard trap crop in his 2012 kale crop.
Areas needing additional study
The chemical ecology of a trap crop system should be investigated further to better understand the impact on other insects in cole crops. There is potential of reducing the population of other brassica specialists, such as diamondback moth or imported cabbageworm. However, it should made certain that an attractive trap crop, such as mustard, does not become a “source” of pest pressure rather than the “sink” it is intended to be. A direct link should be drawn between the survival of natural enemies and their impact on lepidopteran pests. Better understanding of these considerations would add greatly to future cost/benefit analyses.