METHODOLOGY Open Access

An aeroponic culture system for the study of rootherbivory on Arabidopsis thalianaMartha M Vaughan1,3, Dorothea Tholl1*, James G Tokuhisa2

Abstract

Background: Plant defense against herbivory has been studied primarily in aerial tissues. However, complexdefense mechanisms have evolved in all parts of the plant to combat herbivore attack and these mechanisms arelikely to differ in the aerial and subterranean environment. Research investigating defense responses belowgroundhas been hindered by experimental difficulties associated with the accessibility and quality of root tissue and thelack of bioassays using model plants with altered defense profiles.

Results: We have developed an aeroponic culture system based on a calcined clay substrate that allows insectherbivores to feed on plant roots while providing easy recovery of the root tissue. The culture method wasvalidated by a root-herbivore system developed for Arabidopsis thaliana and the herbivore Bradysia spp. (fungusgnat). Arabidopsis root mass obtained from aeroponically grown plants was comparable to that from other culturesystems, and the plants were morphologically normal. Bradysia larvae caused considerable root damage resulting inreduced root biomass and water absorption. After feeding on the aeroponically grown root tissue, the larvaepupated and emerged as adults. Root damage of mature plants cultivated in aeroponic substrate was compared tothat of Arabidopsis seedlings grown in potting mix. Seedlings were notably more susceptible to Bradysia feedingthan mature plants and showed decreased overall growth and survival rates.

Conclusions: A root-herbivore system consisting of Arabidopsis thaliana and larvae of the opportunistic herbivoreBradysia spp. has been established that mimics herbivory in the rhizosphere. Bradysia infestation of Arabidopsisgrown in this culture system significantly affects plant performance. The culture method will allow simple profilingand in vivo functional analysis of root defenses such as chemical defense metabolites that are released in responseto belowground insect attack.

BackgroundBelowground herbivory affects plant performance in sev-eral ways. For example, insect feeding on plant rootsreduces uptake of water and nutrients, limits carbohy-drate storage, and changes the production of phytohor-mones [1-3]. Such alterations in the physical,biochemical, and physiological state of plants can influ-ence surrounding organismal communities both above-and belowground [2,4].Plants have developed multiple strategies such as tol-

erance and direct and indirect defenses to cope with ordefeat herbivore attack [5-7]. Direct defense mechanismsinvolve the production of defense proteins and second-ary (specialized) plant metabolites, which directly affect

herbivores, whereas indirect defenses help attract naturalenemies of herbivores [5-11]. In contrast to the manyplant defense responses investigated aboveground, fewerstudies have focused on plant defenses against root-attacking herbivores [12]. For example, feeding of thecabbage and turnip root maggot (Delia radicum andDelia floralis) was shown to induce the production ofglucosinolate defense metabolites in the roots of severalBrassica species [13,14]. Schmelz et al. [15] demon-strated that phytoecdysteroids accumulating in spinachroots serve as inducible defense compounds thatdecrease root feeding of Bradysia spp. (fungus gnat) lar-vae. In addition to direct defenses, some indirect defenseresponses have been demonstrated belowground. Whenattacked by larvae of the western corn rootworm (Dia-brotica virgifera virgifera), maize roots release the* Correspondence: tholl@vt.edu

1Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USAFull list of author information is available at the end of the article

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

PLANT METHODS

© 2011 Vaughan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

sesquiterpene volatile (E)-b-caryophyllene, which attractsinsect parasitizing nematodes [16].Detailed investigations of molecular and chemical

defense responses in plant roots are limited, which canbe largely attributed to experimental shortfalls asso-ciated with the accessibility of root tissue or the interfer-ence of soil particles with root metabolite analysis [1,17].Moreover, research on belowground defenses has beenhindered by the lack of bioassays using model plantsdeficient in herbivore-induced root defenses such as theformation of defense metabolites. In response to thesechallenges, we have developed a root-herbivore systemusing Arabidopsis thaliana and larvae of the root herbi-vore Bradysia spp.Dark-winged fungus gnats are generalist opportunistic

herbivores, whose larvae feed on organic matter andfungi, but upon depletion of this food source, larvaeactively feed on root tissue of a variety of plants includ-ing Arabidopsis [18,19]. Larvae chew on roots and stripaway the cortex, thereby negatively impacting water andnutrient absorption [18]. The two most common spe-cies, Bradysia coprophila (Lintner) and Bradysia impa-tiens (Johannsen), are both considered importantgreenhouse pests [20], which, at extreme infestations,lead to loss of plant vigor and even mortality [21]. Forexample, 90% of alfalfa seedlings are killed at densitiesof less than one larva per seedling [18]. Young soybeanplants have been shown to survive Bradysia coprophilafeeding but produce less seed [18]. These damagingeffects of Bradysia species can be attributed to theirshort life cycle of 20 to 25 days with females layingbetween 250 to 1000 eggs in approximately three days.Emerging larvae feed in the soil for 12 to 14 days priorto pupating [20].Bradysia larvae have been used to investigate hor-

mone-dependent defense responses in Arabidopsis. Lar-vae caused high mortality of soil-grown Arabidopsismutants deficient in the biosynthesis of jasmonic acid,but also affected the growth of wild-type seedlings [19].Effects on mature plants were described as minor, butthis assessment was based only on observations of foli-age and survival without investigating the severity ofroot damage [19]. Bradysia feeding was also employedto analyze root-specific defense activities of phytoecdys-teroids in spinach [15]. However, this study used invitro feeding assays, instead of an in vivo approach, uti-lizing diets of powdered root tissue containing differentphytoecdysteroid concentrations.In this paper, we describe a culture system based on

commercially-available, porous calcined clay pellets (Ser-amis®) to investigate belowground herbivory on Arabi-dopsis roots. The system maintains a soil-typeenvironment for the herbivore while providing easyaccess to root tissue for analysis. Furthermore, we

evaluate feeding by fungus gnat larvae on seedlingsgrown in soil in comparison to feeding on roots ofmature plants in aeroponic culture. We propose thisbioassay to be a useful tool for studying chemical- andmolecular-defense responses of Arabidopsis to below-ground herbivory. Moreover, the system has broader uti-lity to investigate many aspects of root biochemistry,physiology, and ecology.

MethodsPlant materialArabidopsis thaliana ecotype Columbia (Col-0, ABRCStock no. 6000) was grown under controlled conditionsat 22°C to 25°C, 150 μmol m-2 sec-1 photosyntheticallyactive radiation and a 10 h light - 14 h dark photoper-iod. Seeds were stratified for 24 h at 4°C prior to plant-ing in potting mix [90% Sunshine Mix No. 1 (Sun GroHorticulture, Bellevue, WA) and 10% sand].

Arabidopsis aeroponic culture systemSeramis® clay granules http://www.seramis.de are readilyavailable in Europe with currently limited distribution inNorth America (Ace Gardening Products, Kitchener,Ontario, Canada; http://www.seramis.de/weltweit.html).Fifty ml polypropylene conical tubes (Fisher Scientific,Suwanee, GA) were prepared by drilling five 3.5 mmdiameter holes around the bottom and a 25 mm hole inthe cap (Figure 1).Tubes were then filled with Seramis® clay granules

and wrapped with aluminum foil to reduce light pene-tration and algal growth. The top of each tube wascovered with plastic wrap and the caps were reat-tached. A small hole large enough for the root mass topass through was punctured in the plastic wrap usingthe tip of a scalpel. The tubes were then placed in atest tube rack and submerged in Hoagland’s solution[22] to moisten the substrate. Four-week-old Arabidop-sis plants grown in potting mix were carefully removedfrom their pots (Figure 1). Roots were repeatedly sub-merged in water to remove as much of the pottingsubstrate as possible. A 1.25 cm cube of Rockwool wasdivided and placed beneath the rosette, around the topof the roots. Plants were then transferred to Seramis®-containing tubes and allowed to grow. The Rockwooland plastic wrap stabilized the plant until its roots hadgrown into the clay granulate. The clay granulate waskept moist by submerging the culture tubes in Hoag-land’s solution for 10 to 15 min every other day. Afterfour weeks, when the plants started to bolt, roots hadgrown to the bottom of the tube. During the feedingexperiments, plants were watered every other day byapplying 10 ml of Hoagland’s solution to the surfaceand allowing excess media to drain freely from thetube.

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 2 of 10

Bradysia (fungus gnat) cultureFungus gnat larvae were collected from greenhouse soilby the “potato disk” method [23] and subsequently usedto establish a laboratory colony. The colony was main-tained in 8 L plastic containers with screened openingsfor ventilation. The culture medium consisted of 4 L ofmoist Sunshine Mix No. 1 enriched with 1.5 kg ofshredded potato tubers [24]. The cultures were kept atambient greenhouse conditions (21°C to 23°C; indirectsunlight). To maintain the colony, 0.5 L of medium(containing Bradysia larvae, pupae, adults, and eggs)was transferred to a fresh container of soil and potatomix every three to four weeks. Specimens were identi-fied by Dr. Raymond J. Gragné (USDA Systematic Ento-mology Laboratory, Washington, DC) as a mixed colonyof Bradysia coprophila (Lintner) and Bradysia impatiens(Johannsen).

Isolation of Bradysia larvaeLarvae were collected from the culture using a modi-fied flotation/extraction method previously describedby Cloyd and Zaborski [24]. Approximately 1 L of cul-ture medium containing Bradysia larvae was trans-ferred to a 2 L wide-mouth Erlenmeyer flask. The flaskwas filled with room temperature tap water and agi-tated to break up the culture substrate and release thelarvae. The flask was then inverted and placed on topof a 2 L graduated cylinder filled with cold water, sothat the mouth of the flask opened just below thewater surface in the cylinder (Figure 2A). In this posi-tion the flask remained filled with water with the grad-uated cylinder serving as both a container and a rack

to hold the flask. Denser material including fungusgnat larvae sank from the flask into the cylinder whilemost of the potting mix remained floating in the flask.After approximately 5 min, the flask was removed, andits contents were discarded. Water, larvae, and theremaining organic matter were poured from the gradu-ated cylinder into a plastic container. After the larvaehad settled to the bottom of the container, the waterwas decanted and a 1.5 M MgSO4 solution was added.

A B C

D E F

Figure 1 Technique for growing Arabidopsis in aeroponic culture. (A) Four-week-old Arabidopsis plants removed from pots. (B, C) Repeatedrinsing of roots in water to remove potting mix. (D) Plants prior to transplant into clay granulate with a small cube of Rockwool (1.25 cm) to beplaced around the base of the rosette. (E) Transfer of plants to plastic tubes containing 50 ml of Seramis® clay granules saturated in Hoagland’ssolution. (F) Plants grown for four weeks under short day conditions following transfer to aeroponic culture tubes. At this stage, plant shootsbegan to bolt.

A

B 3B

12

3

4

Figure 2 Floatation method for the collection of Bradysialarvae. (A) A 2 L wide-mouth Erlenmeyer flask inverted on agraduated cylinder and containing fungus gnat larvae and larvalculture substrate suspended in tap water. The larvae were collectedat the bottom of the cylinder while most of the culture substrateremained floating in the flask. (B) Instars of Bradysia larvae collectedby this method. Numbers indicate the different instars. Scale bar: 1mm.

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 3 of 10

Larvae floated to the top because of their lower densitycompared to the MgSO4 solution, while most of theremaining organic matter sank to the bottom. Larvaewere then collected in a sieve (1 mm mesh size), rinsedin water, and placed in a Petri dish with moist filterpaper without food for 20 to 24 h prior to their use infeeding assays. The described isolation method col-lected approximately 1000 Bradysia larvae of all fourinstars from one liter of culture medium (Figure 2B).

Bradysia feeding experiments in aeroponic cultureFour weeks after their transfer to aeroponic culturetubes, Arabidopsis plants were infested with fungus gnatlarvae. To this end, the plastic tube caps were carefullyremoved to avoid leaf damage. Approximately 200 to300 second- and third-instar larvae, collected asdescribed above, were transferred to a single aeroponicculture tube. First instar (~1 mm) and fourth instar lar-vae were excluded from the feeding experiments sincethey were either too difficult to apply or too close topupation. Larvae were submerged in 1 mL of Hoagland’ssolution (Hoagland’s solution was used instead of waterto avoid washing nutrients from the clay pellets) andthen released with a pipette into the Seramis® substrate.After two and four days of larval feeding, roots wereremoved from the tubes by submerging the plant andthe tube in water. Roots were then separated from thegranulate by simply holding the plant rosette and mov-ing it back and forth in the water. The heavier claygranules fell to the bottom of the container, while theroots stayed attached to the rosette. The shoots androots were then blotted dry with Kimwipes (FisherScientific, Suwanee, GA) and their fresh weights wererecorded. Tissue dry weight was measured after two tothree days of air drying at room temperature. Under labconditions, this time was sufficient to completely dehy-drate the root mass and additional drying did not signif-icantly alter weight measurements. The shoot and rootbiomass and percentage of water weight for each tissuewere calculated. For these measurements a T-test wasperformed (n = 12, a = 0.05) to test the null hypothesisof no biomass change for the variables. To determine ifthere was a significant difference in root mass after lar-val feeding, a one-way analysis of variance (ANOVA)followed by Tukey-Kramer HSD (n = 8) was performed.

Bradysia feeding experiments with seedlings grown inpotting mixTo validate the feeding damage by Bradysia larvae onroots of aeroponically-grown mature Arabidopsis plants,plants were challenged with larval feeding at the seed-ling stage in potting mix. Individual seedlings weregrown in pots (8 cm × 8 cm × 10 cm) under thedescribed conditions. Bradysia larvae (second and third

instar, Figure 2B), collected as described above, weredislodged from the bottom of the Petri dish with 1 mLof water, and ten larvae were transferred with a Pasteurpipette into each pot. Larvae that did not move into thepotting substrate within 5 min were removed andreplaced. Larvae were transferred to pots at seed germi-nation, ten and 14 days after germination. Infested andcontrol plants were kept in net enclosures. The percen-tage of surviving seedlings from three independentexperiments was determined seven days after inocula-tion. Plants were removed from the pots, roots wererinsed in water, and fresh and dry weights of the entireseedling were recorded. For data analysis, the weightswere log transformed before performing a one-wayANOVA and Tukey-Kramer HSD (n = 8-10).

Statistical AnalysisStatistical data analysis was performed as described forthe individual experimental procedures. Analysis of var-iance was accomplished by the JMP program (Version 8,SAS Institute Inc., Cary, NC, USA) statistical software.

ResultsArabidopsis growth in an aeroponic root culture systemTo cultivate Arabidopsis under aeroponic conditions,plants were first grown on potting mix for approxi-mately four weeks prior to their transfer to aeroponicculture tubes. By four weeks after transition to the claygranulate, roots had grown from the residual pottingsubstrate particles to the bottom of the plastic tube. Incomparison to Arabidopsis plants grown in potting sub-strate, aeroponically grown plants appeared morphologi-cally normal in primary and secondary root growth aswell as in the formation of root hairs [25] (Figure 3 andsee below). The Rockwool was removed without diffi-culty since roots had not grown into the Rockwool sub-strate. Approximately 1 g of healthy root tissue wasobtained from each individual plant (Table 1). The root-to-shoot ratio for fresh and dry weight was 0.40 and0.45, respectively (Figure 4C).

Larval feeding behavior on aeroponically grownArabidopsis rootsApproximately 200 to 300 second and third instar larvaewere released into each aeroponic culture tube. Within24 h, larvae, observed through the clear plastic tube,were actively feeding on the root tissue. Two days afterthe release of the larvae, severe feeding damage was visi-ble on most roots (Figure 5A and 5B). The majority ofroot consumption was observed within the upper 5 cmof the tube (Figure 5A) indicating that most larvae fedfrom the top and gradually moved toward the bottom ofthe tube as the food source became depleted. Larvaestripped away the root epidermis and cortex, but

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 4 of 10

generally avoided consuming the vascular tissue (Figure6E-H). Some fine roots were completely severed. Feed-ing damage was most severe on root tips, young roots,and root hairs suggesting a preference by larvae forthese cells and tissues. Thick tap roots displayed little orno feeding damage.

Effects of Bradysia larval feeding on roots ofaeroponically grown Arabidopsis plantsLarval root consumption significantly reduced Arabidop-sis root biomass. On day two and four of larval feeding,the root fresh weight was reduced by 58% and 55%,respectively (Figure 5B). These values reflect the highestreduction of root mass observed in one out of ten inde-pendent experiments; the average loss of root mass forall experiments was 37%. In addition to root consump-tion, stems were poorly supported and rosette leaveswere beginning to wilt (Figure 5A). To determinewhether leaf wilting was caused by a decreased uptakeof water, shoot fresh and dry weight were measured andthe weight attributed to water was determined (Figure7). After four days of larval feeding, shoots showed sig-nificantly reduced water content, while shoot dry weightremained unaffected. By contrast, both root fresh anddry weight were significantly reduced with no significantchange in the water content of roots due to feedingdamage (Figure 7).

Bradysia larval feeding on seedlings grown in potting mixBradysia larvae reduced Arabidopsis seedling establish-ment. Only 24 to 33% of seedlings survived whenexposed to ten Bradysia larvae at germination (Figure 8).When 10-day-old seedlings were treated with larvae,survival rates increased to 45 and 51%. However, morethan 95% of seedlings survived when larval treatmentstarted after the first pair of true leaves had developedten to 14 days after germination. Nevertheless, all of thesurviving plants were considerably affected by herbivoredamage since they had significantly reduced biomass incomparison to unchallenged seedlings and were notice-ably stunted in growth (Figure 9).

DiscussionArabidopsis aeroponic root cultures provide conditionssuitable for root herbivoryInvestigating root herbivory has remained a challenge inthe study of belowground plant-organism interactionsbecause of limited access to root tissue for subsequentanalyses. Several culture methods have been developedpreviously to make Arabidopsis roots more accessible formolecular and biochemical studies. These methods areprimarily based on growing plants in liquid culture foroptimal root biomass production, but they are not suitablefor investigating root herbivory [22,26]. We tested whetherhydroponically grown roots removed from liquid culture

A B C

Figure 3 Arabidopsis roots in potting substrate and aeroponic culture. Root morphology of Arabidopsis plants grown in potting mix (A) andunder aeroponic culture conditions (B). (C) Close-up of roots growing in the aeroponic clay substrate. Arrows indicate root tissue.

Table 1 Arabidopsis thaliana growth in aeroponic culture (this study) compared to axenic and hydroponic liquidcultures reported in previous studies

Root Shoot

Age (Days) Fresh weight Dry Weight Fresh Weight (mg) Dry weight

Aeroponic 52(28a) 984 ± 61 92 ± 6 2,824 ± 180 203 ± 10

Hydroponicb 32 498 ± 57 32 ± 3 1,160 ± 69 121 ± 37

Hydroponicb 48 2,916 ± 164 211 ± 10 10,940 ± 499 1,195 ± 37

Axenicc 24 1,100 - - -a Number of days in aeroponic culture. b[22]. c[26]

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 5 of 10

could be used for Bradysia feeding experiments; however,larvae did not actively feed on these roots. Similarly, larvaedid not feed on roots of plants grown in solid sterilemedia, but instead burrowed into the agar and suffocated.Arabidopsis hairy root cultures have been previously usedto investigate root-aphid interactions and the emission ofvolatile organic compounds in response to aphid attack[27]. However, in this study aphid feeding was confined tofloating root tissue, which simulates the natural habitat ofthe herbivore only marginally and prevents analyzingresponses of the whole plant.

To study herbivory on Arabidopsis roots, it was neces-sary to establish a culture system that could accommo-date belowground herbivores while still providing easyaccess to the root tissue for further molecular or bio-chemical analysis. Initial attempts to grow Arabidopsis

15002000250030003500

Ah

Wei

ght (

mg)

0500

1000

Shoot Root

200

250

mg)

B

Fres

h

0

50

100

150

200

Shoot Root

Dry

Wei

ght (

m

0.20.30.40.50.6

ot to

Sho

ot R

atio

C

00.1

Fresh Weight Dry Weight

Roo

Figure 4 Biomass analysis of Arabidopsis grown in aeroponicculture. (A) Average shoot and root fresh weight and (B) dryweight are shown. (C) Root-to-shoot ratios of fresh and dry weight.Values represent means ± standard error (n = 12). Shoots includeleaves and stems.

A

Control Larval Feeding

600

800

1000 Control

Larval feeding

B W

eigh

t (m

g)

a

a

0

200

400

Roo

t Fre

sh

b

b

2 d 4 d Larval Feeding

Figure 5 Feeding damage and root consumption by Bradysialarvae on Arabidopsis grown in aeroponic culture. (A)Arabidopsis plants removed from clay substrate after four days oflarval feeding in comparison to a non-feeding control. Root biomasswas visibly reduced and aerial tissues were beginning to wilt. (B)Reduction of root mass after two and four days of herbivory. Valuesrepresent averages ± standard error. Letters indicate significantdifferences between days and treatments (one-way ANOVA andTukey-Kramer HSD, n = 8, P < 0.0001).

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 6 of 10

in sand and vermiculite resulted in poor plant growth incomparison to soilless potting mix. By contrast, optimalgrowth conditions were achieved by growing plants innonsterile perlite or Seramis® clay granules. Seramis®

substrate was preferred over perlite since roots couldeasily be removed from the clay granules by submersionin water without causing any substantial tissue damage.Optimal survival of plants was achieved when plants

were first grown on potting mix for approximately 4weeks prior to their transfer to aeroponic culture tubes.Transfer of younger plants was avoided due to inconsis-tent growth performance under aeroponic conditions.Growing seedlings directly on Rockwool placed on top ofthe clay substrate is less suitable since plants are easilystressed by over- or under-watering. A similar problemhas been encountered in establishing hydroponic culturesand is considered a major limitation in the success of

germination and seedling survival in this system [22,26].Within two to three days after transplanting, plants adaptto the aeroponic conditions and grow as phenotypicallyhealthy mature plants with no obvious signs of impairedroot growth or damage after 4 weeks (Figure 3 and 6A-D). The culture requires eight weeks to progress fromseed to mature plants, but beyond transferring the plantsfrom soil to aeroponic substrate, only watering withHoagland’s solution is necessary. The harvest of rootsfrom the aeroponic system is rapid, complete, and causesnegligible damage to roots (Figure 5A and 6A-D) in con-trast to removal of potting mix (Figure 1B-D and 9).Aeroponically-grown plants produce a root mass com-

parable to that obtained in other previously reportedArabidopsis culture systems (Table 1). For example,plants grown for 24 days in axenic liquid culture supple-mented with 1 to 3% sucrose yield approximately 1 g ofroot fresh weight [26], which is similar to that of rootsgrown in the aeroponic system (Table 1). When com-pared to Arabidopsis grown under hydroponic condi-tions, the average root-to-shoot ratio in aeroponicculture (0.45) is approximately twice the ratio observedfor hydroponically-grown plants [22]. Thirty percent ofthe total aeroponic plant biomass (dry weight) is com-prised of root tissue while only 15 to 25% of total hydro-ponic plant biomass has been attributed to root tissue[22] (Table 1). The higher percentage of root mass underaeroponic conditions may be the result of increased aera-tion in the root environment [28,29]. Moreover,enhanced root growth might be caused by the intermit-tent nutrient supply in aeroponic culture as opposed tocontinuous nutrient availability in hydroponic systems.Taken together, the technique shown here represents a

simple culture method, which can be established underordinary growth conditions without the use of sophisticatedhydroponic equipment such as bubble stones, air pumps orsprayers. In comparison to hydroponic cultivation, the cul-ture system reflects more closely the conditions of the soilenvironment. Since clay granules such as Seramis® are usedas a common plant medium in home gardens and interiorlandscapes http://www.seramis.de, aeroponic cultures simi-lar to the one presented here can be established for a vari-ety of different plants including those with a longer lifespan than Arabidopsis. Because of the loosely packed con-sistency of the clay substrate, it seems possible that rootherbivores of larger size than Bradysia larvae can beaccommodated in this substrate, which will allow testingthis method for other root-herbivore systems.

Bradysia larval feeding significantly reduces root biomassof mature Arabidopsis plants and affects survival andgrowth of seedlingsFungus gnats are common greenhouse pests and fre-quently infest Arabidopsis growth rooms. While feeding

A B

C D

rh

rh

E F

G H

rh

Figure 6 Roots of aeroponically-grown Arabidopsis with andwithout feeding damage by Bradysia larvae. (A-D) Undamagedroots of eight-week-old Arabidopsis grown in aeroponic culture.Primary and secondary roots and root tips (D) are shown. (E-H)Roots damaged by feeding of 200 to 300 Bradysia larvae over fourdays. (E-G) Larvae chewed and stripped away the root epidermisand the cortex but avoided feeding on the vascular tissue (arrows).(H) Feeding damage of root hairs and root tips (rh, root hairs). Scalebar: 500 μm.

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 7 of 10

of Bradysia larvae on Arabidopsis seedlings has beenpreviously reported [19], the extent of feeding damageon mature wild-type plants has not been investigated.When placed in aeroponic clay substrate deprived ofother food sources, larvae quickly began to feed on roottissue. Within two to four days of feeding, larvae signifi-cantly reduced root mass by an average of 37%. Despitethe considerable damage of tissue belowground, plantsappeared to be less affected aboveground. However, Bra-dysia-damaged plants showed reduced shoot mass,which was due to the loss of water, suggesting thatwater absorption and most likely nutrient absorptionwere compromised by root herbivore damage.In the presented experiments, approximately 200 to

300 larvae at the second and third instar were appliedper plant. Considering that only 20 to 25 gnats releasedinto an area with 15 pots containing Arabidopsis plantscan yield 300 to 500 larvae per pot within 14 days [19],it was within reason that this density can occur undergreenhouse conditions. The observed root damage inaeroponic culture may be magnified due to the absenceof other food sources such as organic matter. Bradysia

2000

3000

150

200

250

95

100

*

ght (

%)

ht (m

g)

ht (m

g)

A B C

*

0

1000

2000

Control Larval Feeding0

50

100

150

Control Larval Feeding80

85

90

Control Larval Feeding

Wat

er W

eig

Dry

Wei

gh

Fres

h W

eigh

50

75

100

125

400

800

1200

90

95

100

r Wei

ght (

%)

Wei

ght (

mg)

Wei

ght (

mg)

D E F

**

0

25

Control Larval Feeding0

400

Control Larval Feeding80

85

Control Larval FeedingW

ater

Dry

Fres

h

Figure 7 Shoot and root biomass and water content in response to larval feeding in aeroponic culture. Shoot fresh weight (A) and dryweight (B) upon four days of larval feeding in comparison to control plants. (C) The percentage of water in the shoot tissue was significantlyreduced by root herbivore damage. (D, E) Change in fresh and dry weight of roots in response to larval feeding. (F) The percentage of waterweight remained the same. Values represent averages ± standard error. Asterisks above bars indicate significant differences (t-test, n = 12, P <0.02).

100 b

Sur

viva

l (%

)

20

40

60

80

100

a

a

Seedling Age

0

20

0 Days 10 Days 14 Days0 d 10 d 14 d

Figure 8 Survival of Arabidopsis seedlings challenged byBradysia larval feeding increased with age. Arabidopsis seedlingswere challenged with ten Bradysia larvae at germination (0 days),ten days, and 14 days after germination. The average (± standarderror) percent of seedling survival was determined from threeindependent experiments (n = 15) after seven days of larval feeding.Letters indicate significant differences (one-way ANOVA and Tukey-Kramer HSD, P < 0.0001).

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 8 of 10

larvae primarily consumed root tips, root hairs, andyoung lateral roots (Figure 6E-H). Less feeding wasobserved on older roots, where larvae preferablyremoved the epidermis and cortex but none of the vas-cular tissue and only parts of the endodermis. This feed-ing preference may be caused in part by increased rootsecondary growth. An increase in cell wall lignificationcan affect root herbivore activity as shown for wirewormfeeding on tobacco roots [30]. Moreover, differences information and concentration of constitutive and inducedsecondary defense metabolites such as glucosinolatesand terpenoids can contribute to the observed

differential feeding behavior and overall root consump-tion (Vaughan, Tokuhisa, and Tholl, unpublishedresults). The role of direct defense metabolites was alsodemonstrated in spinach roots, where Bradysia larvaeled to a dramatic up-regulation of defense compounds[15]. A recent study investigating glucosinolate distribu-tions in canola (Brassica napus) roots showed that thehighest concentration of glucosinolates occurs in theouter layer of roots with secondary growth [31,32]. Towhat extent Bradysia feeding is affected by the distribu-tion of glucosinolates in Arabidopsis roots requiresfurther investigation.Depending on the developmental stage, Arabidopsis

seedlings grown in potting mix were severely affected byBradysia larval feeding. A moderate infestation [18] often larvae per plant reduced seedling survival by 50% orless depending on the plant age at the time the bioticstress was introduced. Surviving seedlings were muchsmaller and appeared to have reduced growth. Thisreduction in plant growth can be attributed to larvalroot consumption and limited uptake of nutrients butmay also reflect fundamental changes in the physiologi-cal state of the plant due to trade-offs between resourceallocation to defense and growth [33,34] or changes inthe potting mix due to frass deposition.To our knowledge, a bioassay for studying below-

ground herbivory on intact Arabidopsis plants has notpreviously been reported. We propose that Arabidopsisgrown in aeroponic culture and the generalist herbivoreBradysia (B. coprophila and B. impatiens) can be usedas a novel system to investigate belowground plantdefense responses to herbivore attack such as the releaseof secondary metabolites. The chemistry of plantdefense against root herbivores is one of the mostneglected aspects of root biology [12]. Using this system,Arabidopsis mutants with altered profiles of secondarymetabolites, such as glucosinolates [35] or terpenoids,can be applied to study the biological effect of individualdefense compounds belowground. Finally, knowledgegained from these and various other mutants may pro-vide closer insight into the still poorly understood mole-cular and physiological mechanisms of belowground andaboveground interactions [36].

ConclusionsWe have established a method of growing Arabidopsis ina clay granulate culture system, which can be used toinvestigate root biology including belowground herbivoryand allows for easy preparation of roots for chemical andmolecular analyses upon insect feeding. We have shownthat Bradysia larvae actively feed on mature Arabidopsisin aeroponic culture and cause severe root damage thataffects both root biomass and water absorption. Further-more, Arabidopsis seedlings challenged by Bradysia

A

Control

200)

Larval Feeding

B

c

40

80

120

160

200Control

Larval Feeding

Fres

h W

eigh

t (m

g)

aa

b

c

010 d 14 d

Seedling AgeFigure 9 Effect of Bradysia feeding on Arabidopsis seedlinggrowth in potting mix. (A) Developing Arabidopsis seedlingschallenged by root herbivore feeding were noticeably stunted ingrowth. (B) Ten and 14-day-old Arabidopsis seedlings treated withten Bradysia larvae were significantly reduced in plant biomass.Values represent averages ± standard error. Letters indicatesignificant differences between seedling ages and treatments (one-way ANOVA and Tukey-Kramer HSD on log(x) transformed data, n =8-10, P < 0.0001).

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 9 of 10

larvae under conventional cultivation show decreasedsurvival and growth. This root-herbivore system can beused to study the biochemistry, molecular regulation, andfunction of root defense compounds in response tobelowground herbivory.

Acknowledgements and FundingWe thank Ashley Spencer for maintaining plant cultures. This work wassupported by a National Science Foundation AdvanceVT research seed grantand by the Thomas and Kate Jeffress Memorial Trust grants (J-850) (to DT)and (J-871) (to JGT).

Author details1Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.2Department of Horticulture, Virginia Tech, Blacksburg, VA 24061, USA.3USDA Agricultural Research Service Center for Medical, Agricultural andVeterinary Entomology, Chemistry Research Unit, Gainesville, FL 32608, USA.

Authors’ contributionsMMV carried out all experiments and drafted the manuscript. JGTcontributed to intellectual conception and design of the method, helpedwith drafting the manuscript, and edited the manuscript. DT participated inthe study design and coordination, helped with drafting the manuscript,and edited the manuscript. All authors read and approved the final versionof the manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 14 December 2010 Accepted: 10 March 2011Published: 10 March 2011

References1. van Dam NM: Belowground herbivory and plant defenses. Annu Rev Ecol

Evol Syst 2009, 40:373-391.2. Van Der Putten WH: Plant defense belowground and spatiotemporal

processes in natural vegetation. Ecology 2003, 84:2269-2280.3. Blossey B, Hunt-Joshi TR: Belowground herbivory by insects: Influence on

plants and aboveground herbivores. Annu Rev Entomol 2003, 48:521-547.4. De Deyn GB, Van der Putten WH: Linking aboveground and belowground

diversity. Trends Ecol Evol 2005, 20:625-633.5. Strauss SY, Agrawal AA: The ecology and evolution of plant tolerance to

herbivory. Trends Ecol Evol 1999, 14:179-185.6. Kessler A, Baldwin IT: Plant responses to insect herbivory: The emerging

molecular analysis. Annu Rev Plant Biol 2002, 53:299-328.7. Arimura G, Kost C, Boland W: Herbivore-induced, indirect plant defences.

Biochim Biophys Acta Mol Cell Biol Lipids 2005, 1734:91-111.8. Unsicker SB, Kunert G, Gershenzon J: Protective perfumes: the role of

vegetative volatiles in plant defense against herbivores. Curr Opin PlantBiol 2009, 12:479-485.

9. Zhu-Salzman K, Luthe DS, Felton GW: Arthropod-inducible proteins: Broadspectrum defenses against multiple herbivores. Plant Physiol 2008,146:852-858.

10. Pechan T, Cohen A, Williams WP, Luthe DS: Insect feeding mobilizes aunique plant defense protease that disrupts the peritrophic matrix ofcaterpillars. Proc Natl Acad Sci USA 2002, 99:13319-13323.

11. Chen H, Wilkerson CG, Kuchar JA, Phinney BS, Howe GA: Jasmonate-inducible plant enzymes degrade essential amino acids in the herbivoremidgut. Proc Natl Acad Sci USA 2005, 102:19237-19242.

12. Rasmann S, Agrawal AA: In defense of roots: A research agenda forstudying plant resistance to belowground herbivory. Plant Physiol 2008,146:875-880.

13. Birch ANE, Griffiths DW, Hopkins RJ, Smith WHM, McKinlay RG: Glucosinolateresponses of swede, kale, forage and oilseed rape to root damage byturnip root fly (Delia floralis) larvae. J Sci Food Agric 1992, 60:1-9.

14. van Dam NM, Raaijmakers CE: Local and systemic induced responses tocabbage root fly larvae (Delia radicum) in Brassica nigra and B. oleracea.Chemoecology 2006, 16:17-24.

15. Schmelz EA, Grebenok RJ, Ohnmeiss TE, Bowers WS: Interactions betweenSpinacia oleracea and Bradysia impatiens: A role for phytoecdysteroids.Arch Insect Biochem 2002, 51:204-221.

16. Rasmann S, Köllner TG, Degenhardt J, Hiltpold I, Toepfer S, Kuhlmann U,Gershenzon J, Turlings TCJ: Recruitment of entomopathogenic nematodesby insect-damaged maize roots. Nature 2005, 434:732-737.

17. Hunter MD: Out of sight, out of mind: the impacts of root-feedinginsects in natural and managed systems. Agric For Entomol 2001, 3:3-9.

18. Harris MA, Gardner WA, Oetting RD: A review of the scientific literature onfungus gnats (Diptera: Sciaridae) in the genus Bradysia. J Entomol Sci1996, 31:252-276.

19. McConn M, Creelman RA, Bell E, Mullet JE, Browse J: Jasmonate is essentialfor insect defense in Arabidopsis. Proc Natl Acad Sci USA 1997,94:5473-5477.

20. Jagdale GB, Casey ML, Canas L, Grewal PS: Effect of entomopathogenicnematode species, split application and potting medium on the controlof the fungus gnat, Bradysia difformis (Diptera : Sciaridae), in thegreenhouse at alternating cold and warm temperatures. Biol Control2007, 43:23-30.

21. Meers TL, Cloyd RA: Egg-laying preference of female fungus gnatBradysia sp. nr. coprophila (Diptera : Sciaridae) on three different soillesssubstrates. J Econ Entomol 2005, 98:1937-1942.

22. Gibeaut DM, Hulett J, Cramer GR, Seemann JR: Maximal biomass ofArabidopsis thaliana using a simple, low-maintenance hydroponicmethod and favorable environmental conditions. Plant Physiol 1997,115:317-319.

23. Harris MA, Oetting RD, Gardner WA: Use of entomopathogenicnematodes and a new monitoring technique for control of fungusgnats, Bradysia coprophila (Diptera, Sciaridae), in floriculture. Biol Control1995, 5:412-418.

24. Cloyd RA, Zaborski ER: Fungus gnats, Bradysia spp. (Diptera : Sciaridae),and other arthropods in commercial bagged soilless growing media androoted plant plugs. J Econ Entomol 2004, 97:503-510.

25. Darrah PR, Jones DL, Kirk GJD, Roose T: Modelling the rhizosphere: areview of methods for ‘upscaling’ to the whole-plant scale. Eur J Soil Sci2006, 57:13-25.

26. Hetu MF, Tremblay LJ, Lefebvre DD: High root biomass production inanchored Arabidopsis plants grown in axenic sucrose supplementedliquid culture. Biotechniques 2005, 39:345-349.

27. Steeghs M, Bais HP, de Gouw J, Goldan P, Kuster W, Northway M, Fall R,Vivanco JM: Proton-transfer-reaction mass spectrometry as a new toolfor real time analysis of root-secreted volatile organic compounds inArabidopsis. Plant Physiol 2004, 135:47-58.

28. Padgett PE, Leonard RT: Contamination of ammonium-based nutrientsolutions by nitrifying organisms and the conversion of ammonium tonitrate. Plant Physiol 1993, 101:141-146.

29. Spek LY: Influence of nitrate and aeration on growth and chemicalcomposition of Zea mays L. Plant Soil 1981, 63:115-118.

30. Johnson SN, Hallett PD, Gillespie TL, Halpin C: Below-ground herbivoryand root toughness: a potential model system using lignin-modifiedtobacco. Physiol Entomol 2010, 35:186-191.

31. van Dam NM, Tytgat TOG, Kirkegaard JA: Root and shoot glucosinolates: acomparison of their diversity, function and interactions in natural andmanaged ecosystems. Phytochem Rev 2009, 8:171-186.

32. McCully ME, Miller C, Sprague SJ, Huang CX, Kirkegaard JA: Distribution ofglucosinolates and sulphur-rich cells in roots of field-grown canola(Brassica napus). New Phytol 2008, 180:193-205.

33. Zavala JA, Patankar AG, Gase K, Baldwin IT: Constitutive and inducibletrypsin proteinase inhibitor production incurs large fitness costs inNicotiana attenuata. Proc Natl Acad Sci USA 2004, 101:1607-1612.

34. Agrawal AA, Strauss SY, Stout MJ: Costs of induced responses andtolerance to herbivory in male and female fitness components of wildradish. Evolution 1999, 53:1093-1104.

35. Gigolashvili T, Berger B, Mock HP, Müller C, Weisshaar B, Fluegge UI: Thetranscription factor HIG1/MYB51 regulates indolic glucosinolatebiosynthesis in Arabidopsis thaliana. Plant J 2007, 50:886-901.

36. van Dam NM, Heil M: Multitrophic interactions below and above ground:en route to the next level. J Ecol 2011, 99:77-88.

doi:10.1186/1746-4811-7-5Cite this article as: Vaughan et al.: An aeroponic culture system for thestudy of root herbivory on Arabidopsis thaliana. Plant Methods 2011 7:5.

Vaughan et al. Plant Methods 2011, 7:5http://www.plantmethods.com/content/7/1/5

Page 10 of 10