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Belowground Resistance to Western Corn Rootworm inLepidopteran-Resistant Maize GenotypesAuthor(s) :Torrence A. Gill, German Sandoya, Paul Williams, and Dawn S. LutheSource: Journal of Economic Entomology, 104(1):299-307. 2011.Published By: Entomological Society of AmericaDOI: 10.1603/EC10117URL: http://www.bioone.org/doi/full/10.1603/EC10117

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PLANT RESISTANCE

Belowground Resistance to Western Corn Rootworm inLepidopteran-Resistant Maize Genotypes

TORRENCE A. GILL,1,2 GERMAN SANDOYA,1 PAUL WILLIAMS,3 AND DAWN S. LUTHE1

J. Econ. Entomol. 104(1): 299Ð307 (2011); DOI: 10.1603/EC10117

ABSTRACT Several maize,ZeamaysL., inbred lines developed from an Antiguan maize populationhave been shown to exhibit resistance to numerous aboveground lepidopteran pests. This study showsthat these genotypes are able to signiÞcantly reduce the survival of two root feeding pests, westerncorn rootworm, Diabrotica virgifera virgifera LeConte, and southern corn rootworm, Diabroticaundecimpunctata howardi Barber. The results also demonstrated that feeding by the abovegroundherbivore fall armyworm, Spodoptera frugiperda (J.E. Smith), before infestation by western cornrootworm reduced survivorship of western corn rootworm in the root tissues of some, but not all,genotypes. Likewise, the presence of western corn rootworm in the soil seemed to increase resistanceto fall armyworm in the whorl in several genotypes. However, genotypes derived from the Antiguangermplasm with genetic resistance to lepidopterans were still more resistant to the fall armyworm andboth rootworm species than the susceptible genotypes even after defense induction. These resultssuggest that there may be intraplant communication that alters plant responses to aboveground andbelowground herbivores.

KEY WORDS rootworm, fall armyworm, Lepidoptera, maize, plantÐherbivore interactions

For half a century, the western corn rootworm, Di-abrotica virgifera virgifera LeConte, has been a majorpest of maize,ZeamaysL. Farmers in the United Statesspend �US$1 billion annually implementing pestmanagement strategies that include applying soil in-secticides to minimize crop losses to western cornrootworm (Sappington et al. 2006, Gray et al. 2009).Since the introduction of this insect pest into Europein 1992 (Baca 1994) and its consistent presence inCanada (Madder et al. 1988), the worldwide estimatesof the cost to control this pest well exceed US$1 billionannually (Kaster and Gray 2005, Kiss et al. 2005).

Several methods have been used to control westerncorn rootworm. The most important methods are croprotation (Baca et al. 2003), insecticide application(Hills and Peters 1971, Lee et al. 1997, Hoffmann et al.2000, Wright et al. 2000), use of resistant maize vari-eties developed by classical plant breeding methods(Flint-Garcia et al. 2009, Gray et al. 2009), or use oftransgenic maize expressingBacillus thuringiensis(Bt)toxins (e.g., Cry3Aa) (Onstad et al. 2001, Hibbard etal. 2005, Nowatzki et al. 2006, Storer et al. 2006, Huanget al. 2007). Nevertheless, western corn rootworm hasbeen able to either adapt or develop some level ofresistance to each control method (Levine et al. 2002,Gray et al. 2009). However, continued research can

improve all of these methods, which can then be usedin integrated pest management strategies. The breed-ing of resistant genotypes yields economically andenvironmentally friendly alternative methods of pestcontrol that could be widely used and easily combinedwith other control methods.

Maize inbred lines have recently been screened toidentify new mechanisms of resistance against west-ern corn rootworm damage to roots (Flint-Garcia et al.2009). This is important because currently used meth-ods have a speciÞc mode of action against westerncorn rootworm. In many cases, development of resis-tance to one type of insecticide or protein minimizesthe effectiveness of subsequent methods that use asimilar mode of action. Therefore, it would be usefulto identify additional mechanisms of resistance torootwormpests.Although it is knownthat root feedingdamage ultimately affects yield, less is known aboutthe interaction between above- and belowground de-fenses and their role in protecting the plant fromherbivore damage (Erb et al. 2009b). More knowledgeabout this interaction and its role in resistance couldprovide information that would facilitate the devel-opment of novel control strategies.

Previous research has shown that a group of maizeinbred lines developed from an Antiguan landracehave resistance to feeding by several species of Lep-idoptera (Davis et al. 1989). These inbreds are Mp496,Mp704, Mp705, Mp707, Mp708, Mp713, and Mp716(Wiseman et al. 1981; Davis et al. 1988, 1989; Williams1992; Davis et al. 1999; Chang et al. 2000; Williams andDavis 2000). The inbred lines Mp704 and Mp708 are

1 Department of Crop and Soil Science, 116 ASI Bldg., The Penn-sylvania State University, University Park, PA 16802.

2 Current address: USDAÐARS, Department of Agronomy, PurdueUniversity West Lafayette, IN 47907-2054 (e-mail: tagill@purdue.edu).

3 Corn Host Plant Research Laboratory, USDAÐARS, MississippiState University, Box 9555, Mississippi State, MS 39762.

resistant to feeding by fall armyworm, Spodoptera fru-giperda (J.E. Smith); southwestern corn borer, Di-atraea grandiosella Dyar; European corn borer, Os-trinia nubilalis (Hubner); tobacco budworm,Heliothisvirescens (F.); corn earworm, Heliocoverpa zea (Bod-die); and many other lepidopteran pests (Davis et al.1988). In response to larval feeding in the whorl theinbred lines Mp704 and Mp708 accumulate maizeinsect resistance 1-cysteine protease (Mir1-CP).Mir1-CP is defensive cysteine protease that perme-abilizes the lepidopteran peritrophic matrix (PM),thereby making the midgut more accessible to damageand pathogen attack (Chang et al. 2000; Pechan et al.2000, 2002; Mohan et al., 2006).

When Mp708 is attacked by caterpillars, Mir1-CPaccumulates at the feeding site within 1 h (Pechan etal. 2000). During the Þrst 24 h of feeding, transcriptlevels for Mir1-CP remain low but subsequently in-crease (Harfouche et al. 2006). This suggests thatMir1-CPÕs accumulation during the Þrst 24 h is nottranscriptionally regulated but rather is due to someother mechanism. In addition to accumulating in thewhorl, Mir1-CP has been found in the roots (Lopez etal. 2007). Surprisingly, its abundance in this below-ground organ seems to increase after 24 h of foliarfeeding by fall armyworm larvae (Lopez et al. 2007).Because immunolocalization indicated thatMir1-CP ispresent in the maize vascular tissues, including thethick-walled sieve elements of the phloem and in theroot metaxylem elements (Lopez et al. 2007), it hasbeen proposed that Mir1-CP may be translocated fromthe roots to the whorl in response to abovegroundlarval feeding. This was supported by an experimentshowing that root excision decreases the amount ofMir1-CP that accumulates in the whorl in response tolarval infestation (Lopez et al. 2007).

Prior evaluations of Mp708 indicated that it alsoexhibited resistance to two root pests, southern(Meloidogyne incognita) and peanut root-knot nema-todes (Meloidogyne arenaria) (Davis et al. 1988, Wil-liams et al. 1989). This, in addition to the evidencesuggesting that Mir1-CP accumulates in the roots andmight be mobile, led to the hypothesis that Mp704 andMp708 could potentially exhibit resistance to below-ground herbivores such as the western corn root-worm. Together, these results suggest that there couldbe above to belowground signaling in maize that leadsto the induction of root defenses that, in turn, protectthe plant from root-feeding herbivores.

The opposite phenomenon of belowground feedingaffecting resistance to aboveground herbivory hasbeen demonstrated in maize (Erb et al. 2009b). In themaize variety Delprim, belowground infestation bywestern corn rootworm enhanced resistance to Spo-doptera littoralis Boisduval damage or developmentand to the necrotrophic fungus Setosphaeria turcica(Erb et al. 2009b), indicating that there was an inter-action between belowground Ð aboveground her-bivory in maize. Also single infestations by westerncorn rootworm, S. littoralis, and S. turcica resulted inthe expression of a unique set of host genes for each

pest, suggesting that there are specialized resistancemechanisms for each attacker (Erb et al. 2009b).

In view of the previous reports, this study was un-dertaken to determine whether the lepidopteran-re-sistant maize inbreds developed from Antiguan germ-plasm also exhibited resistance to root-feedingherbivores and whether there were interactions be-tween belowground feeding by western corn root-worm and aboveground feeding by fall armyworm.

Materials and Methods

Insects and Plant Material. For all experiments,western corn rootworm and southern corn rootworm,Diabrotica undecimpunctata howardi Barber, were or-dered from French Agricultural Research Incorpo-rated (http://www.frenchagresearch.com/products.htm). Fall armyworm eggs and seed for the maizeinbreds were supplied by the U.S. Department of Ag-ricultureÐAgriculture Research Service (USDAÐARS)Corn Host Plant Resistance Research Laboratory atMississippi State University (Mississippi State, MS).For this study the maize inbreds (Mp496, Mp704,Mp705, Mp707, Mp708, Mp713, Mp714, and Mp716)developed by Williams and colleagues (Scott et al.1982; Williams and Davis 1982, 1984, Williams et al.1990; Williams and Davis 2000, 2002) were used as theresistant inbred lines. All of these lines except Mp713and Mp714 share ancestry with Antigua Gpo2 germ-plasm. Although they also are resistant to fall army-worm and other Lepidoptera, the lines Mp713 andMp714 are not derived from Antigua Gpo2. B73 andTx601 are inbred lines that are susceptible toaboveground damage by several lepidopteran pests.Tx601 does not accumulate Mir1-CP in the whorl(Pechan et al. 2002). Seeds (one to two seeds per pot)were planted in Haegerstown loam in pots of two sizes(small, �2.8 liters or large, �5.0 liters). Plants weregrown in the greenhouse of the Department of Cropand Soils Sciences at The Pennsylvania State Univer-sity (University Park, PA).Preliminary Western Corn Rootworm Bioassay.

Mp708 and Tx601 plants in the V5 stage (Ritchie et al.1986) were infested with 50 western corn rootwormÞrst-instar larvae in the small pots. Five pots, eachcontaininga singleplant,wereused foreachgenotype.Small cracks were made in the soil by squeezing thesides of the pot by hand. Then, rootworm larvae wereplaced in these cracks and allowed to disperse in thesoil.

After 14 d, the soil contents of Þve pots were com-bined and sifted by hand until the collection of root-worms was infrequent. At this time, the soil was shiftedthrough two mesh screens of different sizes (2.00 and0.833 mm) to Þnd remaining larvae. After the soil wassifted, it was checked for the presence of rootwormsuntil none were found after 30 min of continuoussearching. This bioassay was replicated three times.

The recovered western corn rootworm larvae werecounted, weighed, and their instar was determined.The same procedure was performed for all genotypes.The percentage of survivorship was the number of

300 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 104, no. 1

rootworm larvae collected at end of trial divided bynumber of rootworm larvae initially infested.Western Corn Rootworm Bioassay. A similar ex-

periment to test for western corn rootworm resistancewas conducted with several inbred lines with differinglevels of resistance to fall armyworm. The genotypesused were B73, Tx601, Mp496, Mp704, Mp705, Mp707,Mp708, Mp713, Mp714, and Mp716.

Two seed per genotype were planted into each pot,and there were four to Þve pots per genotype for eachexperiment. Of these genotypes, B73, Tx601, andMp716 had a single plant per pot and Þve pots pergenotype. Mp705 had two plants per pot. For Mp496,Mp704, Mp707, and Mp713, there were three pots ofgenotype with two plants per pot. For Mp714, therewere four pots, with two plants per pot; and Þnally,Mp708 had Þve pots with two plants per pot. For eachpot 50, Þrst-instar western corn rootworm larvae wereadded per plant. For example, if there were two plantsper pot, then 100 Þrst-instar western corn rootwormlarvaewereused.Maize in theV4stagewasused in thisexperiment. After 16 d in the greenhouse, all of the soilcontents from each genotype were combined, the soilwas sifted by hand, and rootworms were counted andthe percentage of survivorship was determined.Southern Corn Rootworm Bioassay. A similar ex-

periment was conducted with southern corn root-worm, but only the genotypes that exhibited high,medium, and low resistance to western corn rootwormwere used in this experiment. The genotypes usedwere B73, Tx601, Mp496, Mp704, Mp707, Mp708, andMp714. Each pot contained one to two plants in the V4stage, and there were four to Þve pots per genotype.Out of these, B73, Tx601, Mp708, and Mp714 had asingle plant per and Þve pots per genotype. Mp496 hada single pot with two plants, and Mp704 and Mp707both had two pots with two plants per pot. Southerncorn rootworm larvae were applied to the plants asdescribed for western corn rootworm. After 16 d, thesoil contents from each genotype were combined fol-lowed by hand shifting of the soil as described above,and percentage of survivorship was determined.Effect of AbovegroundHerbivory on BelowgroundResistance. An experiment was conducted to deter-mine whether 16 d of aboveground herbivory by fallarmyworm affected resistance to western corn root-worm. In large pots, two seed of genotypes Tx601,Mp496, Mp708, and Mp714 were planted in each ofthree pots. When plants were at the V7 stage, theywere infested with western corn rootworm larvae onlyor with fall armyworm and western corn rootwormlarvae (western corn rootworm fed and fall army-worm/western corn rootworm fed). On set of plantswas infested infested with 250 Þrst-instar larvae perplant. Fall armyworm/western corn rootworm-fedtreatments were initially infested with three third-instar fall armyworm larvae for 24 h before adding 250Þrst-instar western corn rootworm larvae per plant.Fall armyworm larvae remained on the plants for theentire 17 d of the trial. After 17 d, the western cornrootworm larvae were counted from each individualpot.

Whorl tissues from these plants were excised andused for bioassays to assess the performance of fallarmyworm(Wisemanetal. 1983,Williamset al., 1985).If the whorls were not used immediately, they werestored at 4�C. Whorl segments were cut and placed indiet cups along with a single Þrst-instar fall armywormlarva. Insects were allowed to feed for 10 d at 27�C anda photoperiod of 14:10 (L:D) h. After 10 d, weights andpercentages of mortality were assessed. There were 60bioassay cups per treatment.Statistical Analysis.All data were analyzed as a com-

pletely random design by using PROC GLM (SASInstitute, Cary, NC). Mean comparisons were madeusing FisherÕs protected least signiÞcant difference(LSD) test; an LSD test was performed only when theF value was signiÞcant to the probability level � 0.05.

Results

Western Corn Rootworm Survivorship on Mp708andTx601.Three separate bioassays replicated in timewere conducted to assess the resistance of maize in-breds Mp708 and Tx601 to the belowground herbivorewestern corn rootworm. The number of larvae col-lectedperplantwas25.07 forTx601and6.16 forMp708(P � 0.05; LSD � 12.53). In both genotypes, theweights of the recovered larvae were statistically thesame and they were predominantly in the third instar.Western Corn Rootworm Resistance in AdditionalMaize Inbred Lines. Several additional maize inbredlines were analyzed for resistance against westerncorn rootworm larvae. In the fall armyworm suscep-tible genotypes B73 and Tx601, 18 western corn root-worm larvae were recovered per plant, which wassigniÞcantly higher than the number collected fromthe resistant genotypes (Fig. 1A). Within the resistantgenotypes tested, Mp704, Mp705, Mp707, Mp708, andMp713 exhibited the highest level of resistance towestern corn rootworm survivorship reducing popula-tions to approximately eight larvae per plant (Fig. 1A).

In this experiment, western corn rootworm larvalweights were signiÞcantly higher in B73 than in theother genotypes tested (Fig. 1B). All of the remainingweights were signiÞcantly lower than those of larvaecollected from B73. The lowest weights were found inlarvae collected from Tx601 and Mp496 (Fig. 1B).Thus, it seems that B73 has both the most western cornrootworm survivorship and largest larval weights of allthe genotypes tested.Resistance to Southern Corn Rootworm. All insect

resistant maize inbred lines were analyzed for poten-tial resistance against southern corn rootworm larvaeexcept Mp705 and Mp713. In this experiment, theinbred lines B73, Tx601, and Mp714 exhibited the leastsuppression of southern corn rootworm survivorshipcompared with the remaining genotypes (Fig. 2A). Inaddition, the weights of the recovered southern cornrootworm larvae were larger on the control genotypesB73 and Tx601 compared with the remaining insectresistant inbreds (Fig. 2B).

February 2011 GILL ET AL.: BELOWGROUND RESISTANCE TO WCR IN MAIZE GENOTYPES 301

Effect of Aboveground Fall ArmywormFeeding onWestern Corn Rootworm Survival. A preliminaryexperiment was conducted to determine whetheraboveground fall armyworm feeding in the whorl forone day before the addition of western corn rootwormaffected the western corn rootworm population inTx601or Mp708. The results of this experiment (datanot shown) indicated that there was no signiÞcanteffect of short-term aboveground feeding on below-ground resistance to western corn rootworm survi-vorship, larval weight, or instar. Therefore, a long-term experiment was conducted in which plants werecontinuously infested with fall armyworm larvae for17 d. The results showed that signiÞcantly higher num-bers of western corn rootworm (32 per plant) wererecovered from Tx601 that was not infested with fallarmyworm compared with Tx601 fed on by fall army-worm for 16 d (19 per plant) (Fig. 3). Mp496 exhibiteda similar trend as Tx601, averaging 10 larvae per plantcollected from Mp496 control plants and Þve larvaefrom plants infested with fall armyworm. However,the number of western corn rootworm larvae col-lected from Mp708 control and fall armyworm-in-fested plants was similar with an average of two andthree larvae per plant, respectively (Fig. 3). Althoughthe number of larvae collected (20 per plant) washigher for Mp714, there were no differences betweenthe treatments. The inbred Mp708 exhibited the high-est resistance to western corn rootworm survivorship,

but there were no differences between the treat-ments. The results from Tx601 and Mp496 indicatedthat aboveground feeding by fall armyworm signiÞ-cantly reduced the belowground survivorship of west-ern corn rootworm in these lines, which suggests thatthere could be some type of above to belowgroundcommunication occurring.

After 16 d of infestation, whorl tissue was col-lected and used in bioassays to determine whetherfeeding by western corn rootworm or western cornrootworm and fall armyworm in combination in-creased the level of induced defenses in the whorl(Fig. 4). In Tx601 and Mp714, 16 d of feeding by thebelowground herbivore alone resulted in smallerfall armyworm larvae and greater mortality thanthose from plants that were infested with both in-sects. Fall armyworm larvae fed on whorls fromTx601 or Mp714 plants infested with both insectswere larger and the mortality was less. However, inMp496 there was no difference between the treat-ments in fall armyworm weight or survivorship. Bio-assay data for Mp708 indicated that the mortality ofthe insects on this genotype was �95% and its causeis unknown (Fig. 4). In separate experiments rou-tinely conducted in our laboratory, fall armywormlarvae of equivalent instars (third to fourth) that fedon control Tx601 or Mp708 whorls had an averageweight of 45.6 and 77.6 mg, respectively (data notshown). Because the weights of fall armyworm lar-

Fig. 1. Western corn rootworm bioassays testing maize inbred lines with varying resistance to aboveground feeding byfall armyworm. (A) Average number of western corn rootworm larvae collected per plant. (B) Average weight of westerncorn rootworm larvae collected from each genotype; LSD value (P� 0.05) � 0.67. Means for the bars followed by the sameletter are not signiÞcantly different (P � 0.05) according to LSD test.

302 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 104, no. 1

vae in this experiment were smaller than those thatare typical for larvae reared on control plants, it ispossible that both treatments enhanced resistanceto fall armyworm in the whorl.

Discussion

Several studies over the past few years have shownthat systemic resistance in the aerial regions of plants

can be induced by foliar herbivory (Soler et al. 2007;Kaplan et al. 2008; Erb et al. 2009a,b; Rasmann et al.2009). Less is known about the effect of foliar feedingon belowground resistance or root feeding onaboveground resistance. In this study, we used maizegenotypes with varying levels of resistance to fall ar-myworm feeding to assess the interaction betweenabove- and belowground feeding and their effects onthe survivorship of western corn rootworm.

Fig. 2. Southern corn rootworm bioassays testing maize inbred lines with varying resistance to aboveground feeding byfall armyworm. (A) Total number of southern corn rootworm larvae collected from each genotype. (B) Average weight ofsouthern corn rootworm larvae collected from each genotype. Means for the bars followed by the same letter are notsigniÞcantly different (P � 0.05) according to LSD test.

Fig. 3. Effect of 16 d of continuous aboveground feeding by fall armyworm larvae on western corn rootworm survivalin the roots. Means for the bars followed by the same letter are not signiÞcantly different (P � 0.05) according to LSD test(LSD � 22.92). On the x-axis, genotypes with no annotation were infested with western corn rootworm larvae for 16 d;genotypes with the annotation “FAW fed” were infested with fall armyworm and western corn rootworm larvae for 16 d (fordetails, see Materials and Methods).

February 2011 GILL ET AL.: BELOWGROUND RESISTANCE TO WCR IN MAIZE GENOTYPES 303

Previous studies (Pechan et al. 2000) have shownthat the fall armyworm resistant genotype Mp708 ac-cumulates the insecticidal protein Mir1-CP at thefeeding site. The accumulation is in part due to theexpression of Mir1-CP in the tissues adjacent tothe feeding site (Pechan et al. 2000) and the possibletranslocation from the roots via the xylem after fallarmyworm damage (Lopez et al. 2007). The presenceof Mir1-CP is signiÞcant because it has been shown todegrade type I PMs of insects from different orders(Mohan et al. 2006). Therefore, the genotypes devel-oped from Antiguan germplasm that express Mir1-CPand are resistant to aboveground lepidopteran pestsalso might be resistant to the belowground herbivoreswestern and southern corn rootworms.

In the Þrst experiment, the number of western cornrootworm larvae collected from Tx601 plants was 4times higher than recovered from Mp708 and thedecreased recovery suggests that Mp708 has resis-tance against the survival of this insect. The low sur-vivorship might be due to the accumulation ofMir1-CP in Mp708 roots. Because Mir1-CP attacksproteins in type I PMs (Pechan et al. 2002, Mohan etal. 2006), it might be effective against the PMs foundin rootworms. Although PM type of the western cornrootworm is unknown, it has been shown that south-ern corn rootworm has a type I PM (Ryerse et al.1994); hence, it is likely that western corn rootwormhas a similar PM structure. However, until feedingbioassays are done with puriÞed Mir1-CP, we cannotascertain whether the protein itself causes the re-duced survivorship, or whether other factors are in-volved.

Additional experiments were conducted to deter-mine whether other lepidopteran-resistant inbred

lines were resistant to belowground feeding by west-ern corn rootworm and southern corn rootworm.When the insect resistant genotypes were comparedwith the two susceptible inbreds Tx601 and B73, sig-niÞcantly fewer western corn rootworm larvae wererecovered from resistant inbreds. With the exceptionof Mp496 and Mp716, inbreds derived from the An-tiguan germplasm tended to be the most resistant toboth rootworm species. Mp714, which was not derivedfrom Antiguan germplasm, had intermediate resis-tance and was more resistant to western corn root-worm than the susceptible checks Tx601 and B73, butwas far less resistant to the southern corn rootworm.Hence, it seems that selection for aboveground resis-tance in the inbreds derived from the Antiguan germ-plasm might have resulted in constitutive levels ofbelowground resistance to the two types of rootworm.

Because the inbreds derived from the Antiguangermplasm exhibited resistance to aboveground andbelowground herbivores, an experiment was designedto determine whether there was communication be-tween the whorl and roots that could alter the defenseresponses of these organs. An experiment was con-ducted in which fall armyworm larvae were placed inthe whorl 24 h before the pots were infested withwestern corn rootworm, and they remained on theplants for 16 d. In addition to Tx601 and Mp708, twoinbred lines, Mp496 and Mp714, with intermediatelevels of western corn rootworm resistance were in-cluded in the experiment. Tx601 plants that were in-fested with both insects had a lower western cornrootworm survivorship than those infested with west-ern corn rootworm alone. A similar trend was appar-ent for Mp496, but the differences were not statisti-cally signiÞcant. There was no signiÞcant difference

Fig. 4. Fall armyworm bioassay using whorl material collected from plants infested with western corn rootworm aloneor western corn rootworm with fall armyworm. The bar graph shows the average weight of fall armyworm larvae after a 10-dbioassay. Means for the bars followed by the same letter are not signiÞcantly different (P� 0.05) according to LSD test. Thebox below the graph indicates the percent mortality of the fall armyworm larvae during the bioassay. Mean percentages ofmortality followed by the same letter are not signiÞcantly different (P � 0.05), and the LSD value was 16.87. On the x-axis,genotypes annotated “WCR fed only” were infested with western corn rootworm larvae for 16 d and whorls were thencollected for bioassays with neonate fall armyworm larvae; genotypes annotated “WCR & FAW” were infested with both fallarmyworm and western corn rootworm larvae for 16 d and whorls were then collected for bioassays with neonate fallarmyworm larvae.

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between the treatments for Mp708 or Mp714. Al-though aboveground feeding by fall armyworm sig-niÞcantly decreased the survivorship of western cornrootworm larvae in Tx601, more larvae were recov-ered from Tx601 and Mp714, the two lines that werenot derived from Antiguan germplasm. Fewer westerncorn rootworm larvae were recovered from Mp708regardless of the treatment. The lack of a signiÞcantdifference between the two treatments in this geno-typemightbe the resultof strongconstitutivedefensesthat mask the effects of induced defense responses(Shivaji et al. 2010).

To determine whether belowground feeding af-fected aboveground defenses, whorl tissue was col-lected from plants that were subjected to feeding bywestern corn rootworm alone or both fall armywormand western corn rootworm and used for fall army-worm feeding bioassays. For Tx601 and Mp714, be-lowground feeding by western corn rootworm alone,compared with feeding by both insects, seemed toenhance aboveground resistance as measured by fallarmyworm larval weight and mortality. A similar casemay have occurred in Mp708, but the mortality in bothtreatments was very high. Because caterpillar feedingtypically induces defenses in the whorl (Williams andBuckley 2008), this Þnding was unexpected. It is pos-sible that the plant is unable to mount two effectivedefenses simultaneously, thus plants that were in-fested with both insects had compromised resistance.

In both treatments, the weights of the fall army-worm larvae were less than those that we typicallyobtain for larvae reared on control plants that have notbeen subjected to herbivory. So, it seems that insectfeeding, both above and belowground might enhanceherbivore defenses. In related maize genotypes, priorwhorl feeding by either fall armyworm or southwest-ern corn borer, Diatraea grandiosella Dyar, improvedresistance to these insects when the lyophilizedwhorls were then used in feeding bioassays (Williamsand Buckley 2008). In some cases, reductions in larvalweights �75% were observed and in general, thegrowth inhibition effect was more pronounced in ge-notypes with lower innate levels of herbivore resis-tance. In genotypes derived from Antiguan germ-plasm, prior herbivory had little effect on larvalweights, but the weights of larvae reared on thesegenotypes were still signiÞcantly lower than thosereared on “induced” susceptible genotypes (Williamsand Buckley 2008).

It seems that Mp708, Mp714, and to a degree Mp496exhibit constitutive belowground defenses that di-rectly affect survivorship of western corn rootworm.Tx601, however, clearly has lower constitutive de-fenses to western corn rootworm, but abovegroundfeeding by fall armyworm seems to enhance below-ground resistance in this genotype. Furthermore,whorls collected from Tx601 infested with westerncorn rootworm exhibited more resistance to fall ar-myworm larvae than whorls from plants infested withboth insects. These results are comparable to thosereported for the maize variety Delprim where below-ground feeding by western corn rootworm seemed to

enhance whorl resistance to S. littoralis (Erb et al.2009b). They also are similar to those reported for rice,Oryza sativa L., where root feeding by the rice waterweevil, Lissorhoptrus oryzophilus Kuschel, caused areduction in the growth rate of fall armyworm feedingon the leaves (Tindall and Stout 2001). Another ex-ample of the interaction between above and below-ground herbivory occurs in Brassica nigra L. wherefoliar herbivory by Pieris brassicae L. (Lepidoptera:Pieridae) lowered the survivorship of root herbivoreDelia radicum (L.) (Diptera: Anthomyiidae) (Soler etal. 2007).

In summary it seems that there is some level ofabove and belowground communication in maizeplants. Long-term feeding by fall armyworm larvaeenhanced resistance to western corn rootworm sur-vivorship in Tx601 but not other genotypes. Likewise,belowground feeding by western corn rootworm en-hanced resistance to fall armyworm feeding in thewhorl. The effect of above and belowground signalingseems to be greater in this susceptible genotype thanin those derived from Antiguan germplasm. Never-theless, inbreds developed from Antiguan germplasmseem to have higher levels of constitutive defensesthan the susceptible lines that contribute to their re-sistance to herbivory by western corn rootworm andfall armyworm.

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Received 25 March 2010; accepted 14 October 2010.

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