Evaluation of Transgenic Corn for Resistance to Corn Earwonn(Lepidoptera: Noctuidae), Fall Armyworm (Lepidoptera:Noctuidae), and Southwestern Corn Borer (Lepidoptera:

Crambidae) in a Laboratory Bioassayl, 2

W. Paul Williams, Paul M. Buckley, Jonathan B. Sagers,3and John A. Hanten3

U. S. Department or At,"";culture, Agricultur~l ilesearch Ser"i""Corn Host Plant R"sistance Rese~rch Unit, Box 9555

Missi,sippi 5t:ltc. Mississippi 39762 U.S.A.

J. Agric. Entomol. 15(2): 105--112 (April 1998)ABSTRACT The development of crop plants expressing genes that encodeinsecticidal proteins isolated from the bacterium lJacillus thuringiellsisBerliner (Et) has provided a tremendous opportunity for improved control ofinsect pests. Although corn, Zea mays L., gennplasm with resistance in thewhorl stage to fall armyworm, Spodoptera (rugipcrda (J. E. Smith), andsouthwestern corn borer, Diatraca grandiosella Dyar, larval feeding has beenidentified and released, little progress has becn made in identif~'ing

resis~ncc to ear damage by these insects. Husks and silks were collected 3to 4 dafter anthesis from nontransgenic and transgenic corn hybrids andused in laboratory bioassays to determine the effects of the Bt insecticidalprotein on larval survival and growth of fall armyworm, southwestern comborer, and com eaTWorm, Helico~'erpa zca (Boddie). Southwestern corn borerlarvae that fed on diets containing husks of transgenic plants did not survive.Com earworm larvae fed on diets containing husks or silks from transgenicplants died, and those that fed on diets containing silks harvested fromtransgenic plants exhibited reduced survival and growth. Fall armywormlarvae were least susceptible to the Bt protein. but larval survh'al and gro .....thon diets containing husks of transgenic plants were reduced. Larval growthalso was reduced on diets containing silks from transgenic plants. Thebioassays indicated that expression in husks and silks or genes encoding Btinsecticidal proteins could be useful in reducing insect damage in ears of com.

KEY WORDS Lepidoptera, Noctuidae, PFalidac. Spodoplera (rugiperda.Diatroea grandiosella, flelicoverpa zea, Bacillus Ihuringiensis, Zea mays,plant resistance. transgenic hybrids

Tremendous effort has been expended in the past few years on thedevelopment of crop plants expressing genes that encode insecticidal proteins

IAcecp~d for publication 23 February 1998.2'fhi~ article is a contribution of USDA-ARS in cooperation with the l>lississippi Agricultural and

Forestry Experiment Station and is published as Journal No. J·9209 of the Mississippi Agriculturaland Forestry Experiment St:ltion,

3No"artis Seeds. 317 330th 5t.. SI.,.<IIlton. l>linnesota 55018-1308.

105

106 J. Agric. Emomol. Vol. 15, No.2 (1998)

having the property of those found in the bacterium Bacillus thuringiensisBerliner (Bt) (Boulder 1993). Com, Zea mays L., plants expressing o.endotoxininsecticidal proteins have been evaluated in field tests for resistance to Euro­pean corn borer, Ostrinia lIubilolis (Hiibner), a major pest in the Corn Belt anda serious pest in parts of the southern United States. Resistance to both leaffeeding and stalk tunneling werc observed (Armstrong et a1. 1995, Koziel et al.1993).

In the southern United States, fall annyworm, Spodoptcra (rugipereda (J. E.Smith), and southwestern com borer, Diatraea grandiosella Dyar, arc economi­cally more important than European corn borer. Williams ct al. (1997)observed high levels of resistance to leaf feeding by southwestern com borer inwhorl-stage plants of transgenic Bt com hybrids and moderate resistance toleaf feeding by fall armyworm. They also found that when southwestern comborer lanae were fed on laboratory diets containing lyophilized leaf or husk tis­sue of transgenic Bt corn hybrids, the larvae died. Fall annY'vonn larvae feddiets containing lyophilized leaf tissue with the Bt protein exhibited lower sur­vival and reduced weight in comparison with those fed diets that did not con­tain the protein,

In this investigation, survival and growth of fall armywonn. southwesterncorn borer, and corn earworm, Helicouerpa zea (Boddie), larvae were comparedin a laboratory bioassay on diets containing lyophilized husk tissue of twotransgenic com hybrids with Bt insecticidal proteins and nontransgenic ver­sions of the two hybrids lacking the protein. In addition, survival and growth oflarvae were compared on diets consisting of lyophilized leaf tissue of either aleaf-feeding resistant or a susceptible corn hybrid and lyophilized silks of atransgenic or nontransgenic hybrid. The treatments constituted a 2 X 3 factorialset with leaf tissue being the primary factor and no silks, transgenic silks, ornontransgenic silks the secondary factor, Leaf tissue from both resistant andsusceptible hybrids was used in the bioassays to detennine whether the addi­tion of the insecticidal proteins in transgenic silks affected larval survival andgrowth differently when used in combination with resistant leaf tissue thansusceptible leaf tissue.

Materials and Methods

Plant materials. Two experimental transgenic Bt com hybrids, N6800Btand N7634Bt, and nontransgenic versions of these hybrids. N6800 and N7639,were provided by Novartis Seeds. The transgenic single-cross hybrids were pro·duced by crossing Bt lines with elite inbred testers.

The gene used to confer insect resistance was Cry.'!A(b). or Bt 11, from B.thuringiensis var. kurstaki (HtHte & Whiteley 1989). The coding sequence ofthe C",L4'(bJ gene was altered to increase its level of protein expression (Perlaket al. 1991). A 355 promoter from cauliflower mosaic virus (Gardner et aI.1981) was ligated to the CrylA(b) gene and then inserted into a pUC18 cloningvector (Yanisch-Perron et Ill. 1985). The phosphinothricin N.acetyltransferase(PAT) gene from Streptomyces uiridochromogelles (Krainsky) (Wohlleben et al.1988) was inserted into the same pUCl8 cloning vector and used as a selectablemarker. The CryL4.-PAT construct was incorporated into com and selected in

WILLlAMS eta!.: E\'alualion of Transgenic Com in a Laboratolj' Bioassa)' 107

\;tro ....ith a glufosinate·ammonium herbicide. First-generation transfonnedplants were regenerated from calli. Reciprocal crosses were made withNorthrup King Company elite, inbred lines. The progeny were subjected toselective herbicide sprays and enzyme-linked immunosorbent assay (ELISA) toensure that only plants cont:lining the Cf)'IA(b) gene were advanced.

Approximately 100 plants of each hybrid were grown at Mississippi State,j\'lississippi, in 1996 following standard corn production practices to providehusks and silks for laboratory bioassays. Within 3 to 4 d afier silk emergence,primal)' ear shoots were removed from the plant and placed on ice. Husks andsilks were removed from the car, separated, placed in plastic freezer bags. andfrozen at _IScC. The husks and silks were later lyophilized and ground to a finepowder b)' using a laboraloY mill with a I-mOl screen.

Husk bioassay. On 26 February 1997, 20 cups of diet were prepared fromLhe ground, I)'ophilized husk tissue of each hybrid. The transgenic and non­transgenic versions of the two hybrids formed a 2 X 2 factorial seL of Lreat­ments. Following the procedures described by Williams & Buckley (1996), dietswere prepared by combining 250 ml of distilled water, 2.5 g of agar, 528 mg ofascorbic acid, 132 mg of sorbic acid. and 12.5 mg of gentamicin sulfate and heat­ing the mixture to l00"C while stirring_ The mixture was then cooled to 82"C and15 g of lyophilized husk tissue was added. The diet was dispensed into 300mlplastic cups. Each cup was infested v.ith a neonate southwestern com borer andcovered with a paperboard insert cap. Cups were arranged in a randomized com­plete block design with five replications of foUT cups per hybrid and placed in anenvironmental chamber maintained at 29"C and photoperiod of 12:12 (L:D) h. The.laryae were counted and weighed 21 d later. Previous work indicated that south·western corn borer larvae fed on husk tissue of highly susceptible corn genotypesreach the 5th instar, bUL do not pupate within 21 d (P.M.B., unpublished data).

On 4 r-.larch and 10 April. similar experiments were initiated for fall army­wonn and com earworm, respectively. All larvae were obtained from laborato­ry cultures maintained in the Corn Host Plant Resistance Research Unit.Field-collected larvae are infused into the cultures annually to avoid loss of\'igor in the cultures. Because fall annywonn and com earworm grow morerapidly than southwestern com borer, the larvae were weighed after 14 drather than 21 d so larvae would not pupate before weighing.

Silk bioassay. To ensure that the laboratory diets pro\'ided adequate nutri­tion for larval growth, the ground lyophilized silks were combined withlyophilized leaf tissue rather than being used alone in diets. Leaf tissue washarvested from two hybrids: Mp704 X Mp707, which is moderately resistant tofall armyworm and southwestern com borer. and Ab24E x SC229, which is sus­ceptible to both insects. In field evaluations Mp704 X l\"1p707 sustains less leafdamage that Ab24E X SC229 when infested with either fall armyworm orsouthwestern com borer. La....'ae recovered from Mp704 X J\.·lp707 plants weighless than those recovered from Ab24E X SC229 (Williams et al. 1997). Both aresistant and a susceptible hybrid were included to detenrune whether theeffect of a combination of resistant leaf tissue and silks differed from a combina­tion of susceptible leaf tissue and silks. The whorl tissue was haT'\'ested whenplants were in the V7 stage of grov..th (Ritchie et al. 1986) and was processedfollo ....ing the procedures described for husks.

108 J. Agric. Entomol. Vol. 15. No.2 (998)

Diets were prepared following t.he same procedures described for husks except either 9 g of lyophilized leaf tissue or 9 g of lyophilized leaf tissue plus 2 g of either transgenic (N6800Bt) or nontransgenic ( 16800) silks replaced the husks in the diets. With genotypes of leaf tissue as one factor and no silks, non­transgenic silks, or transgenic silks as the second factor, the six treatments con­stituted a 2 X 3 factorial set of treatments. Diet cups in the first experiment. were infested with southwestern corn borer larvae on 12 March, and diet cups in the other experiments were infested with faJ] annywonn and com eanvonn on 21 March. Experimental protocols were the same as for husks except the southwestern corn borer larvae were counted and weighed 14 d after infesta­tion. Fall armyworm and com ean...orm lan'ae .....ere counted and weighed after 10 and 11 d, respectively. Larvae grow more rapidly on leaf tissue than on husk tissue so the larval feeding periods were reduced to avoid pupation on the susceptible hybrid.

Data on larval survival and weight from each experiment .....ere subjected to analysis of variance. The significance of differences between means was deter­mined by Fisher's Protected LSD (P "" 0.05) (Steel & Torrie 1980).

Results and Discussion

The results of bioassays that used lyophilized husk tissue are presented in Table 1. All southwestern corn borer and corn ean\'orrn larvae that fed on husks of the transgenic hybrids were killed. Survival of fall armyworm larvae on the diets containing Bt protein was significantly (F""16.00j df",,3, 12; P<O.OO02) lower (35%) than on the diets lacking Bt. Weights of the surviving larvae also were greatly reduced. Interestingly, mean larval weights for all three species were consistently higher on the nontransgenie hybrid N6800 than on the hybrid N7639. This indicates that N7639 rna)' possess at least some resistance to these insects in the earl)' reproducti\'e stages.

Survival and weights of larvae fed on diets containing mixtures of lyophilized leaves and silks are presented in Table 2. Larval survival was high (~O%) for all species fed on diets containing lYophilized leaf tissue, but no silk tissue, whether the leaf tissue was from the leaf-feeding resistant hybrid jI,·tp704 X Mp707 or from the susceptible h)'brid Ab24E X SC229. The addition of silks from the nontransgenic hybrid N6800 significantly (F",,107.63; df",S, 20; P<O.QOOl) reduced survival of southwestern corn borer larvae. but not fall 3rmywonn or corn earwonn surviv31. Adding silks of the transgenic hybrid N6800Bt to the diet resulted in death of all southwestern com borer lan'3e .....;th no visible sign of larval feeding. significantly (P",,16.59; df"'S. 20; P<O.OOOl) reduced corn earworm survival, but did not affect fall armyworm survival (F""O.55; df",S, 20; P>0.73).

Larvae reared on lyophilized leaf tissue of the resistant hybrid alone weighed about half as much as those reared on lyophilized leaf tissue of the sus­ceptible hybrid. Additions of silks of the nontransgenic hybrid resulted in reduced size. Apparently, something detrimental to larvae of the three species is present in the silks that caused reduced weight of fall armyworm and corn ea"""orm lan'ae and reduced survival of southwestern com borer. Wiseman et :d. (1986) reported that additions of leaf tissue to pintO bean diet resulted in

Table 1. Survival and weight of southwestern corn borer, fall armyworm, and corn earWOl'm larvae fed on lyophilized husk tissue of transgenic and nontransgenic corn hybrids in a laboratory bioassay.

Southwestern corn bore,"" Fall armyworm!> Cal'll earwormb ~ Hybrid Survival

(%) Weight

(mg) Survival

(%) Weight

(mg) Survival

(%) Weight

(mg) ~ UJ

'"... !!:.

N6S00c

N7639c

90 (22)

95 (II)

126 (2)

78 (7)

85 (22)

90 (14)

231 (11)

100 (17)

95(11)

95 (11)

97 (4)

39 (7) t':l ..: !!:. I:

N6S00Btd 0(0) 55 (21) 16 nO) 0(0) ~ cr. o

N7639Btd 0(0) 50 (I8) 15 (12) 0(0) ::l o...,

LSD (0.05) 34 13 16 35 11 25 ~ § II>

Meuns followed by stnndurcl dcvintions in pumntheses.

(l Larvae were connlml und IVci~hcd aftcr rceding 21 <1.

~ ::l;:;.

bLnl'vue were counled on,1 \Vci~h"d arler feeding 14 d,

cNontl'8nsgenic hybrid.

o o :l

dTnlllsgenic (8/) hybrid. ::l to l' l» IT o .... l» S Q to (5' to til til

~

..... o

'"

-Table 2_ Survival and weight of southwestern cOl-n borer, fall armyworm, nnd corn earworm larvae fed in a labo· ratory bioassay on n diet containing lyophilized leaf tissue of genetically resistant (lU nnd susceptible

:0

(S) corn hybrids with additions of IYOllhilized silks of transgenic (T) and non transgenic (NT) hybrids.

Southwestern corn borer' Fall armywormh Corn em'worm':

Diet

S S + NT silks

S+Tsilks

H

Survival (%)

100 (0)

60 (22)

0(0)

lOO (0)

Weight (mg)

120(8)

94 (11)

-60 (8)

Survival (%)

9S{ll)

100(0)

100 (0)

95 (11)

Weight (mg)

1166 (35)

255 (33)

45 (5)

285 (75)

Survival (%)

95 (II)

85 (22)

55 (21)

90(14)

Weight (mg)

250 (71)

40 (12)

4 (0.8)

109(3)

,. >­'1 e '" S 3 ~

n + NT silks

R .. T silks

LSD (0.05)

85(4)

0(0)

13

59 (B)

-9

95 (11)

100 (0)

NS

67 (29)

8 (4)

50

100 (0)

40 (14)

18

20 (3)

I (0.5)

40

~ -". z ? ~-

Menn~ flll1owl!d by ~tnndnrd dev;nlilln~ in Pl'renIM$eol.

1I1~''''''ne ...·ere amnltd nnd weighed nfter 14 d.

~

~ "1.11 ..... 11C ....ere CGulllcd lind ....eillhcd lifter 10 d.

"'l,.nrvnll were tounled nnd wl!illhed "fter 11 d,

WLLLlAMS et al.: E\'aluation of Transgenic: Com in a Laboratory Bioassay III

increased fall armyworm larval weight. but additions of silk resulted in signi.fi~

cant reductions in larval weight of fall armyworm and com earwonn laryae. The addition of transgenic silks to the diets resulted in marked reductions in larval weights whether the leaf tissue came from a resistant or susceptible hybrid. However, fall armyworm and com eanvorm larvae fed on a combina­tion of leaves from a resistant hybrid and silks of a transgenic hybrid were extremel)' small.

Of the three insects, the southwestern com borer was most sensitive to the Bt insecticidal protein. All larvae were killed in any diet containing transgenic tissue, whether husk or silk. The corn earwonn was the second most sensitive. The larvae were killed when fed on diets of husk tissue, and when silk and leaf tissue were combined, larval survival and weight were significantly (F=51.47; df=5, 20; P<O.OOOl) reduced. The fall armywonn was the least sensitive oftbe three insects to the Bt proteins. Survival was reduced on Bt-containing husk diets, but 50% of the larvae survived. On the silk-leaf tissue mixtures, sunrival was unaffected. The greatly reduced weights of fall armyworm larvae fed on any of the diets containing the St insecticidal proteins indicates the potential usefulness of this technology in controlling fall armyv:orrn as well as the other two insects.

Combining native resistance with the Bt insecticidal proteiD in a hybrid could result in hetter control of less susceptible insects such as fall annywonn. Also, the enhancement from native resistance could hinder the development of resistance to Bt proteins in an insect population. Sachs et a1. (1996) suggested pyramiding CrylA(b) insecticidal protein with conventional plant. resistance in cotton to delay the development of resistant tohacco bud worm, Heliothis uirescens (FJ, populations in cotton, Gossypium hirsutum L. Pyramiding could be especially useful with polyphagous insects such as fall armyworm and com eanvorm that attack more than one crop in which transgenic hybrids and vari­eties have alread): been developed.

The effecth'eness of the Dr protein in silks and husks against southwestern com borer. fall armyworm. and corn earworm in these assays indicates the potential for reduced ear damage that would be important in eliminating the entrance of fungi into the caTS and reducing contamination of grain with myco­toxins. This could be especially important in the southern United States where aflatoxin contaminated grain is a chronic problem in corn.

Although the bioassays described herein used husks and silks harvested soon after anthesis, other plant tissues or tissue collected at other growth stages also could be used. The bioassays also could be adapted for use with other insect pests and other crops.

References Cited

Armstrong, C. L., G. B. Parker, J. C. Pershing, S. l'tI. Brown, P. R. Sanders, D. R. Dunean, T. SlOne, D. A. Dean, D. L. DeBoer, J. Hart. A. It. Howe, F. M.Morrish, M. E. Pajeau, W. L. Petersen, B. J. Reich. R. Rodrigue:t, C, G. Santino. S. J. Salo, W. Shuler, S. R. Sims, S. Stehling, L. J. Tarocbione & /It. E. Fromm. 1995. Field evaluation of European corn borer control in progeny of 173 transgenic corn events expressing an insecticidlll protein from Bacillus inuringiensis. Crop Sci. 35: 550-557.

112 J. Agric. Entomo!. Vol. 15. No.2 (1998)

Boulter, D. 1993. Insect pest control b)' copying nature using geneticall)' engineered crops. PhytochemistI'}' 34: 1453-1466.

Gardner, R. C., A. J. Howarth, P. Hahn, M. Brown·Luedi, R. J. Shepherd & J. Messing. 1981. The complete nucleotide sequence of an infectious clone of the cauli­no.....er mosaic \irus by M13mp7 shotgun sequencing. Nucleic Acids Res. 9: 2871-2888.

narte, H. & H. IL Whiteley. 1989. Insecticidal C1j'stal proteins ofBocillw; Ihuringrensis. Microbial. Re\·. 53: 172-275.

Ko:tiel.l\L G., G. L Beland, C. Bowman. N. B. Caro:t:ti, R. Crenshaw, L. Crossland. J. Dawson, N. Des,,"i, M. Hill. S. Kadwell. K. Launis, K. Lewis, D. Maddox. K. McPherson, 1\1. R. Mcghji. E. Merlin. It. Rhodes, G. W. Warren, l'tl. Wright & S. V. E\'ola. 1993. Field perfonnance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Biol1'echnology 11: 194-200.

Perlak. F. J., It. L. Fuchs, D. A. Denn, S. 1... McPherson & O. A. Fischoff. 1991, Modification of the coding sequence enhances plant expression of insect control protein genes. Proc. Nat!. Acad. Sci. USA 88; 3324-3328.

Ritchie, S. W.. J. J. Hnnwa)', E. &: G. O. Benson. 1986. Ho..... a corn plant de\·e)ops. (re\ised), Special Repon No_ 48, IOW3 State University of Science and Technology Cooperath'e Extension Service, Ames. 21 pp.

Sachs, E. S.. J. H. Benedict., J. F. Taylor, 0.1\1. St:e.ll)·, S. K. Davis &: D. W. Altman. 1996. Pyramiding Cf)'L\{b) insecticidal protein and terpenoids in cotton 10 resist 1(Ibacro budwonn (1A!pidoptera; Noctuidae). Environ. Entomol. 25: 1257-1266.

Steel, R. D. G. & J. R. Torrie. 1980. Principles and procedures of statistics. McGraw-Hill. New York.

Williams, W. P. &: P. M. Buckley. 1996. Southwestern corn borer growth on lyophilized com husks in a laboratol'}' bioassay. Crop Sci. 36: 462-164.

Williams, W. P., J. 8. SlIgcrs, J. A. Rnnten, F.M. Davis & P.M. Buckley. 1997. Transgenic corn evaluated for resistance to fall armyworm and southwestern corn borer. Crop Sci. 37; 957-962.

Wiseman, B. R., R. E. Lynch, K. L. Mikolajczak & R. C. Gueldner. 1986. Advance­ments in the use of n laboratory bioassay for basic hos~ plan~ resistance studies. Fill. Entomol. 69: 559-565.

WobJleben., W.. W. Anlold.l. Broer. D. BilJemann. E. S~rnuch &: S. PuebJer. 1988. Analysis of a phosphinothricin N-acetyltransfcrase gene from StlTpwm_w:u dridochro­mogenes and its use for the construction of herbicide resistant plants. Heredity 61: 299.

YllIlisch·Perron, C.. J. Vieira & J.l\tessing. 1985. Impro\'ed M13 phage clonine: vec· tors and host strains; nucleotide sequences of the M13mp18 and pUC19 \·ectors. Gene 33: 103-119.