TREATMENT THRESHOLDS FOR BOLLWORM (Helicoverpa zea) IN DUAL-GENE Bt COTTON

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1 Clemson University TigerPrints All Theses Theses TREATMENT THRESHOLDS FOR BOLLWORM (Helicoverp ze) IN DUAL-GENE Bt COTTON Kristen Crter Clemson University, Follow this nd dditionl works t: Prt of the Entomology Commons Recommended Cittion Crter, Kristen, "TREATMENT THRESHOLDS FOR BOLLWORM (Helicoverp ze) IN DUAL-GENE Bt COTTON" (212). All Theses. Pper This Thesis is brought to you for free nd open ccess by the Theses t TigerPrints. It hs been ccepted for inclusion in All Theses by n uthorized dministrtor of TigerPrints. For more informtion, plese contct wesole@clemson.edu.

2 TREATMENT THRESHOLDS FOR BOLLWORM (Helicoverp ze) IN DUAL-GENE Bt COTTON A Thesis Presented to the Grdute School of Clemson University In Prtil Fulfillment of the Requirements for the Degree Mster of Science Entomology by Kristen M. Crter December 212 Accepted by: Dr. Jeremy K. Greene, Committee Chir Dr. Frncis P. F. Rey-Jones Dr. Peter H. Adler Dr. Michel A. Jones

3 ABSTRACT Dul-gene Bt cotton hs reduced the need for insecticide tretments for bollworm, Helicoverp ze (Boddie), compred with originl single-gene Bt technology. Bollgrd II (Monsnto, St. Louis, MO) nd WideStrike (Dow AgroSciences, Indinpolis, IN), both produce the Cry1Ac protein nd second protein, Cry2Ab or Cry1F, respectively. These dul-gene Bt cottons provide enhnced control of lepidoptern pests, but remin less thn 1% effective ginst bollworm, prticulrly when popultion pressure is high. Current recommended tretment thresholds for bollworm on cotton in South Crolin re s follows: tret with insecticides when three or more lrge lrve re found per 1 plnts or when 5% boll dmge is detected. Studies were conducted in n re prone to high bollworm pressure ner Blckville, South Crolin, in 21 nd 211 to develop pproprite thresholds in Bollgrd II nd WideStrike cotton. Plots contining non-bt, WideStrike, nd Bollgrd II cotton vrieties were exmined weekly nd treted ccording to tretment threshold protocols for one of the following: bollworm eggs, lrve in white blooms, or boll dmge. Although yields incresed with insecticide pplictions in non- Bt cotton, sttisticl differences in yield mong thresholds were not evident within the Bt technologies. The conclusion drwn from this limited study ws tht insecticide pplictions exclusively trgeting bollworm were not necessry in dul-gene Bt cotton. Higher levels of bollworm infesttion nd dmge occurred in WideStrike cotton, however, WideStrike lint yields in this study did not differ mong vrying thresholds nd so did not support the conclusion tht protection strtegies be mended for ech technology. ii

4 ACKNOWLEDGMENTS I would like to cknowledge the support of my dvisor, Dr. Jeremy Greene, nd the members of my committee, Drs. Frncis Rey-Jones, Mike Jones, nd Peter Adler. Thnk you lso to the other grd students nd the members of Edisto REC s bug crew for ll of the hrd work put in under the hot Crolin sun. A specil thnks to Ginger Devinney for your lbor in the field, morl support throughout this project, nd for your friendship. And finlly, I would like to cknowledge the South Crolin Cotton Bord for funding this project. iii

5 TABLE OF CONTENTS Pge TITLE PAGE... i ABSTRACT... ii ACKNOWLEDGMENTS... iii LIST OF TABLES... vi LIST OF FIGURES... vii INTRODUCTION...1 LITERATURE REVIEW...5 MATERIALS AND METHODS...15 Species Determintion...15 Overview of Trils...16 Test 1- Egg Density Threshold...17 Test 2- Lrve Density Threshold...17 Test 3- Boll Dmge Threshold...18 Plnt Mesurements...18 Sttisticl Anlysis...19 RESULTS AND DISCUSSION...2 Overview...2 Egg Density Threshold...27 Lrve Density Threshold...33 Boll Dmge Threshold...39 Plnt Mpping...45 iv

6 Tble of Contents (Continued) Pge CONCLUSIONS...54 REFERENCES...56 v

7 LIST OF TABLES Tble Pge 1 Trget ction thresholds for bollworm eggs, lrve in blooms, nd boll dmge in cotton ner Blckville, South Crolin, in 21 nd Node bove white flower counts (±SEM) nd sttisticl comprisons for 21 nd 211 by technology, verged cross threshold nd test in cotton ner Blckville, South Crolin, 21 nd Sttisticl comprisons of bollworm egg nd lrvl densities nd boll dmge in cotton ner Blckville, South Crolin, 21 nd Sttisticl comprisons of cotton lint yield ner Blckville, South Crolin, 21 nd Sttisticl comprisons for plnt-mpping vribles for bollworm egg density threshold trils on cotton ner Blckville, South Crolin, Sttisticl comprisons for plnt-mpping vribles for bollworm lrve in blooms threshold trils on cotton ner Blckville, South Crolin, Sttisticl comprisons for plnt-mpping vribles for bollworm boll dmge threshold trils on cotton ner Blckville, South Crolin, vi

8 LIST OF FIGURES Figure Pge 1 Numbers of bollworm (BW) nd tobcco budworm (TBW) dults cught in pheromone trps bited weekly ner Blckville, South Crolin, in 21 (A) nd 211(B) Men bollworm eggs per 1 plnts (±SEM) from egg thresholds by cotton technology ner Blckville, South Crolin, in 21 (A) nd 211 (B) Cotton lint yields (±SEM) compring bollworm egg threshold tretments in non-bt cotton ner Blckville, South Crolin, in 21 (A) nd 211 (B). Brs with the sme letter re not significntly different. Numbers indicte number of insecticide pplictions received. UTC, untreted control; SWKLY, spryed weekly Men bollworm lrve per 1 blooms (±SEM) by cotton technology nd smpling dte ner Blckville, South Crolin, in 21 (A) nd 211 (B). Brs of the sme cotton technology with the sme letter re not significntly different. NBT, non-bt; WS, WideStrike; BGII, Bollgrd II Men bollworm lrve per 1 blooms (±SEM) in WideStrike cotton by lrvl tretment threshold nd dte ner Blckville, South Crolin, in 21. UTC, untreted control; SWKLY, spryed weekly...36 vii

9 List of Figures (Continued) Figure Pge 6 Men bollworm lrve per 1 blooms (±SEM) by threshold tretment nd cotton technology in WideStrike (WS), Bollgrd II (BGII), nd non-bt (NBT) cotton ner Blckville, South Crolin, in 21. Brs of the sme cotton technology with the sme letter re not significntly different. Numbers bove brs indicte number of insecticide pplictions received. UTC, untreted control; SWKLY, spryed weekly Comprison of 21 yield (±SEM) by technology in WideStrike (WS), Bollgrd II (BGII), nd non-bt (NBT) cotton ner Blckville, South Crolin. Brs of the sme cotton technology with the sme letter re not significntly different. Numbers bove the brs indicte number of insecticide pplictions tretment received. UTC, untreted control; SWKLY, spryed weekly Percent boll dmge (±SEM) cused by bollworm to non-bt (NBT), WideStrike (WS), nd Bollgrd II (BGII) cotton during July nd August 21 (A) nd 211 (B) ner Blckville, South Crolin. Brs of the sme technology with the sme letter re not significntly different Percent boll dmge (±SEM) cused by bollworm verged cross smpling dte by boll dmge threshold for WideStrike (WS) nd Bollgrd II cotton (BGII) ner Blckville, South Crolin, in 21. Brs of the sme technology with the sme letter re not significntly different. Numbers indicte number of insecticide pplictions received. UTC, untreted control; SWKLY, spryed weekly...41 viii

10 List of Figures (Continued) Figure Pge 1 Percent boll dmge (±SEM) cused by bollworm verged cross smpling dte by boll dmge threshold for non-bt (NBT), Bollgrd II (BGII), nd WideStrike (WS) cotton ner Blckville, South Crolin, in 211. Brs of the sme technology with the sme letter re not significntly different. Numbers indicte number of insecticide pplictions received. UTC, untreted control; SWKLY, spryed weekly Cotton lint yield (±SEM) verged by tretment for 21 nd 211 in non-bt cotton ner Blckville, South Crolin. Brs of the sme yer with the sme letter re not significntly different. Numbers indicte number of insecticide pplictions received. UTC, untreted control; SWKLY, spryed weekly Percent retention t the first position (±SEM) in non-bt cotton from bollworm lrve in blooms test ner Blckville, South Crolin, in 211. UTC, untreted control; SWKLY, spryed weekly. Numbers indicte number of insecticide pplictions the tretment received. Brs with the sme letter re not significntly different Percent retention t the second position (±SEM) in non-bt cotton from bollworm lrve in blooms test ner Blckville, South Crolin, in 211. UTC, untreted control; SWKLY, spryed weekly. Numbers indicte number of insecticide pplictions the tretment received. Brs with the sme letter re not significntly different...48 ix

11 List of Figures (Continued) Figure Pge 14 Men number of first position bolls (±SEM) in non-bt cotton from bollworm boll dmge test ner Blckville, South Crolin, in 211. UTC, untreted control; SWKLY, spryed weekly. Numbers indicte number of insecticide pplictions the tretment received. Brs with the sme letter re not significntly different Percent retention t the first position (±SEM) in non-bt cotton from bollworm boll dmge test ner Blckville, South Crolin, in 211. UTC, untreted control; SWKLY, spryed weekly. Numbers indicte number of insecticide pplictions the tretment received. Brs with the sme letter re not significntly different Men number of second position bolls (±SEM) in non-bt cotton from bollworm boll dmge test ner Blckville, South Crolin, in 211. UTC, untreted control; SWKLY, spryed weekly. Numbers indicte number of insecticide pplictions the tretment received. Brs with the sme letter re not significntly different Percent retention t the second position (±SEM) in non-bt cotton from bollworm boll dmge test ner Blckville, South Crolin, in 211. UTC, untreted control; SWKLY, spryed weekly. Numbers indicte number of insecticide pplictions the tretment received. Brs with the sme letter re not significntly different...53 x

12 INTRODUCTION Historiclly, the bollworm, Helicoverp ze (Boddie), nd tobcco budworm, Heliothis virescens (F.), hve been mjor pests of cotton in the southestern United Sttes. The bollworm/budworm complex ws the most dmging nd costly of ll the cotton insect pests for 13 yers between 1979 nd 1996 (Diffie et l. 24). In 22, the complex ws responsible for reducing cotton yields cross the US by 613 thousnd bles (2.31%) (Willims 23). Until the introduction of geneticlly engineered cotton, the primry mens of controlling lepidoptern pests ws chemicl insecticides. However, resistnce to orgnophosphtes nd pyrethroids during the 199s reduced the effectiveness of chemicl control (Gore nd Admczyk 24). In 1996, Monsnto Corportion (St Louis, MO) ws the first to commercilize geneticlly engineered cotton. Bollgrd cotton expressed Cry1Ac proteins from gene found in the soil bcterium Bcillus thuringiensis kurstki Berliner (Bt). The Bt gene ws introduced into cotton to enble engineered plnts to produce their own insecticidl Cry1Ac endotoxin, thus reducing the need for insecticide pplictions (Perlk et l. 21, Gore nd Admczyk 24). Bollgrd cotton ws found to be highly effective on H. virescens nd modertelyto-highly effective ginst H. ze. In most situtions, nnul pplictions of insecticide remined necessry to prevent yield loss from bollworm becuse the species is less susceptible thn tobcco budworm nd often voids mortlity through lrvl behvior such s feeding on blooms which contin lower levels of the toxin (Gore et l. 23). 1

13 Becuse Cry1Ac is vribly expressed in the cotton plnt, some plnt prts (such s the blooms) hve lower concentrtions of the toxin (Gore nd Admczyk 24). In ddition to differences in titer of toxin by plnt structure, crop mturity lso ffects the level of Cry1Ac expression (Gore et l. 23). Greenplte et l. (1998) found tht expression of the Cry1Ac toxin ws non-uniform throughout the plnt, ws often lower in cotton blooms, nd decresed in squres nd bolls s the growing seson progressed (Greenplte 1999). In diet choice studies, bollworm lrve were ble to discriminte between diet contining Cry1Ac nd untreted diet nd showed preference for the untreted diet (Greenplte et l. 1998). Behvior modifiction, differentil survivl on blooms, nd overll generl reduced susceptibility to Bt proteins were cited s resons why bollworm were ble to survive on Bt cotton (Gore nd Admczyk 24). Action thresholds bsed on the number of eggs, number nd size of lrve, nd on observed boll dmge were refined becuse Bollgrd ws not 1% effective in controlling bollworms (Sullivn et l. 1998). In 23, Monsnto Compny relesed dul-bt gene cotton technology clled Bollgrd II, which produces the originl Bt protein (Cry1Ac) nd second protein (Cry2Ab). Two yers lter, Dow AgroSciences (Indinpolis, IN) relesed WideStrike cotton, which lso produces the originl Bt protein (Cry1Ac) combined with different Bt protein (Cry1F). These dul-gene Bt cotton vrieties provide better control of bollworm thn the originl, single-gene technology, in Bollgrd vrieties (Gore et l. 28). Although dul-bt gene technologies further enhnce control of cterpillrs nd reduce the need for insecticides, Bollgrd II 2

14 nd WideStrike cotton vrieties do not offer 1% control of bollworm (Greene nd Robinson 21) nd continued refinement of tretment thresholds is wrrnted. In lbortory studies conducted by Stewrt et l. (21), the greter toxicity of dul-gene Bt cotton on lepidopterns compred with single-gene Bt cotton ws demonstrted. Survivl nd growth rte were reduced in multiple species, including bollworm, fll rmyworm, Spodopter frugiperd (Smith), nd beet rmyworm, Spodopter exigu (Hübner). Stewrt et l. (21) concluded tht dul-toxin technologies would be more effective nd hve wider rnge of ctivity thn first-genertion Bt cotton. Differentil toxin expression in the plnt between Cry2Ab (Bollgrd II) nd Cry1F (WideStrike) re issues tht exceed the rgument whether two endotoxins re more effective controlling lepidopterns thn one endotoxin. Results from field cge experiments conducted in Mississippi to determine bollworm impct on Bollgrd II nd WideStrike cotton suggested tht bollworm would rrely cuse yield loss in either technology (Gore et l. 28). In study in North Crolin, Bollgrd II showed greter efficcy thn WideStrike or Bollgrd when bollworm pressure ws high (Bcheler et l. 26). Under light or moderte pressure, however, the dul-gene Bt technologies did not differ in bollworm control (Bcheler et l. 26). Greene nd Robinson (21) reported differences between Bollgrd II nd WideStrike in lint yield potentil, sustined boll dmge, nd compenstory bility from trils conducted in South Crolin. Both technologies benefited from supplementl control of bollworm when exposed to high numbers of bollworm (Greene nd Robinson 3

15 21). Becuse bollworms hve the bility to cuse economic dmge, nd neither technology demonstrtes 1% control of the species, ction thresholds my need to be developed specificlly for ech dul-gene Bt technology. Studies compring efficcy between Bollgrd, Bollgrd II, nd WideStrike cotton vrieties under nturlly occurring high pressure hve been conducted (Bcheler et l. 26, Greene nd Robinson 21), s hve trils to investigte injury levels from rtificilly infested dul-gene Bt cotton (Gore et l. 28). However, current threshold recommendtions for dul-bt gene cotton in South Crolin remin similr to thresholds used for bollworm on single-gene Bt technologies, less the egg threshold recommendtion (Greene nd Robinson 21). The objective of this study ws to refine ction thresholds for ech dul-gene Bt technology in order to better enble growers to mnge bollworms. These studies ddress the development of thresholds for bollworm in Bollgrd II nd WideStrike cotton in res prone to historiclly high nturl infesttion by the species. Thresholds bsed on egg density, lrve in blooms, nd percent boll dmge were investigted during the 21 nd 211 growing sesons. 4

16 LITERATURE REVIEW Cotton, Gossypium hirsutum L., is perennil plnt of tropicl origin tht is grown s n nnul crop in the United Sttes. Cotton hs n indeterminte fruiting pttern nd produces more fruit ech seson thn cn be mtured (Guinn 1982). From study in Louisin, only 24-36% of flowers produced during growing seson mtured to hrvestble bolls (Kennedy et l. 1991), nd other studies hve shown higher nd lower rtes of boll production from blooms. First position bolls on sympodil brnches re the most vluble fruits in terms of yield (Gore et l. 2) nd, under idel conditions, the first position sympodil loctions my produce s much s 35% more hrvestble bolls thn sites t or beyond the second position (Jenkins et l. 199, Jones nd Snipes 1999). Cotton s indeterminte growth hbit llows it to withstnd the loss of fruiting structures without significnt reduction in yield. Fruit bscission is nturl occurrence tht brings the fruit lod into blnce with the vilble nutrient, crbohydrte, nd wter supply (Guinn 1982). Fruit cn be bscised due to biotic cuses such s nutrient deficiency, wter stress, temperture, nd mechnicl injury, s well s from biotic cuses such s insects nd pthogens (Guinn 1982). Cotton cn compenste for bscised bolls; however, if the pressure is t high enough level, economic dmge will occur. A wide spectrum of insect pests cn cuse economic dmge nd yield loss in cotton, such s thrips, plnt bugs, stink bugs, flehoppers, nd cterpillrs (Gore et l. 2, Admczyk nd Burris 24). After erdiction of the boll weevil, Anthonomus grndis grndis Bohemn, from the Southest nd before the relese of trnsgenic cotton vrieties contining genes from the bcterium Bcillus thuringiensis (Bt), some of the 5

17 primry pests of the crop in the United Sttes were pink bollworm, Pectinophor gossypiell (Sunders), bollworm, Helicoverp ze (Boddie), nd tobcco budworm, Heliothis virescens (F.) (Gore et l. 2). In the 22 cotton insect losses presented t the Beltwide Cotton Conferences, Willims (23) reported tht the bollworm/budworm complex reduced US cotton yields by 613, 12 bles (2.31%). On cotton, femle bollworm nd tobcco budworm moths deposit their eggs on young leves nd points of growth (Guinn 1982). In generl, bollworm eggs re deposited on the top third of the cotton plnt nd most re concentrted ner plnt terminls (Gore et l. 22). After the eggs htch, lrve move down the plnt nd feed on young tissue, squres, nd bolls nd progress to feed on more mture bolls s they grow. Cterpillr feeding dmge stimultes the plnt to produce ethylene which cn trigger shedding of dmged squres or bolls (Guinn 1982). In 1996, Monsnto Corportion (St. Louis, MO) ws the first to commercilize geneticlly engineered Bt cotton. Bcillus thuringiensis (Bt) is grm-positive soil bcterium tht nturlly produces protein crystl structure during sporultion. Insects tht ingest this crystlline structure solubilize it with proteses of the midgut where the environment is t the right lkline ph level: Solubilized proteins relese δ-endotoxins which then interct with the midgut epithelium. Membrne integrity is compromised in sensitive individuls nd those insects my die from strvtion, prlysis, or septicemi (Gill et l. 1992). Severl Bt genes hve been identified, which code for the production of toxic proteins. The primry structure is dependent on the coding gene nd the specific endotoxin relesed is toxic to different insect groups. The Cry1 or Cry2 proteins re toxic 6

18 to lepidopterns (Gill et l. 1992). Bollgrd cultivrs expressed the Cry1Ac endotoxin which reduced the need for insecticide pplictions for lepidoptern pests such s the tobcco budworm nd bollworm. These Bt proteins were found to be sfe for humn use nd trget-specific to the insect order Lepidopter (Perlk et l. 21, Gore nd Admczyk 24). Trnsgenic Bt cotton hs demonstrted very good control of H. virescens nd P. gossypiell (Willims 2). While Bt cotton is toxic to both the tobcco budworm nd the bollworm, it is more ctive ginst tobcco budworm. Soon fter the introduction of Bt cotton, it ws determined tht bollworms often required supplementl tretment (Lyton et l. 1997). The Cry1Ac Bt toxin suppressed bollworm popultions, but economic injury still occurred under pressure from lrge popultions (Pitts et l.1999, Gore et l. 23, Greene nd Robinson 21). Smith (1997) noted tht bollworm numbers peked twice during the 1996 growing seson in Albm. During mid-to-lte July, the pek ws ttributed to the movement of moths from mturing corn into cotton. In erly September, high survivl of bollworm on cotton ws ttributed to elevted numbers erly in the seson nd lter to loction where eggs were lid on the plnts (Smith 1997). This pttern continues to be present in the Southest. Pheromone trp numbers for bollworm from 27 to 29 in Brnwell County, South Crolin, supported the July/August pek in numbers reported previously (Greene nd Robinson 21). Egg loction on the plnt ffects rtes of lrvl survivl becuse the Bt toxin is not uniformly expressed throughout the plnt (Admczyk et l. 21). Bollworm hs brod 7

19 host rnge nd erly-seson lrve primrily develop on weed hosts (Hed et l. 21). The complex of bollworm nd tobcco budworm hs been reported to feed collectively on over 13 plnt hosts (Diffie et l. 24). Corn nd sorghum re mjor hosts for the complex from mid-june to mid-july, nd movement (clled flights) to hosts such s cotton, soybens, nd penuts occurs lter in the seson (Hed et l. 21). In the Southest, the criticl flight of bollworm moths generlly occurs in mid-july (Sullivn et l. 1993, Smith 1997). Using certin brod-spectrum insecticides just before lrge bollworm flight cn ctully increse crop dmge becuse predceous rthropod popultions re decimted (Turnipseed nd Sullivn 1999). Nturl enemies of bollworm such s lcewings, ldy beetles, geocorids, nd other predceous bugs cn reduce bollworm popultions nd their ssocited crop injury (Lopez et l. 1976, Hutchinson nd Pitre 1983). Pheromone trps re used to monitor moth ctivity, but trp numbers re often poorly ssocited with lrvl densities in the field (Diffie et l. 24) becuse moths re extremely mobile nd the specific crops within loclized re hve little impct on popultions of H. ze (Jckson et l. 23). However, corn my impct the totl popultion on lrger scle. Diffie et l. (24) found significnt correltion between corn crege nd popultions of bollworm (Diffie et l. 24). The wide host rnge nd mobility of H. ze mke it difficult to chrcterize wht fctors in the groecosystem re contributing to popultion numbers (Jckson et l. 23). The expression of Cry1Ac in Bollgrd cotton vried with the structure nd mturity of tht structure (Gore et l. 23). Admczyk et l. (21) used n ELISA test 8

20 to corroborte with erlier studies (Fitt 1998, Holt 1998, Schs et l. 1998, Greenplte 1999) showing tht levels of toxin decresed in mny plnt prts s the seson progressed. The more thn 25 different Bt vrieties expressed dissimilr levels of Cry1Ac δ-endotoxin (Admczyk et l. 21). Cterpillrs tht survived Bt toxins (clled escpes ) from Bollgrd cotton were observed in Albm (Smith 1997) nd were originlly thought to be from extremely high bollworm numbers. However, escpes were lso observed under moderte pressure, nd further investigtion showed tht eggs lid on dried blooms (bloom tgs) led to the incresed rte of survivl (Smith 1997). The bloom tgs did not express lethl mount of Bt toxin, llowing cterpillrs to feed nd increse in size. Although there were no observed preferences for bloom tgs in Bt cotton versus conventionl vrieties (Smith 1997), bollworm lrve were ble to discriminte between diet contining Cry1Ac nd untreted diet nd consequently showed preference for the untreted diet (Greenplte et l. 1998). In field trils conducted by Admczyk et l. (21) in Mississippi, it ws reported tht bollworms were predominntly found feeding on flowers, squres, nd bolls s opposed to meristemtic tissue where Bt protein levels re highest. Bollworm my preferentilly oviposit ner flowers in Bt cotton. Gore et l. (22) found more bollworm feeding on white blooms in Bollgrd cotton versus conventionl, non-bt cotton in Louisin. Tobcco budworms disperse differently on Bt cotton versus conventionl vrieties, with more cterpillrs moving wy from the plnt terminls nd t fster rte on Bt cotton compred with movement on non-bt vrieties (Prker nd Luttrell 1999). Gore et l. (22) found tht bollworm lrve begn to migrte wy from 9

21 Bollgrd terminls within the first hour of eclosion. Lrve of bollworm nd tobcco budworm might detect the Bt proteins nd exhibit n voidnce response. Studies by Greenplte (1999) found decresed levels of Cry1Ac δ-endotoxins in squres nd bolls, s well s incresed feeding by tobcco budworm s the growing seson progressed, supporting the hypothesis tht differences in Bt expression re bsed on structure nd mturity. Bollworm lrve tht feed lower on the plnt nd on older reproductive components re more likely to survive thn those tht feed on fresh tissue such s white blooms. The feeding hbits nd subsequent reduced susceptibility of bollworm to Cry1Ac endotoxins mke H. ze more likely to survive nd dmge Bollgrd cotton (Gore et l. 23). During the first three yers of commercil-use of trnsgenic Bt cotton, dditionl bollworm control, in the form of folir insecticide pplictions, ws required in order to prevent economic injury (Smith 1997; Lyton et l. 1997, 1998; Leonrd et l 1997, 1998; Roof nd Durnt 1997; Gore et l. 2). In survey conducted cross Mississippi in 1997, it ws reported tht trnsgenic cotton ws effective in controlling tobcco budworm, but high popultions of bollworm still hd the cpcity to cuse excessive dmge in some cses (Lyton et l. 1998). Roof nd Durnt (1997) found tht t lest one insecticide ppliction ws required for Bt cotton compred with 4.8 pplictions in conventionl cotton fields in South Crolin. Despite reduced insecticide use, yield increses of 11 nd 23% were observed in Bt cotton treted with dditionl insecticide pplictions in Louisin (Leonrd et l. 1998). Although Bt toxins specificlly trget lepidoptern pests, 1

22 supplementl folir insecticides re lso necessry to control bollworm escpes nd other insect pests (Leonrd et l. 1997, 1998). The trgets of insecticide pplictions in trnsgenic Bt cotton in South Crolin were initilly defined s stink bugs nd bollworm (Roof nd Durnt 1997). Across the Cotton Belt, secondry insects such s stink bugs, plnt bugs, nd rmyworms becme more prominent pests in lte seson Bollgrd cotton (Pitts et l. 1999). Mi et l. (1998) reported tht monitoring eggs on plnts to nticipte feeding dmge from cterpillrs ws no longer useful in trnsgenic cotton becuse the Bt technology should kill newly htched lrve. However, Sullivn et l. (1998) recommended n egg threshold of 75 eggs per 1 plnts becuse smll lrve feeding underneth bloom tgs could survive nd were difficult to detect. At two loctions in South Crolin (one using disruptive insecticides to decimte nturl enemies nd the other left undisturbed), insecticide pplictions using the egg threshold s opposed to the escped worm threshold (8 lrge lrve per 1 plnts) incresed lint yields by 65 nd 93 kg/h (58 nd 83 lb/cre), respectively (Sullivn et l. 1998). Trnsgenic technology nd the use of more selective insecticides hve mde insect pest mngement decisions more complex. Sttic thresholds bsed on the experience of the pest mnger or recommendtions from the locl Coopertive Extension Service do not reflect chnges in production costs, crop prices, or physiologicl susceptibility of cotton vrieties (Mi et l. 1998). Becuse one or two nnul insecticide pplictions my be necessry to prevent economic loss from bollworm, ction thresholds were estblished in most sttes (Gore et 11

23 l. 28). Action threshold recommendtions for bollworm in single-gene Bt technology in South Crolin were: 75 eggs, 3 smll (<6.35 mm [.25 in]) lrve, or 3 lrge (>6.35 mm [.25 in]) lrve per 1 plnts, or 5% boll dmge (Greene 21). However, single- Bt gene technology (Bollgrd) ws no longer commercilly vilble fter the 21 growing seson (Greene nd Robinson 21). Guidelines were developed to prevent or postpone the development of resistnce mong trget insects to Bt cotton. The strtegy ws to combine the plnting of cultivrs with high doses of the toxin with refuge plntings tht contined no toxin (Gould 1998). Mndting refuge plnting of non-bt cotton ws intended to produce susceptible individuls to mte with resistnt dults nd thereby prevent the production of resistnt offspring (Cprio 1994). The high fitness costs relted to Cry1Ac resistnce could, however, dely or inhibit field popultions of bollworm from developing resistnce to Bollgrd cotton (Anilkumr et l. 28). Dul-toxin cultivrs re more toxic nd hve wider rnge of ctivity on lepidoptern pests (Stewrt et l. 21) nd my further dely or inhibit the development of resistnce. In 23, Monsnto relesed dul-toxin Bt cotton clled Bollgrd II tht expresses the originl Cry1Ac protein s well s Cry2Ab. In 25, Dow AgroSciences (Indinpolis, IN) relesed dul-toxin technology clled WideStrike tht expresses Cry1Ac nd Cry1F (Gore et l. 28). Dul-gene technologies provide enhnced control of lepidoptern pests, but do not offer 1% control of bollworm, nd dditionl insecticide might still be needed (Greene nd Robinson 21). 12

24 Lbortory studies conducted by Stewrt et l. (21) clerly demonstrted the greter toxicity of dul-gene Bt cotton on lepidopterns over expression of only single insecticidl protein. In biossys, lrve fed plnt tissues contining both Cry1Ac nd Cry2Ab experienced higher mortlity thn lrve fed on cultivrs contining Cry1Ac (Stewrt et l. 21). In nother study, the dditionl gene in Bollgrd II tht codes for the Cry2Ab protein ws lso found to increse the mortlity of bollworm lrve (Gore et l. 21). The combintion of genes nd toxins ffected the survivl nd growth rte of multiple species, including H. ze, Spodopter frugiperd (Smith), nd Spodopter exigu (Hübner). Dul-toxin technologies re more effective nd hve wider rnge of ctivity thn first genertion Bt cotton (Stewrt et l. 21). Second genertion Bt cotton is generlly considered 1% effective ginst tobcco budworm. It hs lso enhnced protection ginst bollworm compred with single Bt gene vrieties, yet Bollgrd II nd WideStrike still produce yield gins when there re dditionl insecticide pplictions (Greene nd Robinson 21). Bollgrd II nd WideStrike cotton vry in efficcy becuse of the different Cry proteins expressed between technologies. Dt from field-cge experiments conducted in Mississippi suggested tht bollworm would rrely cuse yield loss in either technology (Gore et l. 28). Bcheler et l. (26) indicted tht Bollgrd II hd greter efficcy thn WideStrike or Bollgrd cotton when grown under high bollworm pressure in North Crolin. Dul-Bt gene technologies did not differ in controlling light or moderte infesttions of bollworm (Bcheler et l. 26). 13

25 Wheres lrve tend to migrte wy from terminls in Bollgrd nd Bollgrd II cotton vrieties, bollworm re more often found feeding on terminls in WideStrike vrieties (Jckson et l. 21). Bollworm nd tobcco budworm hve been observed migrting down the plnt nd wy from terminls in Bt cotton vrieties contining the Cry1Ac gene (Prker nd Luttrell 1999, Gore et l. 22). However, Jckson et l. (21) concluded tht the combintion of Cry1Ac nd Cry1F proteins in WideStrike did not hve ny mesurble effect on lrvl movement wy from plnt terminls s compred with lrvl movement on non-bt cotton vriety. Efficcy trils conducted by Greene nd Robinson (21) from 26 to 29 in South Crolin found differences in boll dmge cused by bollworm between Bollgrd II nd WideStrike, nd both technologies benefited from supplementl control when exposed to extreme bollworm pressure. Although greter losses in lint yield were sustined in some WideStrike vrieties thn those incurred in Bollgrd II vrieties, it ws speculted tht the extended optiml growing conditions llowed the full-seson WideStrike vriety to compenste for the reltively erly nd elevted dmge cused by bollworm feeding (Greene nd Robinson 21). According to Pitts et l. (1999), the re of South Crolin where this reserch took plce is in the mngement region, Svnnh River Vlley: Estern Georgi-South Crolin below the lkes nd tht there is no region in the Southest tht hs the intensity or predictbility of bollworm pressure thn this region. Becuse neither technology demonstrtes 1% bollworm control, nd these pests hve the bility to cuse economic dmge, ction thresholds need to be modified specificlly for ech technology. 14

26 MATERIALS AND METHODS Species Determintion Popultions of bollworm nd tobcco budworm were monitored three times per week by counting moths cught in pheromone-bited Hrtstck-type trps (Hrtstck et l. 1979) plced in undisturbed loctions (e.g. ner power poles, etc) round row-crop production fields t the Edisto Reserch nd Eduction Center ner Blckville, South Crolin. Pheromone lures (Luretpe lures, Hercon Environmentl, Phildelphi, PA) for bollworm nd tobcco budworm were replced in ech trp (1 trps for ech species) every week from My to erly October in 21 nd 211. Trpping dt were used to estimte proportions of the two species tht were ovipositing nd feeding ner the loction of the trils conducted in this study. Cterpillrs were collected from non-bt, WideStrike, nd Bollgrd II cotton vrieties on 2, 6, nd 16 August 211, nd lte instrs were identified using dissecting scope bsed on distinguishing chrcter of the mndibles. Tobcco budworms hve tooth-like projection on the inner surfce of the mndibles, wheres bollworms do not hve this projection (Boyer et l. 1977, Ji et l. 27). Becuse erly instrs re difficult to mnipulte nd mndibulr chrcters re indistinguishble under the dissecting scope, erly instrs were kept nd held on rtificil diet until lrge enough to exmine s lte instrs. The combintion of dt from pheromone trps nd the dissections served to determine bundnce of ech species. Voucher specimens of one Helicoverp ze nd one Heliothis virescens lrve were collected 9 August 211from cotton t the Edisto REC ner Blckville, South 15

27 Crolin. Specimens were preserved in 8% ethyl lcohol nd deposited in the Clemson University Arthropod Collection. Overview of Trils Three seprte replicted trils were conducted in 21 nd 211 t the Edisto Reserch nd Eduction Center ner Blckville, South Crolin. Ech tril consisted of non-bt (DP174RF), WideStrike (PHY565WRF), nd Bollgrd II (DP949B2RF) cotton vrieties plnted on 14 My 21 nd 18 My 211. Plots were eight rows by 12.2 m (4 ft) nd tretments were replicted four times using rndomized complete block design. Stndrd cotton production prctices were followed s outlined in the Clemson University Coopertive Extension Service Cotton Production Guide (Jones et l. 211). Acephte (Orthene 97), folir orgnophosphte, ws pplied t 1.9 kg (AI)/h (1 lb/cre) during the first week of bloom to eliminte predceous rthropods nd mximize bollworm pressure. Insecticides ineffective on lepidopterns, but efficcious on hemipterns were pplied twice cross the entire test re ech seson to minimize yield impct. In 21, thimethoxm (Centric 4 WG) ws pplied t.7 kg (AI)/h (2.5 oz/cre) on 22 July nd dicrotophos (Bidrin 8 EC) ws pplied t.56 kg (AI)/h (8 oz/cre) on 9 August. In 211, methyl prthion (Methyl 4 EC) ws pplied t.84 kg (AI)/h (1.5 pt/cre) on 18 July to both control hemiptern popultions nd lso disrupt beneficil rthropods. Dicrotophos (Bidrin 8 EC) ws pplied t.56 kg (AI)/h (8 oz/cre) on 4 August. Plots meeting or exceeding trgeted ction thresholds for bollworm 16

28 (Tble 1) were spryed weekly lternting between bet-cyfluthrin t.23 kg (AI)/h (2.6 oz/cre) nd lmbd-cyhlothrin t.45 kg (AI)/h (5.12 oz/cre). Tble 1. Trget ction thresholds for bollworm eggs, lrve in blooms, nd boll dmge in cotton ner Blckville, South Crolin, in 21 nd 211. Threshold Tretments type Egg density (Test 1) Untreted control Spryed weekly 25 eggs per 75 eggs per (1)* eggs per Lrve in white blooms (Test 2) Boll dmge (Test 3) Untreted control Untreted control Spryed weekly Spryed weekly *Prentheses indicte modified threshold for plnts 4 or 5 lrve per 1 blooms 4 or 5% boll dmge Test 1- Egg Density Threshold plnts 15 lrve per 1 blooms 1% boll dmge 1 plnts 25 lrve per 1 blooms 2% boll dmge Following first bloom, plots were monitored weekly for bollworm eggs. Becuse bollworm eggs re deposited on the top third of the cotton plnt nd most concentrted ner the plnt terminls (Gore et l. 22), egg density ws determined by visully exmining the top 2% of 25 plnts per plot. Plnts smpled were locted in the middle four rows nd wy from the plot edge. Eggs were counted on leves, terminls, preflorl buds (squres), brcts, nd stems. Test 2- Lrve Density Threshold At bloom initition, plots were monitored weekly for cterpillrs by visully exmining 25 blooms (in situ) per plot nd clssifying lrve present s smll, <6.35mm (.25 in), or lrge, >6.35mm (.25 in). Blooms were chosen from the middle four rows nd wy from plot edges. When fewer thn 25 white blooms were observed per plot, the 17

29 numbers of cterpillrs in vilble blooms were extrpolted. If no blooms were present in plot, lrve density ws ssumed to hve reched the highest threshold. Lrve were initilly ctegorized s smll or lrge, but numbers of smll nd lrge cterpillrs were totled per plot for nlysis. Test 3- Boll Dmge Threshold After the first cohort of bolls reched dime size in ll vrieties, pprox mm (.5 in) in dimeter t widest point, plots were exmined weekly by visully exmining 25 bolls (in situ) per plot for bollworm feeding injury. Bolls were chosen from the middle four rows nd wy from plot edges. Bolls were considered dmged when t lest one site on the boll wll ws compromised or penetrted by lepidoptern feeding injury. When there were fewer thn 25 bolls per plot, missing bolls from fruiting positions were considered dmged nd those tretments were considered bove tretment threshold. Plnt Mesurements In 21 nd 211, stnd counts were tken to monitor stnd uniformity nd verify tht plot yield would not be impcted by non-uniform stnds. During 21, numbers of plnts in one meter of row were counted in four loctions in ech plot (4 m totl). In 211, totl number of plnts in rows four nd five were counted (ech row being 12.2 m). Nodes bove white flower (NAWF) counts were tken three times ech seson to ssess plnt mturity nd determine physiologicl cutout, indicting mturing crop nd lst cohort of hrvestble bolls (Bernhrdt et l. 1986). In the Southest, 18

30 physiologicl cutout is generlly thought to hve occurred when plnts verge five or fewer nodes bove the highest first position white flower (Bernhrdt et l. 1986). Before the 211 hrvest, plnt mpping ws done in response to dt from 21 tht suggested significnt yield compenstion in response to bollworm injury occurred. Five plnts per plot were mesured, exmined, nd mpped to look for compenstory growth behvior. All bolls were counted nd considered hrvestble, worm-dmged, unhrvestble, or bscised. Node nd brnch position were lso noted. Following plnt mpping, cotton ws mechniclly hrvested nd plot yields were clculted ssuming 4% lint turnout. Sttisticl Anlysis Dt for ech test were subjected to two-wy repeted mesures nlysis of vrince with dte nd tretment threshold s fixed effects nd repliction s rndom effect (PROC MIXED, SAS Institute Inc. 211). Dt filing the Shpiro-Wilkes test for norml distribution were trnsformed prior to ANOVA. Egg dt were trnsformed using log(x+1), lrve dt were trnsformed using (x+1), nd boll dmge dt trnsformed using rcsin (proportion of dmge). Tukey men seprtion tests were lso performed using SAS 9.3 (SAS Institute Inc. 211). Node bove white flower dt were subjected to one-wy repeted mesures nlysis of vrince with dte s fixed effect (SAS Institute Inc. 211). 19

31 RESULTS AND DISCUSSION Overview Species Determintion Eggs were estimted to be primrily those of bollworm nd not tobcco budworm bsed on pheromone moth trp dt (Figure 1) nd cterpillr collection nd identifiction dt. Moth popultions peked in lte August for both species in 21 nd 211. However, bollworm nd tobcco budworm pek numbers were lower in 211 thn in 21. Fctors such s overwintering conditions for pupe, nd other sesonl vrition my lrgely hve ccounted for this difference. The months of December preceding the 21 nd 211 cotton sesons were mrkedly different in temperture nd precipittion. In December 29, the verge minimum temperture ws 2.5 C (36.4 F), with n extreme low of -3.5 C (25.7 F). The following December hd sustined period of cold tempertures, with verge lows of -2.8 C (27. F) nd n extreme low of -9.9 C (14.2 F). December 29 ws both wrmer nd wetter with recorded rinfll of 24.6 cm (9.7 in) compred to December 21 nd 6.22 cm (2.45 in) of recorded rinfll (Ntionl Climtic Dt Center). Soil conditions impct bollworm survivl becuse bollworm pupe overwinter in the soil. The hrsher winter conditions in December 21 my hve been significnt fctor in reducing the overwintering popultion of bollworm thus reducing the numbers found on cotton during the 211 growing seson. 2

32 Figure 1. Numbers of bollworm (BW) nd tobcco budworm (TBW) dults cught in pheromone trps bited weekly ner Bl Blckville, South Crolin, in 21 (A) nd 211 (B). 21

33 Of the cterpillrs collected (31) in 211 from Bt cotton vrieties, ll were bollworm. Only 2 of the 7 cterpillrs found in non-bt cotton were tobcco budworm. It ws expected tht tobcco budworm lrve would not be found on dul-gene Bt cotton becuse Bt endotoxins exhibit complete field control of tobcco budworm (Stewrt et l. 21). Tobcco budworm represented only bout 3% of the two-species when considering only dt from non-bt cotton. The pheromone trp dt lso showed lrger number of bollworm dults compred with tobcco budworm dults (Figure 1). All eggs, lrve, nd plnt injury counted were therefore presumed predominntly from bollworm. Stnd Counts The recommended plnt stnd for cotton in South Crolin is plnts per row meter (2-3 plnts per row foot) (Jones et l. 211). Stnd verges per technology in 21 were s follows: non-bt, 9.28 ±.1; WideStrike, 9.1 ±.11; nd Bollgrd II, 8.85 ±.12 plnts per row meter. In 211 non-bt, 6.5 ±.1; WideStrike, 6.8 ±.11; nd Bollgrd II, 6.4 ±.14 plnts per row meter. Stnds in 211 were thinner thn in 21; however, differences within cotton technologies were not significnt (P >.5). Lint yield differences within ech technology were therefore likely ttributble to bollworm feeding dmge nd vrible levels of insecticide protection rther thn to vritions in stnd density. Crop Mturity (NAWF Counts) Although comprble mturing vrieties were chosen for these trils, mesurements were tken to detect potentil mturity differences due to multiple fctors 22

34 including insect injury. Unprotected cotton my experience delyed mturity s resources re diverted to vegettive growth becuse of insect dmge. In 21, some plots hd cterpillr feeding dmge so severe tht first position white flowers were scrce. Mny NAWF dt in plots of non-bt cotton could not be determined due to the high level of dmge nd bsence of blooms. Becuse of high dmge nd missing dt in non-bt cotton in 21, verge NAWF clcultions re unrelible mesures of plnt mturity. Node bove white flower trends should decrese over the seson s cotton plnts mture (Gore et l. 2). In 211, cotton mturtion ws observed over the three dtes in ll vrieties (Tble 2). 23

35 Tble 2. Node bove white flower counts (±SEM) nd sttisticl comprisons for 21 nd 211 by technology, verged cross threshold nd test in cotton ner Blckville, South Crolin, 21 nd 211. Yer Dte Cotton technology Non-Bt WideStrike Bollgrd II July 4.25 ±.31 A 4.33 ±.15 A 3.97 ±.12 A 5 August 4.76 ±.57 A 4.22 ±.15 A 3.65 ±.12 A 12 August 4.54 ±.28 A 3.9 ±.15 A 3.9 ±.12 B F.49 b 2.38 c d P>F < July 7.1 ±.11 A 6.91 ±.12 A 6.88 ±.11 A 29 July 4.6 ±.11 B 4.65 ±.12 B 4.38 ±.11 B 15 Aug 2.52 ±.11 C 2.49 ±.12 C 2.14 ±.11 C F e f g P>F <.1 <.1 <.1 NAWF counts in the sme column nd yer with different letter re significntly different b df= 2, 46 c df= 2, 155 d df= 2, 136 e df= 2, 175 f df= 2, 174 g df= 2,

36 Tble 3. Sttisticl comprisons of bollworm egg nd lrvl densities nd boll dmge in cotton ner Blckville, South Crolin, 21 nd 211. Yer Mngement fctor combintion Egg density Lrve density Boll dmge df F P > F df F P > F df F P > F 21 NBT Threshold NBT Dte NBT Threshold*Dte 4, , , < , , , < , , , <.1.31 WS Threshold WS Dte WS Threshold*Dte 4, , , < , , 68 24, <.1 <.1 <.1 4, , , BGII Threshold BGII Dte BGII Threshold*Dte 4, , 71 32, < , , , , , 5.6 2, NBT Threshold NBT Dte NBT Threshold*Dte 4, , , < , , , , , 45 16, WS Threshold WS Dte WS Threshold*Dte 4, , , < , , , < , 2.8 4, , BGII Threshold BGII Dte BGII Threshold*Dte 4, , 6.1 2, < , 2.4 4, 42 16, < , 32 4, ,

37 Tble 4. Sttisticl comprisons of cotton lint yield ner Blckville, South Crolin, 21 nd 211. Yer Mngement fctor Egg density test Lrve density test Boll dmge test df F P > F df F P > F df F P > F 21 NBT Threshold WS Threshold BGII Threshold 4, 15 4, 14 4, < , 14 4, 14 4, < , 15 4, 14 4, < NBT Threshold WS Threshold BGII Threshold 4, 14 4, 14 4, , 14 4, 14 4, , 15 4, 15 4,

38 Egg Density Threshold In 21, the highest threshold of 125 eggs per 1 plnts ws never reched in ny of the vrieties (Figure 2A). The threshold of 75 eggs per 1 plnts ws met or exceeded three times in WideStrike nd twice in Bollgrd II. The non-bt control never reched 75 eggs per 1 plnts, most likely becuse it suffered high cterpillr feeding dmge. Bollworm egg density is not good predictor of future dmge in dul-bt gene cotton becuse lrge number of the lrve do not survive. However, most lrve from eggs on conventionl cotton do survive nd feed on the cotton plnt until puption. Lower egg numbers on non-bt cotton were likely the result of diminished florl cues (Cllhn 1958), incresed plnt voltiles, reduced lef re nd fruiting structures, or combintion of ll which likely discourged femles from ovipositing fter initil infesttion nd dmge. Egg numbers peked in ll three cotton vrieties on 21 July (Figure 2A). There ws second, smller pek between 11 nd 18 August. Peks were similr to those of dult moth numbers in 21 (Figure 1A). In Albm, Smith (1997) ttributed pek in bollworm numbers during mid-to-lte July to moth movement from mturing corn into cotton. The pttern observed during the current study (Figures 1) nd in Albm (Smith 1997) hs been consistent over the pst severl yers in the Brnwell County re of South Crolin (Greene nd Robinson 21). In 21, egg densities were not significntly ffected by threshold nor ws there n interction between threshold nd dte for ech cotton technology. The lck of significnt tretment effect ws probbly becuse the insecticide hd little ovicidl effect 27

39 nd did not deter femle moths from ovipositing. For these resons, ppliction decisions were bsed on egg density numbers verged cross ech vriety insted of verged within threshold. Insecticides were not considered to hve hd significnt effect on the number of eggs on the plnts one week fter ppliction. 28

40 (A) Men bollworm eggs per 1 plnts (B) Smpling Dte Figure 2. Men bollworm eggs per 1 plnts (±SEM) from egg thresholds by cotton technology ner Blckville, South Crolin, in 21 (A) nd 211 (B). 29

41 In 211, overll egg numbers were lower thn in the previous yer (Figure 2B). Hot nd dry conditions lso cused plnts to mture fster nd shortened the smpling period. Despite lowering the highest egg density threshold from 125 to 1 eggs per 1 plnts, the lowered threshold ws not reched. Furthermore, the 75 eggs per 1 plnts threshold ws not met in ny vriety during 211. At this sme loction in 21, Jenkins et l. (22) lso filed to rech their bollworm thresholds of 75 eggs per 1 plnts or four lrve per 1 plnts. They concluded tht bollworm ws not problem on Bollgrd II cotton. A similr conclusion could be drwn from the observtions of the current study for both Bollgrd II nd WideStrike. However, bollworm pressure ws considered moderte during 1997 experiment in Blckville, where the 75 eggs per 1 plnts threshold ws met (Sullivn et l. 1998). The moderte pressure in 1997 led to lint yield increses in first genertion Bollgrd cotton when treted t the 75 egg per 1 plnt threshold. Bollworm pressure vries gretly from one loction to the next nd even in the sme loction from yer to yer. Egg density peked on 25 July nd 8 August in 211. The timing of the peks ws similr to those of 21; however, in 21, the lrger pek in egg density occurred in July, with smller pek in August. There were no differences in lint yield (kg/h) between egg threshold tretments within WideStrike nd Bollgrd technologies in 21 or 211. These results suggested tht supplementl insecticide for bollworm bsed on egg density thresholds ws unnecessry in WideStrike nd Bollgrd II. The non-bt control did experience significnt yield gins in both yers (Figures 3) when treted weekly nd following the 3

42 ggressive egg threshold (25 eggs per 1 plnts) when compred with the untreted control nd higher egg thresholds (75 nd 125/1 eggs per 1 plnts) which were not spryed for bollworms ll seson (Figures 3). 31

43 (A) Lint (kg/h) (B) b 8 7 UTC SWKLY 25/1 75/1 125/1 b 6 4 UTC SWKLY 25/1 75/1 1/1 b b b b Tretment threshold (eggs/1 plnts) Figure 3. Cotton lint yields from (±SEM) compring bollworm egg threshold tretments in non-bt cotton ner Blckville, South Crolin, in 21 (A) nd 211 (B). Brs with the sme letter re not significntly different. Numbers indicte number of insecticide pplictions received. UTC, untreted control; SWKLY, spryed weekly. 32

44 Lrve Density Threshold In 21 nd 211, lrvl density ws significntly ffected by dte in ll three cotton technologies (Figure 4). Bollworm density peks lgged slightly behind peks in egg density (Figure 2) nd fell between the first nd second peks found from the pheromone trp dt (Figure 1). Lrvl densities were significntly ffected by the interction of threshold nd dte in non-bt cotton both yers, s well s in WideStrike cotton in 21 (Figure 5). Weekly pplictions of insecticide in WideStrike cotton were effective in mintining low lrvl densities wheres greter vribility ws observed in other lrvl thresholds (Figure 5). The interction suggests tht the timing of insecticide ppliction my be importnt. The end of July hd high lrvl pressure nd cotton my benefit from control t this time in prticulr. Insecticide pplictions hd negtive impct on lrvl density (Figure 6). Differences in lrvl densities correlted with WideStrike lint yield in 21; lower lrvl density in the plots spryed weekly correlted with higher lint yields in these plots thn in the untreted plots (Figure 7). Insecticide pplictions bsed on tretment thresholds did not impct yield in dul Bt-gene cotton in 21. In 211, there ws no significnt difference in yield in ny of the three technologies (Tble 4). Gore et l. (28) hd similr results from field cge experiment conducted in Mississippi. White blooms of Bollgrd II nd WideStrike were infested with bollworm lrve t, 5, nd 1%. Bollworm infesttion hd little impct on yield of Bollgrd II or WideStrike except when 1% of white flowers were infested for t lest one week (Gore et l. 33