INCREASED PLANT PROTEIN, INSECT MORTALITY, AND YIELD WITH CHAPERONE TM

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1 Summries of Arknss Cotton Reserch 2003 INCREASED PLANT PROTEIN, INSECT MORTALITY, AND YIELD WITH CHAPERONE TM D.M. Oosterhuis nd RS. Brown 1 RESEARCH PROBLEM The plnt growth regultor Chperone TM hs been reported to increse plnt nitrogen levels, promote protein constituent trnsport, nd increse overll yields. Field nd growth- chmber studies were conducted in 2002 nd 2003 to quntify 1) the effect of folir pplictions of Chperone on protein nd endotoxin levels of cotton (Gossypium hirsutum L.) leves nd squres, nd 2) the subsequent effect on bollworm mortlity nd yield. BACKGROUND INFORMATION Chperone TM is new protein enhncer tht ws registered by the Environmentl Protection Agency in 2000 nd the ptent is pending. Chperone is combintion of nitrophenols, nmely sodium 5-nitroguicolte, sodium o- nitrophenolte nd sodium p-nitrophenolte. Phenolics ply centrl role in plnt metbolism, e.g., incresed photosynthetic electron trnsport, improved membrne integrity, incresed enzyme/protein production, incresed lignin bio-synthesis, nd incresed fruit retention nd growth (Robinson nd Trevor, 1980). Observnces in trnsgenic cottons hve shown tht endotoxin levels hve occsionlly filed to be fully expressed under vrious conditions, including environmentl fctors nd vrietl differences, thus occsionlly leding to less efficient insect control nd subsequent yield losses. Cotton plnts engineered to express the endotoxin protein, Cry1Ac, from Bcillus thurengiensis (Bt) hve shown significnt declines in efficcy ginst Helicoverp spp. during the seson, prticulrly from flowering onwrds (Fitt et l., 1998). Thompson et l. (1976) reported tht there ws less totl protein in the leves of older plnts s result of three- to five-fold reduction in protein synthesis over the seson. Furthermore, Olsen nd Dly (2000) concluded tht not only is there less Bt protein in older plnts, it ppers tht the protein is either less vilble or less toxic to neontes. The concentrtion of Cry1Ac protein, s proportion of totl protein, lso declines during the seson (Holt 1998). The phenolic properties of Chperone my id in trnsgenic cotton by lleviting non-expression or under-expression of CryIAc or combintion of CryIAc with Cry2Ab, the genes utilized for expression of the endotoxin protein, B. thuringiensis. 1 Distinguished professor nd grdute ssistnt, respectively, Deprtment of Crop, Soil, nd Environmentl Sciences, Fyetteville. 101

2 AAES Reserch Series 521 RESEARCH DESCRIPTION Field nd growth-chmber studies were conducted in 2001, 2002, nd 2003 to quntify t 1)he effect of folir pplictions of Chperone on protein nd endotoxin levels of cotton leves nd squres, nd 2) the subsequent effect on bollworm mortlity nd yield. Growth Chmber Study Cotton (Gossypium hirsutum L.) cv. DP 33B ws plnted in Mrch 2002 t the Altheimer Lbortory, University of Arknss, into 2 L pots contining soilless horticulturl mix. The growth chmber ws set for 12-h photoperiod, with dy/night tempertures of 30 o F/25 o C nd reltive humidities of 60 to 80%. Plnts were rrnged in completely rndomized design with three replictions. All pots received hlf-strength Hoglnd s nutrient solution dily to mintin dequte nutrients nd wter. Chperone tretments were pplied s folir spry with CO 2 bckpck spryer clibrted to deliver 10 gllons H 2 O/cre. The djuvnt, Penetrtor Plus t 0.05% v/v ws used. Chperone tretments were spryed t the seventh true lef nd the upper expnded min-stem lef ws smpled 10 dys lter. Tretments were spryed gin t the seventh true lef +10 dys nd leves nd squres smpled 5 dys lter. Smpled tissues were plced in ziploc bgs nd immeditely tken to the University of Arknss Entomology Deprtment for bollworm mortlity testing. Bollworm mortlity ws ssessed by plcing single one-dy old neonte bollworms on lef sections in individul plstic cups with grose in n incubtor t 26 o C. Mortlity rtes were ssessed t 24, 48, 72, nd 96 hours from the initition of feeding for smples collected following the first spry ppliction nd ssessed t 72 nd 96 hours following the strt of feeding for smples tken fter the second spry ppliction. In 2001 nd 2002, min-stem leves long with the ccompnying petioles nd first-position squres were smpled four min-stem nodes from the terminl t 5 nd 10 dys fter Chperone ppliction, plced immeditely on dry ice, nd shipped to the Agdi testing fcility to be nlyzed for Bt endotoxin levels. Field Studies Cotton (Gossypium hirsutum L.) cultivr Suregrow 215Bt/RR ws plnted in erly My 2002 nd 2003 in Cptin silt lom t Clrkedle in northest Arknss, nd in Fyetteville in northwest Arknss. The design ws rndomized complete block with three replictions. Fertilizer, pesticides, nd irrigtion prctices were ccording to current extension recommendtions. Tretments consisted of n untreted control nd single folir pplictions of Chperone t first flower (FF) pplied t 2.5, 5, 10, nd 20 oz /cre, nd two pplictions of Chperone t mthed squre (MHS) nd FF t 2.5, 5, nd 10 oz/cre in In 2003, Chperone ws 102

3 Summries of Arknss Cotton Reserch 2003 pplied t rte of 5 oz/cre t MHS nd FF. Yields were determined by mechniclly hrvesting the middle two rows of ech four-row plot nd components of yield nd fiber qulity were determined from two-meter smple from ech plot. The methods for testing for neonte mortlity nd the times for testing for mortlity were the sme s in the growth-chmber studies. Mesurements were mde t select times ech seson nd included lef nd squre protein concentrtions nlyzed t the University of Arknss (Brdford, 1976), lef nd squre endotoxin concentrtion (conducted by Agdi), insect mortlity (University of Arknss Deprtment of Entomology), nd yield nd yield components. RESULTS AND DISCUSSION Effect of Chperone on Cotton Yield Folir ppliction of Chperone in field trils t two loctions in Arknss for three yers incresed lint yields by n verge of 61 lb/cre (Fig. 1). This yield increse ws ssocited with incresed plnt protein levels (Fig. 2). Incresed yields hve lso been reported for three yers from numerous consultnt field trils cross the US Cotton Belt (Lckey et l., 2004). Effect of Chperone on Protein Content of Leves nd Squres In 2002, in the field study t Clrkedle, folir ppliction of Chperone t first flower cused n increse (47.7%) in lef protein content (Fig. 2). Similrly, in 2003, there ws numericl but not significnt (P<0.05) increse in lef protein from Chperone pplictions in Fyetteville (+2.1%) nd Clrkedle (+7.7%) compred to the untreted control. Effect of Chperone on Endotoxin Levels in Leves nd Squres In the 2001 field study t Clrkedle, Chperone cused significnt increse in endotoxin levels in leves, petioles, nd squres, prticulrly t the higher concentrtions of Chperone (Fig.2). Similrly, in the 2003 field study in Clrkedle there ws trend for Chperone to increse endotoxin levels of the squres (dt not shown). The increse in endotoxin ws ssocited with the enhnced protein levels observed in growth-chmber nd field studies. It hs been observed tht reduction in the mount of expressed endotoxin protein occurs s plnts mture leding to loss of efficcy in the ltter stges of the growing seson nd thus incresing the probbility of surviving pests which my develop immunity to the endotoxin protein (Greenplte, 1999; Benbrook nd Hnsen, 1997). Chperone ppers to be vible mens for enhncing endotoxin levels nd thereby improving insect mortlity. 103

4 AAES Reserch Series 521 Effect of Chperone on Bollworm Mortlity on Leves nd Squres ( Field Studies) Increses in bollworm mortlity were recorded in the growth-chmber study in Fyetteville in 2002 (Fig. 4). These results indicted tht ll Chperone tretments resulted in higher bollworm mortlity compred to the untreted control, nd lso tht mortlity incresed with incresing rtes of Chperone. Likewise, field studies in Arknss hve confirmed increses in bollworm mortlity following pplictions of Chperone, prticulrly worms feeding on squres (dt not shown). PRACTICAL APPLICATION Dt from the growth-chmber nd field studies in 2002 nd 2003 show tht folir pplictions of Chperone my be vible mens for enhncing lint yields in cotton through the enhncement of plnt protein levels. Furthermore, in trnsgenic (Bt) cultivrs the enhnced protein sttus contributes to improved lteseson endotoxin levels, prticulrly in the squres, resulting in incresed mortlity of neonte bollworms feeding on the treted plnts. LITERATURE CITED Brdford, M.M A rpid nd sensitive method for the quntifiction of microgrm quntities of protein utilizing the principle of protein-dye binding. Anlyticl Biochemistry 72: Benbrook, M. nd M. Hnsen Return to the Stone Age of Pest Mngement. In: Proceedings of Plnt Pesticide Resistnce Mngement, A Public Meeting, Mrch 21, 1997, Wshington. D.C. Kennedy, G.G., nd B.S. Turner Emerging Technologies for Integrted Pest Mngement _ Proceedings, Mrch 8-10, 1999 Rleigh, N.C.: APS Press. Dly, J.C. nd G.P. Fitt Efficcy of Bt Cotton Plnts in Austrli Wht is Going On? Pp In: Gilhm (ed.). New Frontiers in Cotton Reserch. Proceedings World Cotton Reserch Conference-2, Athens, Greece, Sept. 6-12, Published P. Petridis, Thessloniki, Greece. Fitt, G.P., J.C. Dly, C.L. Mres, nd K. Olsen Chnging efficcy of trnsgenic cotton plnt ptterns nd consequences. Sixth Austrlin Entomologicl Reserch Conference, Brisbne, Austrli. Greenplte, J.T Quntifiction of Bcillus thuringiensis Insect Control Protein CryIAc Over Time in BOLLGARD Cotton Fruit nd Terminls. 104

5 Summries of Arknss Cotton Reserch 2003 Journl of Economic Entomology. 92: Holt, H Seson-long monitoring of trnsgenic cotton plnts- development of n ssy for the quntifiction of Bcillus thuringiensis insecticidl protein. Pp , In: Proceedings 9th Austrlin Cotton Grower s Reserch Assocition, Queenslnd, Wee W, Austrli. Lckey, J.H. nd D.M. Oosterhuis, Chperone yield results from the Tri- Stte Delt. In: Proceedings, Beltwide Cotton Conferences. Sn Antonio, Tex., Jn 6-9, Ntionl Cotton Council, Memphis, Tenn. CD-ROM. Olsen, K.M., nd J.C. Dly, Plnt-toxin interctions in trnsgenic Bt cotton nd their effects on mortlity of helicoverp rmiger (lepidopter: Noctuide). Entomology Society Americ 93: Robinson, T The Orgnic Constituents of Higher Plnts, 4th Edition. Cordus Press, N. Amherst, Mss. Thompson, A.C., H.C Lne, J.W. Jones, nd J.D. Hesketh Nitrogen concentrtion of cotton leves, buds, nd bolls in reltion to ge nd nitrogen fertiliztion. Agronomy Journl 68:

6 AAES Reserch Series 521 Lint yield (lb/cre) Control 5 oz/cre 500 Yer 01 Yer 02 Yer 03 Fig. 1. Effect of Chperone on lint yield verged cross loctions in Arknss, Totl Protein (ug/ml) % Neonte Mortlity Leves Squres UTC - Bt Chperone - 5 oz Chperone - 10 oz Tretments Fig. 2. Effects of Chperone on totl soluble protein in leves t two loctions in Ark.,

7 Summries of Arknss Cotton Reserch 2003 % Increse Control Bt 5 oz/cre 10 oz/cre 20 oz/cre Tretment Lef Petiole Squre Fig. 3. Percentge increse in endotoxin level, bove the control, following Chperone pplictions t 5, 10 nd 20 oz/cre. Lef Protein (ug/ml) UTC Chperone 5 MHS UTC 2485 Chperone 5 MHS 1631 UTC 33 o C 36 o C 39 o C Tretment 2092 Chperone 5 MHS Fig. 4. Effects of Chperone on neonte mortlity in leves nd squres. Field study, Clrkedle, Ark.,