Transgenic Cotton for Insect Control

1. Transgenic Cotton for Insect Control Peter C. Ellsworth, Ph.D. IPM Specialist, University of Arizona Maricopa Agricultural Center Maricopa, AZ, USA

2. Disclosure • Those engaged in the dialog on biotechnology should fully disclose their relationships and opinions “up front” so that audiences can consider the context. • Partial support for my research comes from companies with interests in biotechnology. • The balance of support comes from state and federal sources of competitively available public funds.

3. Disclosure (continued) • Biotechnology and its products are neither inherently good nor bad. • The specific process and each of its products should be scientifically and independently evaluated.

4. Transgenic Cotton for Insect Control • What is available now & in the future? • Origin, identity & development • Insect target(s) in the U.S. • Efficacy & utility in the Arizona system (benefits) • Safety (risks) • Resistance • Impact of gene on plant • Biodiversity • non-target effects

5. Products Available for Cotton Insect Control • Only 1 ‘trans’-gene has been commercialized • Based on the crystalline protein produced by Bacillus thuringiensis (Bt) • Developed by Monsanto as Bollgard® and incorporated into commercial varieties by several cotton seed companies (e.g., Delta Pineland Co. & Stoneville Pedigreed Seed Co.) • Sold in the U.S., Australia, Mexico, South Africa, India, China, Argentina, Indonesia

6. Bacillus thuringiensis (Bt) • Common soil bacterium • Present in nature in a variety of forms (species & strains) • Produces proteins that are toxic to insects • Commonly used in garden sprays & for commercial agriculture, including organic farming • Extremely well-known toxin in terms of human health & environmental safety

7. Bacillus thuringiensis (Bt) • Crystalline proteins are classified according to structure & have a specific nomenclature (e.g., Cry1Ac) • Cotton has been transformed with Cry1Ac (narrow spectrum; Lepidoptera only) • Protein binds with receptors in the insect gut causing pores which perforate the midgut & lead to cell leakage & insect death

8. Coker 312 The Transformation • The gene of interest is spliced out of the bacterium using a vector, like Agrobacterium tumefasciens, & transferred to cotton cells grown in tissue culture • The cells are grown into a plant & then, after testing, plants are back-crossed into commercial lines to make new varieties Recurrent back-crossing

9. Excellent Control No Control Spectrum of Activity for BG Tobacco Budworm, the principal pest in the South Trichoplusia ni Spodoptera exigua Heliothis virescens Spodoptera frugiperda Spodoptera ornithogalli Pectinophora gossypiella Bucculatrix thurberiella Pink Bollworm (PBW), our principal pest Beneficial Insects Estigmene acrea Helicoverpa zea (pre-bloom) Agrotis & Feltia spp. Helicoverpa zea (post-bloom) Pseudoplusia includens Marmara spp.

10. AZ’s Primary Lepidopteran Pest • Pink Bollworm • Multiple generations • Adult lays eggs on bolls or susceptible squares(SS) • Larvae hatch & penetrate bolls within 24 hrs

11. Alternatives for PBW Control • Repeated, broad-spectrum sprays are required to prevent moths from invading fields • No effective larvicides or ovicides • Biological controls are limited by the biology of this pest • Little impact of parasitoid or predators • Cultural controls can be very effective • Requires early termination & areawide compliance with plowdown requirements

12. Secondary Lepidopteran Pests • Helicoverpa zea • Heliothis virescens • Occasional pests • Induced pests • Trichoplusia ni • Estigmene acrea(Arctiidae) • Bucculatrix thurberiella • Spodoptera exigua

13. Bt Cotton Questions • Ca. 100% for PBW • Not for PBW • Search for large larvae • No unintended effects • Yes, actively growing • No, some not marketed • > 98% (?) • No unintended effects • Efficacy & economic studies • How effective is the gene? • Are oversprays required for lepidopteran control? • If so, are there new scouting & threshold considerations? • Agronomic studies • Impacts (+/-) on yield & fiber qualities? • Product integrity & stability studies • High-dose through life of plant? • High-dose in all varieties? • Purity? • Ecological studies • Impact on non-target organisms (NTO)

14. BG Cotton Efficacy • Young larvae present regardless of cotton type • Little difference between Bt & non-Bt (-) varieties

15. BG Cotton Kills Small Larvae • PBW larvae must feed in order to be killed. • Large larvae survive mainly in non-Bt varieties.

16. Impact on Arizona Cotton • In 1990, > 6.8 sprays were made against PBW; still, > 5% yield loss • Since 1996 when Bt cotton was introduced, it has never required oversprays for PBW control, AND • Since 1997, only 0.5 sprays have been made against PBW over all cotton acreage (Bt and non-Bt); i.e., an areawide reduction of PBW has occurred • The net reduction in insecticide use has resulted in huge savings to farmers, and large improvements to the agroecosystem in terms of beneficial insect communities & IPM

17. Safety - Resistance • Given time & exposure, insects have the capacity to overcome most insecticides. Bt cotton may be no different, however, there are safeguards: • Refugia • High-Dose Strategy • Development of additional proteins

18. RR RR RR SS RS SS RR Refugia • Objective: provide harborage for susceptible moth production to reduce the chance of resistant (R) moths mating with each other • U.S. growers are required to plant a proportion of their acreage to non-Bt cotton • 5% Refuge, if no lepidopteran-active insecticides are used on it, or else • 20% Refuge

19. High-Dose Strategy, Depends on: • The production of a dose high enough to kill: >99.9% of a susceptible (SS) population, and >95% of the heterozygous (RS) individuals, • A recessive resistance, • Random mating, • A low initial frequency of the ‘R’ allele. • Yes • Yes? • Yes • Yes, *refuges • No (?)

20. Development of Additional Transgenes (Bt’s) • Bollgard II® • 2 Bt gene product, original Bollgard (Cry1Ac) + Cry2Ab • Final stages of US-EPA approval • Limited commercial production in 2003 • Full replacement of BG varieties by 2008? • Bollgard III • Little information on this available at this time; research stages only • Cry1F • Under development by Dow Agrosciences in combination with Cry1Ac

21. C312B Particle gun DP50B (Cry1Ac) DP50II (Cry1AC+Cry2Ab) DP50 • Lines • Cry1Ac+ Cry2Ab • Cry1Ac only • Cry2Ab only • Null DP50 Cry1Ac Cry2Ab Impact of Gene on Plant • Isogenic lines were developed for testing the impact of the gene(s) on agronomic and efficacy characteristics of the plant

22. Isoline Studies of BG & BGII • Replicated studies • Artificial & natural PBW infestations • Sprayed & Unsprayed conditions

23. Warts are often formed at the site of PBW attack Dead 1st instar in Bt cotton

24. BGII Results - PBW, 1st Instars Dead 1st Instars Live 1st Instars

25. BGII Results - PBW, All Instars

26. BGII Results - B. thurberiella • BGII prevented cotton leafperforator development better than BG • Leaves at top of plant (younger) express highest doses of Bt • Older leaves (bottom) have reduced doses of Bt

27. Marmara sp. • Citrus Peel Miner is an incidental lepidopteran that mines the main stem and boll surfaces • Cry2Ab alone (‘X’) is more effective than Cry1Ac (‘B’)

28. Excellent Control No Control Pectinophora gossypiella Beneficial Insects Heliothis virescens Bucculatrix thurberiella Agrotis & Feltia spp. Marmara spp. Helicoverpa zea Estigmene acrea Trichoplusia ni Pseudoplusia includens Spodoptera exigua Spodoptera frugiperda Spodoptera ornithogalli Spectrum of Activity for BG(Cry1Ac)

29. Excellent Control No Control Pectinophora gossypiella Beneficial Insects Heliothis virescens Bucculatrix thurberiella Agrotis & Feltia spp. Marmara spp. Helicoverpa zea Estigmene acrea Trichoplusia ni Pseudoplusia includens Spodoptera exigua Spodoptera frugiperda Spodoptera ornithogalli Spectrum of Activity for BGII(Cry1Ac + Cry2Ab)

30. High Dose and % Efficacy? • Throughout our early work with BG cotton, we often would find low levels of “survivors” from our field plots

31. Source of Survivors • Low expression of Bt in plants? • Low levels of non-Bt contaminants? • In the seedbag • From volunteer seed • Resistance?

32. Before plants are tested for presence of Bt After PBW from non-Bt plants are discarded % Efficacy Against PBW Cry1Ac 100% Cry2Ab 99.67% Both Genes 100%

33. Biodiversity / NTO Studies • The reports of Bt effects on Monarch butterflies have fueled much emotional debate on the use of biotech crops. • Monarch Butterfly, symbol of nature and “wildness” in North America.

34. Non-Target Organisms (NTO) • Over 370 arthropod species have been tracked in 2 years of field studies using a variety of methodologies. • So far, no major or functional differences have been found in Arizona between BG, BGII, and conventional cotton communities… • Except where harsh PBW sprays are needed in conventional cottons. • Thus, Bt cotton ecosystems are not only safe, but safer than conventional cotton ecosystems where insecticidal inputs are higher.

35. Conclusions • The use of Bt cottons in Arizona has provided the first larvicidal and selective approach to controlling PBW. • The control provided by Bt cottons approaches immunity. No survivors have been found in field studies. • Bt cotton has revolutionized our ability to implement IPM in AZ cotton & reduced our insecticide inputs by over 60%. • Future transgenic products for insect control in cotton should be independently & scientifically tested. • Other than new Bt genes/events, there are few, if any, development plans for insect contol products.

36. Information ACIS • All University of Arizona crop production & crop protection information is available on our web site, • Arizona Crop Information Site (ACIS), at • http://ag.arizona.edu/crops