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

Ellsworth/UA 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.

Ellsworth/UA 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.

Ellsworth/UA 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

Ellsworth/UA 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

Ellsworth/UA 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

Ellsworth/UA 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

Ellsworth/UA 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 Coker 312 Recurrent back-crossing

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

Ellsworth/UA 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

Ellsworth/UA 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

Ellsworth/UA Secondary Lepidopteran Pests Occasional pests Induced pests Helicoverpa zea Heliothis virescens Estigmene acrea (Arctiidae) Trichoplusia ni Spodoptera exigua Bucculatrix thurberiella

Ellsworth/UA Bt Cotton Questions 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) 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

Ellsworth/UA BG Cotton Efficacy Young larvae present regardless of cotton type Little difference between Bt & non-Bt (-) varieties

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

Ellsworth/UA 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

Ellsworth/UA 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

Ellsworth/UA 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 RR SS RR RSSS

Ellsworth/UA 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 (?)

Ellsworth/UA 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

Ellsworth/UA 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 C312B DP50 DP50B (Cry1Ac) DP50 Cry1AcCry2Ab DP50II (Cry1AC+Cry2Ab) Particle gun Lines 1.Cry1Ac+ Cry2Ab 2.Cry1Ac only 3.Cry2Ab only 4.Null

Ellsworth/UA Isoline Studies of BG & BGII Replicated studies Artificial & natural PBW infestations Sprayed & Unsprayed conditions

Ellsworth/UA Dead 1st instar in Bt cotton Warts are often formed at the site of PBW attack

Ellsworth/UA BGII Results - PBW, 1st Instars Dead 1st Instars Live 1st Instars

Ellsworth/UA BGII Results - PBW, All Instars

Ellsworth/UA 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

Ellsworth/UA 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’)

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

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

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

Ellsworth/UA Source of Survivors Low expression of Bt in plants? Low levels of non-Bt contaminants? –In the seedbag –From volunteer seed Resistance?

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

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

Ellsworth/UA 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.

Ellsworth/UA 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.

Ellsworth/UA Information All University of Arizona crop production & crop protection information is available on our web site, Arizona Crop Information Site (ACIS), at ACISACIS