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Transgenic Cotton for Insect Control

Peter C. Ellsworth, Ph.D.IPM Specialist, University of ArizonaMaricopa Agricultural CenterMaricopa, AZ, USA

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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.

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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.

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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

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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

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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

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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

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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

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Spectrum of Activity for BGExcellent Control

No Control

Heliothis virescens

Pectinophora gossypiella

Helicoverpa zea(pre-bloom)

Bucculatrix thurberiella

Spodoptera exigua

Estigmene acrea

Trichoplusia ni

Spodoptera frugiperdaSpodoptera 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.

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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

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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

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Secondary Lepidopteran Pests• Occasional pests

• Induced pests

Helicoverpa zea

Heliothis virescens

Estigmene acrea (Arctiidae)Trichoplusia ni

Spodoptera exigua Bucculatrix thurberiella

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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 growingNo, some not marketed> 98% (?)

No unintended effects

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1

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4

E

19-Sep 12-Oct

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100

Infe

sted

Bol

ls (%

)

- BG - BG

19-Sep 12-Oct

- BG - BG

19-Sep 12-Oct

- BG - BG

19-Sep 12-Oct

- BG - BG

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

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19-Sep 12-Oct

0102030405060708090

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Infe

sted

Bol

ls (%

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- BG - BG

19-Sep 12-Oct

- BG - BG

19-Sep 12-Oct

- BG - BG

19-Sep 12-Oct

- BG - BG

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

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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

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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

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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 RR RR SS

RRRS SS

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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.

YesYes?

Yes

Yes, *refuges

No (?)

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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

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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

Cry1Ac Cry2Ab

DP50II(Cry1AC+Cry2Ab)

Particle gun Lines1. Cry1Ac+

Cry2Ab

2. Cry1Ac only

3. Cry2Ab only

4. Null

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Isoline Studies of BG & BGII• Replicated studies• Artificial & natural

PBW infestations• Sprayed & Unsprayed

conditions

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Dead 1st instar in Bt cotton

Warts are often formed at the site of PBW attack

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BGII Results - PBW, 1st Instars

50 50B 50BII0

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50 50B 50BII0

1

2

Pink

Bol

lwor

m p

er b

oll 1st live

1st dead2nd live2nd dead

3rd live3rd dead

4th live4th dead

PupaeExits

Dead 1st Instars

Live 1st Instars

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0

1

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50 50B 50BII0

1

2

Pink

Bol

lwor

m p

er b

oll 1st live

1st dead2nd live2nd dead

3rd live3rd dead

4th live4th dead

PupaeExits

BGII Results - PBW, All Instars

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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

DP50BII DP50B DP500

10

20

30

40

50

CLP

Min

es p

er le

af

Bottom Middle Top

a

bc

a

bc

a

b b

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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’)

(-) (-) B BII 50 50B 50B 50X 50BII0

2

4

6

8

10Citrus Peel Miner Presence (per 10 plants)

NL Sa

bb

a a

b b

cc

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Spectrum of Activity for BG (Cry1Ac)

Excellent Control

No Control

Heliothis virescensPectinophora gossypiella

Helicoverpa zea

Bucculatrix thurberiella

Spodoptera exigua

Estigmene acreaTrichoplusia ni

Spodoptera frugiperdaSpodoptera ornithogalli

Pseudoplusia includens

Agrotis & Feltia spp.

Beneficial Insects

Marmara spp.

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Spectrum of Activity for BGII (Cry1Ac + Cry2Ab)

Excellent Control

No Control

Heliothis virescensPectinophora gossypiella

Helicoverpa zea

Bucculatrix thurberiella

Spodoptera exigua

Estigmene acreaTrichoplusia ni

Spodoptera frugiperdaSpodoptera ornithogalli

Pseudoplusia includens

Agrotis & Feltia spp.

Beneficial Insects

Marmara spp.

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High Dose and % Efficacy?• Throughout our early work with BG cotton, we

often would find low levels of “survivors” from our field plots

12-Aug 31-Aug 11-Oct

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Infe

sted

Bol

ls (%

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- BG - BG - BG1st

2nd

3rd

4th

Exits

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Source of Survivors• Low expression of Bt in

plants?• Low levels of non-Bt

contaminants?– In the seedbag– From volunteer seed

• Resistance?

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% Efficacy Against PBWCry1Ac Cry2Ab Variety

+ - DP50B- + 985X+ + 985BX+ - DP33B+ - DP448B+ - DP458BR+ + DP33BX+ - SG215BR- + SG125X+ + SG125BX

Raw*100

99.59199.324100

99.78899.536100100

99.256100

Adjusted100

99.591100100100100100100

99.758100

Cry1Ac 100%

Cry2Ab 99.67%

Both Genes 100%

Before plants are tested for presence of Bt

After PBW from non-Bt plants are discarded

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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.

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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.

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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.

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Information• 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

ACIS