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DELAYING THE DEVELOPMENT OF RESISTANCE TO BT CORN IN THE POPULATION OF ASIAN CORN BORER , OSTRINIA FURNACALIS Project Leaders:
Belen Morallo-Rejesus, Ph. D. (Nov. 2005 – Feb. 2007) Pio A. Javier Ph. D. (March 2007 – April 2008)

Project Duration
Date Started: November 2, 2005 Date Completed: April 30, 2008

Period covered by this report
November 1, 2005 – April 30, 2008

Project Funding
Amount approved for the three years= PhP 3,258,754.48 Amount released in first year = PhP 1,034,881.18 Amount approved for the second year = PhP 300,000.00

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INTRODUCTION
Approval of the commercialization of transgenic Bt-corn in the Philippines in December 2002 introduces a new technology to control ACB in an ecologically benign way.  Transgenic crops significantly increase the efficacy of the Cry toxins compared to Bt sprays, thus, are more attractive to use than the conventional Bt formulations.  Since the Cry protein engineered into corn is basically the same as those found in natural Bt organisms, and Bt toxins are expressed in high amounts throughout the growing season, the risk of resistance development is more likely to occur.

INTRODUCTION  Several species of insects including ACB and the European corn borer, were reported to developed resistance to Bt corn.  Baseline data on the susceptibility of ACB poplns in some areas where BT corn were grown in the country and the rate of resistance devt to Bt corn of ACB poplns. from Bt corn growing areas, are therefore important in designing proper resistance management tactics adapted to Philippine conditions.

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INTRODUCTION
Transgenic Bt plants typically provide continuous protection against damage from susceptible insect species, thereby, increasing the potential of insect selection for resistance and the probability of negative effects on natural enemies.

• Very few studies have been reported on the effect of Bt corn plant on predators that were fed with Bt-fed preys.

General Objectives:
1. Survey the occurrence of resistant ACB poplns in selected growing areas (Bt corn planted for 2 years)

2. Determine the rate of development of resistance to Cry1Ab of ACB popl’ns in the laboratory
3. Determine the effect of Bt protein on biology of earwigs fed with ACB larvae reared on artificial diet, ACB treated with Bt-protein and dog food. 4. Determine the delaying effect of augmentative releases of predatory earwigs on the increase of ACB population resistant to Bt corn in the field.

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Study 1. Survey of Resistance Development and Rate of Development of Resistance to Bt-corn in Asian Corn Borer (ACB) Population Study Leaders: Dr. Barbara L. Caoili - Nov. 2005 – Oct. 2006
Dr. Pio A. Javier - Nov. 2006 – April 2008

Study 2. Effect of Bt Corn on the Biology of Predatory Earwig, Euborellia annulipes and on the Development of Resistant Asian Corn Borer Population Study Leaders:
Dr. Belen Morallo-Rejesus - Nov. 2005 – Feb. 2007 Dr. Pio A. Javier - March 2007 – April 2008

Study 1. Survey of Resistance Devt and Rate of Devt of Resistance to Bt corn in ACB Population.

STUDY OBJECTIVES Objectives: 1. Determine the susceptibility of ACB popln from Bt corn growing areas to Cry1-Ab and establish the LC99 for monitoring resistance. 2. Determine the rate of devt of resistance of ACB popln from Bt corn growing areas by selection in the laboratory. % Accomplishment

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METHODOLOGY:

1. Determined planting history in each site - interview
2. Collected ACB larvae & pupae (250 from each site):  Bt-corn areas: - Alcala, and Santa Maria (Pangasinan) - Candon City (Ilocos Sur)  Non-Bt corn areas: - San Felix (Sto. Tomas, Batangas) - BIOTECH and CES, UPLB (College, Laguna) - Tranca (Bay, Laguna) - Bunggo (Calamba City) - Sto Tomas (Calauan, Laguna) 3. ACB reared in artificial diet 4. Larvae used for bioassay/maintaining culture.

METHODOLOGY………………. Bioassay studies using lyophilized non-Bt and Bt-corn leaves While sourcing purified Cry1Ab protein from other institution, lyophilized Bt-CL (25-day old ) were used. Lyophilization of Bt and non-Bt leaves done at BIOTECH, UPLB. Cry1Ab protein concentration (freeze-dried sample) estimated through ELISA , at BIOTECH, U.P. Los Baños. Cry1Ab antibody and ELISA reagents by Drs. V. Exconde and T. M. Espino.

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METHODOLOGY……….. Estimation LC values for Cry1Ab on ACB neonates
 Stock solution 1 mg/ml Cry1Ab prepared in 0.1M CAPS

buffer, pH 11.2, stored in a -20ºC freezer until use.  Diet-incorporation bioassay  Initial test used 4 to 5 concentrations diluted into 18 μL artificial diet.  Control group given an artificial diet with 0.1M CAPS buffer.  Treatments replicated 3X; 10 larvae/rep.  Mortality (moribund & dead) observed after 7 days.  Data analyzed with PriProbit ver. 1.63 program.

Accomplishment & Major Findings - Study 1
 ACB larvae reared in the laboratory for several generations were tested on lyophilized Bt corn leaf, exposed on a detached Bt corn leaf in the laboratory and on the Bt-corn potted plants in the screen house.  Use of powdered lyophilized Bt-corn leaves (laborious) may offer an alternative method in the estimation of LC values in the event that purified Cry1Ab protein is unavailable. The Cry1Ab content in the powdered lyophilized Bt-corn leaves was estimated to be 1.25 ng/mg leaves by ELISA.

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Accomplishment and Major Findings- Study 1
Table 1. Percent mortality of ACB neonates treated with Cry1Ab protein.

POPULATION San Felix, Sto Tomas, Batangas Tranca, Bay, Laguna

CONC’N (ng/uL) 0.10- 2.5 0.10- 2.5

% MORTALITY 17.20ab 14.70c

Bungo, Calamba, Laguna Sto Tomas, Calauan, Laguna Pili Drive, CES, UPLB BIOTECH, UPLB
Auburn University, Alabama, USA.

0.10- 2.5 0.10- 2.5 0.10- 2.5 0.10- 2.5

16.88bc 18.80ab 19.50a 19.60a

 Purified Cry1Ab protein obtained from Dr. William Moar, Professor,
 All populations of ACB neonates showed comparable susceptibility against Cry1Ab toxin except Tranca, population.

Accomplishment and Major Findings- Study 1
Table 2. Percent weight reduction on retarded growth of ACB neonates treated with Cry1Ab protein1,2. POPULATION CONC’N (ng/uL) % WEIGHT REDUCTION1,2

San Felix, Sto Tomas, Batangas
Tranca, Bay, Laguna Bungo, Calamba, Laguna Sto Tomas, Calauan, Laguna Pili Drive, CES, UPLB  Significantly highest weight reduction in Sto Tomas, Batangas, and lowest in Calamba & Calauan Lag.
1Weight

0.10- 2.5
0.10- 2.5 0.10- 2.5 0.10- 2.5 0.10- 2.5
A- Retarded larva. B- normal larva

85.88a
82.56ab 72.28c 77bc 82.72ab

reduction was based only on larvae treated with 0.10 to 1.0 ng/uL (amount on Bt-corn leaves) larvae are dead at 2.5 ng/uL treatment.

2All

A

B

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Table 2. Susceptibility of the ACB neonates collected from non-Bt corn field to Cry1Ab protein by surface contamination method.
LC(ng/cm2) POPULATION LC25
0.034 0.089 0.060 0.059 0.020 0.124

Fiducial Limits
0.004 - 0.084 0.019 – 0.174 0.012 – 0.122 0.005 – 0.147 0.002 – 0.057 0.038 – 0.216

LC50
0.138 0.356 0.216 0.216 0.091 0.298

Fiducial Limits
0.044-0.238 0.189-0.569 0.099 – 0.339 0.058 – 0.385 0.021 – 0.177 0.152 – 0.436

LC99
6.191 41.33 17.46 19.39 17.28 6.191

Fiducial Limits
3.045-29.75 9.86 – 1,684 5.35 - 321 6.43 – 377.92 5.28 – 310.33 3.045 – 29.75

San Felix, Sto Tomas, Batangas Tranca, Bay, Laguna Bungo, Calamba, Lag. Sto Tomas, Calauan, Lag Pili Drive, CES, UPLB BIOTECH, UPLB
1Based

on five trials; 30 insects per replicate, replicated 3X (n=150) was done after 7 days. The lower the LC values the more susceptible. The higher the LC values the more tolerant
2Observation

Table 3. Susceptibility of the ACB neonates collected from Bt corn growing areas to Cry1Ab protein by surface contamination method. MEAN MORTALITY POPULATION Asingan, Pangasinan Sta Barbara, Pangasinan Sta Maria, Pangasinan Treated with LC50 14.20 19.2 15.6 Treated with LC99 26.20 28 27.6 LC LC50 0.258 0.180 0.35 LC99 7.08 6.63 6.73 RESISTANCE RATIO 1.145 1.071 1.07

LC50 = 0.216 ng/cm2

LC99 = 6.191 ng/cm2

Resistance ratio = __LC99 population_______ LC99 susceptible population RR = 1.0 – 1.9 = susceptible = 2.0 - 9.9 = tolerant = 10 - 20 = moderately resistant > 20 = resistant

 Based on resistance ratio, all populations collected from three sites in Pangasinan are still susceptible to Cry1Ab toxin.

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Study 2. Effect of Bt corn on the Biology of Predatory Earwig, Euborellia annulipes and on the Development of Resistant Asian Corn Borer Population Study Leaders:
Dr. Belen Morallo- Rejesus Pio A. Javier Project Staff: Evangeline G. Punzalan - University Res. Associate I: Jonathan Dechitan - Laborer

Objectives Specific Study Objectives

% Accomplishment

1. Determine the effect of feeding ACB larvae with Bt corn leaf, Bt corn leaf extracts and Bt corn potted plants.
2. Determine the LD50 & LD99 of Cry1AB on 3rd instar ACB larvae 3. Determine the effect of feeding on tassel w/ pollen & decaying Bt corn leaves/organic matter (additional objectives after the inception meeting). 4. Determine predatory consumption of earwig feed w/ ACB larvae reared on artificial diet only & those fed w/ ACB larvae imbibed w/ Bt-protein & from those fed w/ dog food-corn cob diet. 5. Determine the effect of integrating earwigs & Bt corn in delaying development of resistant ACB popln (budget was not released). 6. To determine popl’n of natural enemies in Bt corn plants & ordinary hybrid corn with & without earwig releases. 7. Determine effect of Cry1Ab protein on the life cycle, fecundity, egg viability, survival, sex ratio & longevity of earwigs fed w/ ACB larvae reared on artificial diet, fed w/ ACB larvae imbibed w/ Bt-protein and from those feed with dog food-corn cob diet .

100
100 100

100

30 30 100

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METHODOLOGY-STUDY 2 Effect of feeding ACB larvae with Bt- CL and Bt corn leaf extracts (Bt-LE)  ACB reared on artificial diet (Ceballo); predatory earwigs on dog food-corn cob combination (Punzalan & Moralllo-Rejesus, 2004).  Indirectly, portion of corn leaf (MON 818) that contained the highest Bt conc’n was determined by allowing the 3rd instar larvae to feed on the detached lower, middle and tip portion.  Third instar larva individually imbibed with the Bt-LE from the lower, middle and tip portion by droplet method. Tests replicated 3X, 10 larvae/rep, mortality recorded at 24 and 48 hours.  Another expt., five larval instars of ACB were allowed to feed separately on detached Bt-CL (on top of moistened tissue) placed inside a pan. Test replicated 3X , 10 larvae/rep. Mortality noted at 24, 48, 72, 96 and 120 hrs.

METHODOLOGY- STUDY 2 Screen house Neonate 2nd and 3rd instar larvae were allowed to feed on Bt corn plant enclosed in Mylar cage (Fig. 2). Larvae were placed on the leaf. Mortality and behavior of larvae noted at 12 and 48 hrs, thereafter every 24 hrs for 3 days, a total of 7 days observation.

Fig. 2. Set-up on the feeding of ACB larvae on Bt corn plants.

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METHODOLOGY – STUDY 2 Predatory capacity  Four nymphal instars of earwigs starved for 48 hrs were introduced with different ACB larval instars 24, 48, and 72 hours.  One earwig released/acrylic pan and fed with varying prey densities of 150 neonates, 100 2nd instar, 60, 40, and 20 for the 3rd, 4th, 5th instars ACB larvae, respectively. Same number of prey was given every six hours.  Predatory consumption of the female earwigs were separately done from the males, expt. replicated 3X.  Number of preys killed/consumed was recorded at 6, 12, 18, 24, 48 and 72 hours.

METHODOLOGY – STUDY 2 Insect rearing
Mass rearing of ACB and Predatory earwigs, Euborellia annullipes

Treatments:
Prey A = earwigs fed with ACB larvae imbibed with LD25 (300 ng) Prey B = earwigs fed with ACB larvae imbibed with LD50 (600 ng) Prey C = earwigs fed w/ ACB larvae imbibed w/ pure Bt corn leaf extracts Prey D = earwigs fed with ACB larvae imbibed 1,250ng (amount of Bt protein expressed in Bt corn leaf obtained by ELISA) Prey E = earwigs fed with untreated ACB larvae (control) Prey F = earwigs fed with DFC diet (control) P1 earwigs were reared until adult stage using artificial diet (dog food & corn cob combination) & soil MC was maintained at 27 to 30%.

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METHODOLOGY – STUDY 2
 Bt & non- Bt corn (IPB 911) tassel & leaves were collected from pots planted with corn in the screen house.  Materials were wet, stored in a covered plastic container for 15 days.  200 g each were placed inside an acrylic pan & one pair of 3rd nymphal instar earwig was released & allowed to feed on it .  20 containers/replicate, replicated 3X (60 pairs/medium)  Artificial diet served as the control.  % survivors computed and compared from the control.
B Fig 3. A- Bt corn tassel with pollen & decaying Bt leaves. A

METHODOLOGY – STUDY 2

Fig. 4. Different varieties of corn planted at CES, UPLB.  Field expt. was set-up July – Oct. 2006 in an area of 5,000 sq. m. at CES, divided into 9 blocks with 6 m. border between each block.  Mon 818 (Bt corn), IPB 911 and IPB supersweet (Asukar) were planted/block at a planting distance of 75 cm between rows and 50 cm between hills.

 Expt. was arranged RCBD with three reps ( 250 sq. m/ block), with 1,500 plants.

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METHODOLOGY – STUDY 2
Earwigs and other NE’s monitored at pre-vegetative, whorl & tassel stages by putting traps and by sweepings Initial collection done 5 days after setting up the traps, thereafter every day for 7 days. Collected earwigs/block were brought to the lab, fed w/ dog foodcorncob diet & then returned in each block from where it was collected & were deducted from the 250 earwigs that were released/ block. Earwigs collected at harvest/ block were pooled according to variety & brought to the laboratory for the determination of their biological differences.

B A

A

B

C

D

Fig. 5. Trapping of predatory earwigs: A- plastic container containing dog food & surrounded w/ corn cob, B- covered with corn husk, C- collection of earwig in the corn husk, D- collection of earwig in the plastic container).

ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2
Table 4. The lethal time (LT50) that caused 50% mortality of the third instar ACB larvae fed w/ the different portions Bt corn leaf & imbibed with its leaf extract1.

Parts of the leaf
Lower Middle Tip
1Ten

LT50 (Hours) Imbibed (pure) 48.95 31.27 11.68 Feed 80.35 63.42 23.12

insects per replicate; replicated 3X (n=90)

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ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2

Table 5. The lethal time (LT50) that caused death of different ACB larval instars fed with whole Bt –corn leaf kept inside an acrylic pan.

Larval instar Neonate (1st instar < 24 hr) 2nd instar 3rd instar 4th instar

LT50 (Hours) 20.35 50.87 85.08 191.27

5th instar
1Ten

260.31

larvae per replicate; replicated 3X (n=90)

ACCOMPLISHMENT AND MAJOR FINDINGS - STUDY 2
200

Average Number of Preys Consumed by the Earwig

150

1st instar nymph 2nd instar nymph 3rd instar nymph 4th instar nymph

100

Adult Male Adult Female

0

50

6h

12h eggmass

18h

24h

6h

12h 1st instar

18h

24h

6h

12h 2nd instar

18h

24h

6h

12h 3rd instar

18h

24h

6h

12h 4th instar

18h

24h

6h

12h 5th instar

18h

24h

-50

Stages of Preys (ACB)

Fig. 6. Average number of the different stages of ACB consumed by the different stages of predatory earwigs in 24 hour.

All the nymphal instars & adult earwigs prefer to fed on early instar prey especially neonates Due to the bigger size of 4th & 5th instar ACB larval preys, adult earwigs have difficulty in paralyzing the larvae through their forceps. Some of the adult earwigs that persist on preying had their notal membrane split, consequently resulting to their death.

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ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2
120 100
Percent Survivor

Bt corn (MON 818) Non-Bt corn (IPB 911) Artificial Diet (Dog Food-Corn Cob mixture)
Percent Survivor

120 100 80 60 40 20 0

Bt corn (MON 818) Non-Bt corn (IPB 911) Artificial Diet (Dog Food-Corn Cob mixture)

80 60 40 20 0 3 6 9 12 15 18 21 Days of Counting 24 27 30

3

6

9

12 15 18 21 Days of Counting

24

27

30

Fig. 7. % earwig survivor when fed with A – Bt corn tassel & B –Bt corn decaying eaves.  No difference in % earwig survivor when the predator was fed with both Bt & non-Bt corn tassel w/ pollen & decaying leaves for 30 days (Fig. 7).  Tassel, pollen and decaying Bt corn leaves were not detrimental to the earwigs indicating that the continuous planting of Bt corn will not reduce the popln of predatory earwig in the field.

ACCOMPLISHMENT & MAJOR FINDINGS - STUDY 2 Predatory earwigs fed with Prey A, B, C, & D had an average of three egg batches per female with 80 to 90 eggs per batch, which is comparable with Prey E (control) (Table 7).  No differences on the life cycle of earwigs was observed when the predator was fed with Cry1Ab-treated and -untreated ACB larvae but feeding them with Prey F showed a slightly l longer life span ( Table 8).

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ACCOMP-LISHMENT AND MAJOR FINDINGS - STUDY 2
Table 7. Fecundity and percent survival of predatory earwigs fed with Cry1Abtreated third instar ACB larvae. Parameters No. of egg batches laid per female Ave. no. of eggs laid per batch Percent hatchability Percent survival 1st to 2nd instar Percent survival 2nd to 3rd instar Percent survival 3rd to 4th instar Percent survival 4th instar to adult Sex Ratio F:M PREYS A 3 90 100 85 81 81 78 3:2 B 3 85 100 84 80 82 78 3:1 C 3 80 100 78 88 84 71 3:2 D 3 84 100 82 85 88 73 3:1 E 3 85 100 84 87 87 78 3:1 F 4 110 100 100 97 92 95 3:2

ACCOMPLISHMENT AND MAJOR FINDINGS - STUDY 2 Table 8. Comparative life cycle1 (days) of predatory earwigs fed with Cry1Ab treated 3rd instar ACB larvae. Stages/Period Eggs Incubation Nymphal Stages First Instar Second Instar Third Instar Fourth Instar Total Adult emergence to first egg laying Adult longevity Egg DIETS A 7 7 7 9 8 31 10 63
101

B 7 7 7 9 8 31 10 65

C 7 7 7 9 8 31 10 63

D 7 7 7 9 8 31 10 65

E 7 7 7 9 8 31 10 65

F 8 5 7 8 8 28 10 72

103

101

103

103

108

1Based on 90 females (5 trials); Prey A - ACB larvae imbibed with LD25 (300 ng); Prey B - ACB larvae imbibed with LD50 (600 ng); Prey C - ACB larvae imbibed with pure Bt corn leaf extracts ; Prey D - ACB larvae imbibed 1,250 ng (amount of Bt protein expressed in Bt corn leaf obtained by ELISA) ; Prey E untreated ACB larvae (control); Prey F – Dog food –corn cob combination diet (control)o death of an adult

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 Abundance of earwig in Bt corn MON 818 was comparable to

hybrid and Asukar variety (Table 8). Table 8. Mean number of predatory earwigs collected from the trap 1 installedd in the cornfield (July – October2006; CES, UPLB).

VARIETY

EARLY WHORL (30-35 DAP) 20.66 27.67 23.67

LATE WHORL (36-40 DAP) 3.33 3.67 3.67

TASSEL STAGE (46 – 50 DAP) 2 1 0.67

Bt corn (MON 818) Hybrid corn (IPB-911) Green corn (Asukar)

1Based

on 50 plants/replication; 25 plants in X-pattern were examined.

ACCOMPLISHMENT AND MAJOR FINDINGS - STUDY 2

Bt corn was totally free from ACB damage throughout the duration of the expt (32 to 60 DAP) while slight damage was noted in IPB 911. In contrast, 16.67% damage was recorded in supersweet corn at 32 DAP andgradually increased towards maturity. An ETL of 25% was reached at 53 DAP and slightly increased at 32 DAP.
Table 9. Mean percent Ostrinia furnacalis infestations on three corn varieties (July – October 2006; CES, UPLB).

VARIETY/ HYBRID Bt corn (MON 818) Hybrid (IPB 911)

PERCENT DAMAGED PLANT1 32 DAP 0.00 8.00 39 DAP 0.00 13.33 46 DAP 0.00 13.33 53 DAP 0.00 16.00 60 DAP 0.00 16.00

Supersweet corn (Asukar)
1Based

16.67

20.67

23.33

26.67

31.33

on 50 plants/replication; 25 plants in X-pattern were examined.

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SUMMARY AND CONCLUSION
 The Cry1ab content of the powdered lyophilized Bt corn leaves was estimated to be 1.25 ng/mg leaves by ELISA.  Collected ACB populations from non-Bt corn growing areas, Calauan and Calamba populations were the most susceptible while Tranca was the least.  The established baseline concentration (LC50) was 0.216 ng/cm2 while the established discriminating concentrations (LC99) was 6.191 ng/cm2.  Based on resistance ratio, all populations collected from three sites in Pangasinan are still susceptible to Cry1Ab, although these sites are near the Monsanto Bt corn planting area. Based on the life cycle, percent survival and fecundity, earwigs preyed with Cry1Ab-treated and –untreated ACB larvae showed no differences. In effect, Cry1Ab is not detrimental to earwigs.

RECOMMENDATION
 Yearly

monitoring on the resistance development of ACB from Bt corn growing areas.

 Intensive study on the determination of the rate of development of resistance of ACB population from Bt corn growing areas should be undertaken.  More field experiments should be conducted to determine the effect of integrating earwigs and Bt corn in delaying the development of resistant ACB population.

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ACKNOWLEDGEMENT

The authors gratefully acknowledge the Department of Agriculture-Bureau of Agricultural Research (DABAR) for funding the project.

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