Production of Spore-δ-entotoxin of Bacillus thuringiensis strains and studies on cross-resistance of Cry1Ac and Bt-based biopesticide resistant Helicoverpa armigera

摘要

The present study elaborates on the results of cross-resistance of Cry1Ac and Bt-based biopesticide resistant Helicoverpa armigera (Hübner) to various Bt strains (HD-1, HD-4, HD-8, HD-57, HD-133, HD-234, HD-263, HD-282, HD-551,HD-744 and Bt-based biopesticide. In this.study, all the bioassays were done on larvae of 55th, 60th and 61st Cry1Ac and 58th, 64th, and 65th Bt-based biopesticide selected generations of H. armigera. The resistance ratios (RR) among Cry1Ac and Bt-based biopesticide selected generations varied from 417 fold in 46th generation to 172 fold in 55th generation of Cry1Ac selection and from 21 fold in 58th generation to 15 fold in 67 generation of Bt-based biopesticide selection.Cry1Ac selection exhibited minimum resistance ratio (2.722, 2.756) against HD-1and HD-8 among all the HD strains tested and the slopes for HD-1 and HD-8were highly significant (1.49 and 1.31 respectively). The test insect also showed low resistance ratios 3.959 and 4.385 for HD-551 and HD-57 respectively with highly significant slopes 1.18 and 2.36 accordingly. The spore-crystal toxins of strains of HD-1, HD-8 and HD-263 and Bt-based biopesticide caused significant reduction in larval wt.(％)with regressions y=0.011x+93.912, R2=0.7269, df=4,p＜0.05, and y=0.03x+85.332, R2=0.6982, df=4, p＜0.05, y=0.0458x+79.58,R2=0.7271, df=4,p＜0.05, and y=0.0776x+59.169, R2=0.8273, df=4, p＜0.05,respectively against Cry1Ac selection. Significant slopes 1.49, 1.31, 2.36, 1.67,1.18, 1.12 and 2.09 were observed for HD-1, HD-8, HD-57, HD-263, HD-551,HD-744 and Bt-based biopesticide respectively which indicated fast interaction between spore-crystal toxins of Bts and the Cry1Ac resistant insect. Bt-based biopesticide selection of H.armigera showed low resistance ratios (1.12, 1.10, 1.82,1.95,2.09, and 1.55 for spore-crystal toxins of HD-1, HD-8, HD-133, HD-263, HD-282,and HD-744 respectively). The linear regressions between larval wt. Reduction(％) and toxin concentrations were significant for HD-263, HD744 and Bt-based biopesticide with equations y= 0.0937x+82.891, R2 =0.7832, df=2, p＜0.05, y=0.0421x+92.930 R2=0.8869, df=4, p＜0.05,and y=01163x+44.382, R2=0.7975, df=2, p＜0.05) respectively. Previous studies conducted by other workers indicated synergistic impact between spores and crystals of Bt strains against resistance insects. Our results suggest that the spore-crystal complex (perhaps due to synergistic interaction between or among the spore and crystal components) may have caused synergism due to which a Cry1Ac selection was knocked down and spore-crystal complex-based formulations of Bts can be developed for use as an effective tool to manage insect populations that have development resistance to single toxin (Cry1Ac) produced in transgenic crops.

目录

英文文摘

论文说明：Glossary of terms and abbreviations

Acknowledgements

Introduction

Review of literature

Evaluation of resistance of different insect strains to various proteins of δ-endotoxin of B. thuringiensis

History, distribution, status as pest and economic importance of Helicoverpa (Heliothis) armigera (Huebn.)

History of Bbacillus thuringiensis Berliner

Mode of action of Bacillus thuringiensis

Insecticidal activity of B. thuringiensis

Materials and Methods

Procurement of research materials

Methodology used in raising Cry1Ac and Bt-based biopesticide resistant selections of H. armigera

Insect selection for developing Bt-based biopesticide resistant H. armigera:

Insect selection for developing Bt-based biopesticide resistant H. armigera:

Laboratory scale production of Spore-δ-endotoxin of Bacillus thuringiensis strains and bioassays

Basic growth medium (BGM) for production of spore-δ- Endotoxin

Preparation of solid BGM for production of Bacillus thuringiensis spore-δ-endotoxin in petri-plates

Inoculation of BGM in conical flask

Centrifugation

Freeze - Drying

General Staining/Dyeing of Bt protein crystal

Physical parameters at which production of individual Bt strains of HD-series and NARC was undertaken

Yield Biomass

Determination of colony forming units (CFU or spore) per mg of freeze dried spore-δ-endotoxin powder

Colonial and crystal morphological characteristics of various HD and NARC Bt strains

Bioassay

Cross Resistance

Data Analysis

Results

Microbiological studies (production of spore-δ-endotoxin of Bts)

Yield biomass of various Bt strains (production of Bt powder) and determination of number of spores (colony forming unit CFU) per milligram of the spore-δ-endotoxin mixture

Evaluation of resistance in Cry1Ac selection of Helicoverpa armigera to spore-δ-endotoxin of various B. thuringiensis

Larval weight, percent larval weight reduction and trend in larval weight reduction in Cry1AC resistant insect due to feeding of spore-δ-endotoxin mixtures of Bts

Disorientation in the development of Cry1Ac resistant H. armigera larvae due to 7 days feeding of spore-crystal complex (diet-Bt-bioassays) of various Bt strains

Larval weight, percent larval weight reduction and trend in larval weight reduction in susceptible H. armigera due to feeding of spore-δ-endotoxin mixtures of Bts

Disorientation in the development of susceptible H. armigera larvae due to 7 days feeding of spore-crystal complex (diet-Bt-bioassays) of various Bt strains

Evaluation of resistance in Bt-based biopesticide selection of Helicoverpa armigera to spore-δ-endotoxin of various B. thuringiensis

Larval weight, percent larval weight reduction and trend in larval weight reduction in Bt-based bio-pesticide resistant H. armigera due to feeding of spore-δ-endotoxin mixtures of Bts

Disorientation in the development of susceptible H. armigera larvae due to 7 days feeding of spore-crystal complex (diet-Bt-bioassays) of various Bt strains

Discussion

Conclusion

References