Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Characterization of Bacillus thuringiensis Having Insecticidal Effects Against Larvae of Musca domestica

  • Oh, Se-Teak (Department of Biotechnology, Konkuk University) ;
  • Kim, Jin-Kyu (Department of Biotechnology, Konkuk University) ;
  • Yang, Si-Yong (Department of Biotechnology, Konkuk University) ;
  • Song, Min-Dong (Department of Biotechnology, Konkuk University)
  • Published : 2004.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

The entomopathogenic bacterium Bacillus thuringiensis is the most widely used biopesticide. Insecticidal proteins, coded by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. We isolated a Bacillus thuringiensis strain having insecticidal activity against larvae of the house fly (M. domestica) from the soils at a pig farm in Korea, and named it Bacillus thuringiensis SM. The culture filtrate from Bacillus thuringiensis SM showed strong lethality (83.3%) against M. domestica larvae. The parasporal crystal is enclosed within the spores' outermost envelope, as determined by transmission electron microscopy, and exhibited a bipyramidal form. The crystal proteins of strain SM consisted of five proteins with molecular weights of approximately ~130, ~80, ~68, ~42, and ~27 kDa on a 10% SDS-PAGE (major band, a size characteristic of Cry protein). Examination of antibiotic resistance revealed that the strain SM showed multiple resistant. The strain SM had at least three different plasmids with sizes of 6.6, 9.3, and 54 kb. Polymerase chain reactions (PCRs) revealed the presence of cry1, cry4A2, and cry11A1 genes in the strain SM. The cry1 gene profile of the strain SM appeared in the three respective products of 487 bp [cry1A(c)], 414 bp [cry1D], and 238 bp [cry1A(b)]. However, the strain SM has not shown the cry4A2 md cry11A1 genes. In in vivo toxicity assays, the strain SM showed high toxicity on fly larvae (M. domestic) [with $LC_{50}$ of 4.2 mg/ml, $LC_{90}$ of 8.2 mg/ml].

Keywords

References

  1. Bechtel, D. B. and L. A. Bulla, Jr. 1976. Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis. J. Bacteriol. 127: 1472-1481.
  2. Ben-Dov, E., M. Einav, N. Peleg, S. Boussiba, and A. Zaritsky. 1996. Restriction map of the 125-kilobase plasmid of Bacillus thuringiensis subsp. israelensis carrying the genes that encode delta-endotoxins active against mosquito larvae. Appl. Environ. Microbiol. 59: 523- 527.
  3. Ben-Dov, E., A. Zaritsky, E. Dahan, Z. Barak, R. Sinai, R. Manasherob, A. Khamraev, E. Troitskaya, A. Dubitsky, N. Berezina, and Y. Margalith. 1997. Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis. Appl. Environ. Microbiol. 63: 4883-4890.
  4. Bourque, S. N., J. R. Valero, J. Mercier, M. C. Lavoie, and L. C. Levesque. 1993. MUltiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis. Appl. Environ. Microbiol. 59: 523-527.
  5. Burges, H. D. 1981. Microbial Control of Pests and Plant Diseases, pp. 193- 280. Academic Press, London.
  6. Cleeland, R. and E. Grunberg. 1986. Laboratory evaluation of new antibiotics in vitro and in experimental animal infections, pp. 825- 876. In V. Lorian (ed.), Antibiotics in Laboratory Medicine. Williams & Wilkins Ltd., Baltimore.
  7. Crickmore, N., E. J. Bone, J. A. Williams, and D. J. Ellar. 1995. Contribution of the individual components of the $\delta$-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis. FEMS Microbiol. Lett. 131: 249- 254.
  8. Crickmore, N., D. R Zeigler, J. Feitelson, A. Schnepf, B. Lambert, D. Lereclus, J. Baum, and D. H. Dean. 1995. Revision of the nomenclature for Bacillus thuringiensis pesticide cry genes, p. 14. In Program and Abstracts of the $28^{th}$ Annual Meeting of the Society for Invertebrate Pathology, Society for Invertebrate Pathology, Bethesta, MD.
  9. Crickmore, N., D. R. Zeigler, J. Feitelson, E. Schnepf, J. VanRie, D. Lereclus, J. Baum, and D. H. Dean. 1998, Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Rev. 62: 807-813.
  10. Crickmore, N. 2000. The diversity of Bacillus thuringiensis ,$\delta$-endotoxins, pp. 65- 79. In Charles, J. F., Delecluse, A., Nielsen-LeRoux, C. (eds.), Entomopsthogenic Bacteria: From Laboratory To Field Application. Kluwer Academic Publishers, Dordrecht.
  11. de Barjac, H. and A. Bonnefoi. 1962. Essai de classification biochimique et seroloque de 24 souches de Bacillus du type B. thuringiensis. Entomophaga 7: 5- 31.
  12. de Barjac, H. and A. Bonnefoi. 1968. A classification of strains of Bacillus thuringiensis berliner with a key to their differentiation. J. Invertebr: Pathol. 11: 335- 347.
  13. de Barjac, H. 1978. Un nouveau candidat a la lutte biologique contre les moustiques: Bacillus thuringiensis var. israelensis. Entomophaga 23: 309- 319.
  14. Federici, B. A., P. Luthy, and J. E. Ibarra. 1990. The paraspora1 body of Bacillus thuringiensis subsp. israelensis: Structure, protein composition and toxicity, pp. 16- 44. In H. de Barjac and S. Sutherland (eds.), Bacterial Control of Mosquitoes and Blackflies: Biochemistry, Genetics and Applications of Bacillus thuringiensis and Bacillus sphaericus. Rutgers University Press, New Brunswick, N.J.
  15. Gonzalez, J. M., Jr. H. T. Dulmage, and B. C. Carlton. 1981. Correlation between specific plasmids and endotoxin production in Bacillus thuringiensis. Plasmid 5: 351- 365.
  16. Hermstadt, C., G. G. Soares, E. R. Wilcox, and D. L. Edwards. 1986. A new strain of Bacillus thuringiensis with activity against coleopteran insects. Bio/Technology 4: 305-308.
  17. Hofte, H. and H. R. Whitley. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbial. Rev. 53: 242-255.
  18. Ibarra, J. E. and B. A. Federici. 1986. Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis. J. Bacterial. 165: 527- 533.
  19. lung, Y. C., S. U. K.m, and S. H. Bok. 1994. Characterization of Bacillus thuringiensis strain Bt-14 having insecticidal activity against Plutella xalostella. J. Microbiol. Biotechnol. 4: 322- 326.
  20. Kalman, S., K. L. Kiehne, J. L. Libs, and T. Yamamoto. 1993. Cloning of a novel crylCa-type gene from a strain of Bacillus thuringiensis subsp. galleriae. Appl. Environ. Microbiol. 59: 1131- 1137.
  21. Lecadet, M. M., E Frachon, V. Durnanoir, H. Ripouteau, S. Hamon, P. Laurent, and I. Thiery. 1999. Updating the H-antigen classification of Bacillus thuringiensis. J. Appl. Microbial. 86: 660- 672.
  22. Lereclus, D., A. Delecluse, and M. Lecadet. 1993. Diversity of Bacillus thuringiensis toxins and genes, pp. 37- 69. In P. F. Entwistle, J. S. Cory, M. J. Bailey, and S. Higgs (eds.), Bacillus thuringiensis, An Environmental Biopesticide: Theory and Practice. John Wiley & Sons Ltd., United Kingdom.
  23. Lopez. J. E., B. A. Federici, W. J. Poehner, and J. E. Ibarra. 1995. Highly mosquitocidal isolates of Bacillus thuringiensis subspecies kenyae and entomocidus from Mexico. Biochem. Syst. Ecol. 23: 461- 468.
  24. Lopez. J. E. and J. E. Ibarra. 1996. Characterization of a novel strain of Bacillus thuringiensis. Appl. Environ. Microbiol. 62: 1306- 1310.
  25. Mahadi, N. M., H. Sugyo, L. Bibiana, and D. H. Donald. 1998. Application of multiplex PCR for rapid determination of cry1 gene profiles of new Bacillus thuringiensis isolates. J. Microbiol. Biotechnol. 8: 517-522.
  26. Meadow, M. P. 1993. Bacillus thuringiensis in the environment: Ecology and risk assessment, pp. 193- 220. In P. F. Entwistle, J. S. Cory, M. J. Bailey, and S. Higgs (eds.), Bacillus Thuringiensis, An Environmental Biopesticide: Theory and Practice. John Wiley & Sons, Ltd., Chichester, United Kingdom.
  27. Poncet, S., A. Delecluse, A. Klier, and G. Rapoport. 1995. Evaluation of synergistic interactions between the CryIVA, CryIVB, and CryIVD toxic components of Bacillus thuringiensis subsp. israelensis crystals. J. Invertebr. Pathol. 66: 131-135.
  28. Rodrigues-Padilla, C., L. Galan-Wong, H. de Barjac, E. Roman-Calderon, R. Tamez-Guerra, and H. Dulmage. 1990. Bacillus thuringiensis subspecies neoleonensis serotype H-24, a new subspecies which produces a triangular crystal. J. lnvertebr. Pathol. 56: 280- 282.
  29. Temeyer, K. B. 1983. Larvicidal activity of Bacillus thuringiensis subsp. israelensis in the Dipteran Haematobia irritans. Appl. Environ. Microbial. 47: 952- 955.
  30. Travers, R. S., D. W. Martin, and C. F. Reichelderfer. 1987. Selective process for efficient isolation of soil Bacillus spp. Appl. Environ. Microbiol. 53: 1263- 1266.
  31. Voskuil, M. Land G. H. Chambliss. 1993. Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis. Appl. Environ. Microbiol. 59: 1138- 1142.
  32. Yamamoto, T. and R. E. Mclaughlin. 1981. Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var. kurstaki toxic to mosquito larvae, Aedes taeniorhynchus. Biochem. Biophys. Res. Commun. 103: 414-421.
  33. Yamamoto, T. and T. Lizuka. 1981. Isolation of a protein from the parasporal crystal of B. thuringiensis kurstaki. Arch. Biochem. Biophys. 227: 233- 241.