Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
권호 표지

A Highly Pathogenic Strain of Bacillus thuringiensis serovar kurstaki in Lepidopteran Pests

  • Kati, Hatice (Department of Biology, Faculty of Arts and Sciences, Giresun University) ;
  • Sezen, Kazim (Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University) ;
  • Nalcacioglu, Remziye (Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University) ;
  • Demirbag, Zihni (Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University)
  • Published : 2007.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.

Keywords

References

  1. Adang, M.J. 1991. Bacillus thuringiensis insecticidal crystal proteins: gene structure, action and utilization, p. 3-24. In K. Maramorosch (ed.), Biotechnology for biological control of pests and vectors. CRC Press, Boston, USA
  2. Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403-410 https://doi.org/10.1016/S0022-2836(05)80360-2
  3. Aptosoglou, S.G., A. Sivropoulou, and S.I. Koliais. 1997. Plasmid patterns of Bacillus thuringiensis strains and isolates. Microbios 91, 203-214
  4. Baum, J.A., T.B. Johnson, and B.C. Carlton. 1999. Bacillus thuringiensis: natural and recombinant bioinsecticide products, p. 189-210. In F.R. Hall, N.J. Totowa, and J.J. Menn (eds.), Biopesticides: use and delivery, Humana Press, Totowa, NJ, USA
  5. Becker, N. 1997. Microbial control of mosquitoes: management of the upper rhine mosquito population as a model programme. Parasitol. Today 13, 485-487 https://doi.org/10.1016/S0169-4758(97)01154-X
  6. Ben-Dov, E., A. Zaritsky, E. Dahan, Z. Barak, R. Sinai, R. Manasherob, A. Khameraev, E. Troitskaya, A. Dubitsky, N. Berezina, and Y. Margalith. 1997. Extended screening by PCR for seven crygroup genes from field-collected strains of Bacillus thuringiensis. Appl. Environ. Microbiol. 63, 4883-4890
  7. Bernhard, K., P. Jarret, M. Meadows, D.J. Ellis, G.M. Roberts, S. Pauli, P. Rodgers, and H.D. Burges. 1997. Natural isolates of Bacillus thuringiensis: worldwide distribution, characterization, and activity against insects pests. J. Invertebr. Pathol. 70, 59-68 https://doi.org/10.1006/jipa.1997.4669
  8. Bohorova, N., A.M. Maciel, R.M. Brito, L. Aguilat, J.E. Ibarra, and D. Hoisington. 1996. Selection and characterization of Mexican strains of Bacillus thuringiensis active against from major lepidopteran maize pests. Entomophaga 41, 153-165 https://doi.org/10.1007/BF02764243
  9. Dean, D.H. and D.R. Zeigler. 1994. Bacillus genetic stock centers and data, 6th ed. Ohio State University Press, Colombus, USA
  10. Donovan, W.P., C.C. Dankocsik, M.P. Gilbert, M.C. Gawron-Burke, R.G. Groat, and B.C. Carlton. 1988. Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki. J. Biol. Chem. 263, 561-567
  11. Honigman, A., G. Nedjar-Pazerin, A. Yawetz, U. Oron, S. Schuster, M. Broza, and B. Snek. 1986. Cloning and expression of the Lepidopteran toxin produced by Bacillus thuringiensis in Escherichia coli. Gene 42, 69-77 https://doi.org/10.1016/0378-1119(86)90151-4
  12. Iriarte, J., V.C. Dumanoir, Y. Bel, M. Porcar, M.D. Ferrandis, M.M. Lecadet, J. Ferre, and P. Caballero. 2000. Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (Serotype 50): Two novel serovars isolated in Spain. Curr. Microbiol. 40, 17-22 https://doi.org/10.1007/s002849910004
  13. Jensen, G.B., A. Wilcks, S.S. Petersen, J. Damgaard, J.A. Baum, and L. Andrup. 1995. The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16. J. Bacteriol. 177, 2914-2917 https://doi.org/10.1128/jb.177.10.2914-2917.1995
  14. Kati, H., K. Sezen, A.O. Beldüz, and Z. Demirbag. 2005. Characterization of a Bacillus thuringiensis subsp. kurstaki strain isolated from Malacosoma neustria L. (Lepidoptera: Lasiocampidae). Biologia 60, 301-305
  15. Kellar, B. and G.A. Langenfruch. 1993. Control of coleopteran pests by Bacillus thuringiensis, p. 171-191. In P.F. Entwhistle, J.S. Cory, M.J. Bailey, and S. Higgs (eds.), Bacillus thuringiensis, an environmental biopesticide: theory and practice. Wiley, New York, USA
  16. Kurt, A., M. Ozkan, K. Sezen, Z. Demirbag, and G. Ozcengiz. 2005. Cry3Aa11: A new Cry3Aa $\delta$-endotoxin from a local isolate of Bacillus thuringiensis. Biotechnol. Lett. 27, 1117-1121 https://doi.org/10.1007/s10529-005-8460-1
  17. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  18. Lereclus, D., A. Delecluse, and M.M. Lecadet. 1993. Diversity of Bacillus thuringiensis toxins and genes, p. 7-70. In P.F. Entwistle, J.S. Cory, M. Bailey, and S. Higgs (eds.), Bacillus thuringiensis an environmental biopesticide: theory and practice. Wiley, New York, USA
  19. Minitab, 1997. User's Guide, Release 11. Minitab, State College, PA, USA
  20. Porcar, M., J. Iriarte, V.C. Dumanoir, M.D. Ferrandis, M.M. Lecadet, J. Ferre, and P. Caballero. 1999. Identification and characterization of the new Bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59). J. Appl. Microbiol. 87, 640-648 https://doi.org/10.1046/j.1365-2672.1999.00863.x
  21. Ritchie, S. 1993. Bacillus thuringiensis subsp. israelensis use in Australia: the opportune moment, p. 111-115. In R.J. Akhurst (ed.), Proceedings of the second canberra Bacillus thuringiensis meeting. CSIRO, Canberra, USA
  22. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning, 2nd ed. Cold Spring, Harbor Laboratory Press, New York, USA
  23. Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Ziegler, and D.H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775-806
  24. Sezen, K. and Z. Demirbağ. 1999. Isolation and insecticidal activity of some bacteria from the hazelnut beetle (Balaninus nucum L.). Appl. Entomol. Zool. 34, 85-89 https://doi.org/10.1303/aez.34.85
  25. Sharif, F.A. and N.G. Alaeddinoglu. 1988. A rapid and simple method for staining of the crystal protein of Bacillus thuringiensis. J. Ind. Microbiol. 3, 227-229 https://doi.org/10.1007/BF01569580
  26. Stackebrandt, E. and B.M. Goebel. 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44, 846-849 https://doi.org/10.1099/00207713-44-4-846
  27. Teakle, R.E. 1994. Present use of, and problems with, Bacillus thuringiensis in Australia. Agric. Ecosyst. Environ. 49, 39-44 https://doi.org/10.1016/0167-8809(94)90020-5
  28. Zhong, C., D.J. Ellar, A. Bishop, C. Johnson, S. Lin, and E.R Hart. 2000. Characterization of a Bacillus thuringiensis $\delta$-endotoxin which is toxic to insects in three orders. J. Invertebr. Pathol. 76, 131-139 https://doi.org/10.1006/jipa.2000.4962