Bacillus thuringiensis BT17 균주를 이용한 인시목 유충 방제용 미생물 살충제 생산

Production of Microbial Insecticide Using Bacillus thuringiensis BT17 for the Control of Lepidopteran Larvae

  • 투고 : 2010.11.22
  • 심사 : 2010.12.24
  • 발행 : 2010.12.31

초록

살충성 독소 결정을 생산하는 Bacillus thuringiensis BT17 균주를 분리하였으며, 16S rRNA 유전자 분석 결과 B. thuringiensis serovar colmeri로 동정하였다. BT17 균주는 인시목 유충에 대해 살충성을 보이는 결정성 독소를 soybean meal과 skim milk를 포함하는 배지에서 $30^{\circ}C$, 280 rpm으로 36시간 진탕배양하였을 때 효율적으로 생산하였다. 200 L fermentor에서 배양하였을 때 균수는 24시간에 최대가 되었으나 독소 결정의 수는 36시간까지 증가하였다. 액상의 미생물 살충제 제품을 제조하였으며, 배양액을 $20^{\circ}C$에서 3개월간 보관시 독소 결정의 수는 보관 초기보다 2배까지 증가하였다. BT17 균주가 포함된 살충제 제품의 효능은 파밤나방보다 배추좀나방에서 좋았으며 동물에 대한 독성은 거의 없었다.

Insecticidal crystalline toxin producing Bacillus thuringiensis BT17 strain was isolated and identified as B. thuringiensis serovar colmeri by 16S rRNA analysis. BT17 strain produced crystalline ${\delta}$-endotoxin against to Lepidopteran larvae effectively on the culture broth of soybean meal and skim milk, $30^{\circ}C$ and 36 h shaking culture of 280 rpm. The maximum colony forming unit achieved when the culture was continued for 24 h, but the number of crystals increased until 36 h in the 200 L fermentor. Liquid type of biological insecticide product was made, and after 3 months storage in $20^{\circ}C$ the number of crystals was increased up to twice than beginning. Biocontrol effect of BT17 insecticide product was better in Plutella xylostella than in Spodoptera exigua, and the toxicity to animals was negligible.

키워드

참고문헌

  1. Agaisse, H. and D. Lereclus. 1995. How does Bacillus thuringiensis produce so much insecticidal crystal protein? J. Bacteriol. 177, 6027-6032. https://doi.org/10.1128/jb.177.21.6027-6032.1995
  2. Baum, J.A., T.B. Johnson, and B.C. Carlton. 1999. Bacillus thuringiensis: natural and recombinant products, pp. 189-210. In F.R. Hall and J.J. Menn (eds.), Biopesticides Use and Delivery. Humana Press, Totowa, NJ, USA.
  3. Berliner, E. 1915. Uber die schlaffsucht der Mehlmottenraupe (Ephestia kuhnielluszell) und ihre Errenger Bacillus thuringiensis n. sp. Z. Angew. Entomol. 2, 29-56.
  4. Bulla, L.A., Jr. 1975. Bacteria as insect pathogens. Ann. Rev. Microbiol. 29, 163-190. https://doi.org/10.1146/annurev.mi.29.100175.001115
  5. Bulla, L.A., Jr., R.N. Costilow, and E.S. Sharpe. 1978. Biology of Bacillus popilliae. Adv. Appl. Microbiol. 22, 1-18.
  6. Crickmore, N., D.R. Zigler, J. Freitelson, E. Schnepf, J. van Rie, D. Lereclus, J. Baum, and D.H. Dean. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Bio. Rev. 62, 807-813.
  7. Dingman, D.W. and D.P. Stahly. 1983. Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl. Environ. Microbiol. 46, 860-869.
  8. Du, C., P.A.W. Martin, and K.W. Nickerson. 1994. Comparison of disulfide contents and solubility at alkaline pH of insecticidal and noninsecticidal Bacillus thuringiensis protein crystals. Appl. Environ. Microbiol. 60, 3847-3853.
  9. Dulmage, H.T. 1981. Microbial control of pests and plant diseases, pp. 193-222. In H.D. Burgess (ed.). Academic Press, London, UK.
  10. Goldberg, L.H. and J. Margalit. 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti and Culex pipens. Mosquito News 37, 355-358.
  11. Griego, V.M. and K.D. Spence. 1978. Inactivation of Bacillus thuringiensis spores by ultraviolet and visible light. Appl. Environ. Microbiol. 35, 906-910.
  12. Guerchicoff, A., A. Delecluse, and C.P. Rubinstein. 2001. The Bacillus thuringiensis cyt genes for hemolytic endo toxin constitute a gene family. Appl. Environ. Microbiol. 67, 1090-1096. https://doi.org/10.1128/AEM.67.3.1090-1096.2001
  13. Helgason, E., O.A. Okstad, D.A. Caugant, H.A. Johansen, A. Fouet, M. Mock, I. Hegna, and A.B. Klosto. 2000. Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis-one species on the basis of genetic evidence. Appl. Environ. Microbiol. 66, 2627-2630. https://doi.org/10.1128/AEM.66.6.2627-2630.2000
  14. Hofte, H. and H.R. Whiteley. 1989. Insecticidal cyrstal proteins of Bacillus thuringiensis. Microbiol. Rev. 53, 242-255.
  15. Miyagawa, E., R. Azuma, and T. Suto. 1979. Cellular fatty acid composition in Gram negative obligately anaerobic rods. J. Gen. Appl. Microbiol. 25, 41-51. https://doi.org/10.2323/jgam.25.41
  16. Obeta, J.A.N. and N. Okafor. 1984. Medium for the production of primary powder of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 47, 863-867.
  17. Perani, M., A.H. Bishop, and A. Vaid. 1998. Prevalence of $\beta$ – exotoxin in natural isolates of Bacillus thuringiensis. FEMS Microbiol. Lett. 160, 55-60.
  18. Priest, F.G., D.A. Kaji, Y.B. Rosato, and V.P. Canhos. 1994. Characterization of Bacillus thuringiensis and related bacteria by ribosomal RNA gene restriction fragment length polymorphisms. Microbiology 140, 15-22.
  19. Pusztai, M., P. Fast, L. Gringorten, H. Kaplan, T. Lessard, and P.R. Carey. 1991. The mechanism of sunlight-mediated inactivation of Bacillus thuringiensis crystals. Biochem. J. 273, 43-47. https://doi.org/10.1042/bj2730043
  20. Scherrer, P., P. Luthy, and B. Trumpi. 1973. Production of $\delta$-endotoxin by Bacillus thuringiensis as a function of glucose concentrations. Appl. Microbiol. 25, 644-646.
  21. Schnepf, E., N. Crickmore, J. van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler, and D.H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775-806.
  22. Vasantha, N. and E. Freese. 1979. The role of manganese in growth and sporulation of Bacillus subtilis. J. Gen. Microbiol. 112, 329-336. https://doi.org/10.1099/00221287-112-2-329
  23. Wei, J.Z., K. Hale, L. Carta, E. Platzer, C. Wang, S.C. Fang, and R.V. Aroian. 2003. Bacillus thuringiensis crystal proteins that target nematodes. Proc. Natl. Acad. Sci. USA 100, 2760-2765. https://doi.org/10.1073/pnas.0538072100
  24. Widner, R.W. and H.R. Whiteley. 1989. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. J. Bacteriol. 171, 965-974. https://doi.org/10.1128/jb.171.2.965-974.1989
  25. Yezza, A., R.D. Tyagi, J.R. Valero, R.Y. Surampalli, and J. Smith. 2004. Scale-up of biopesticide production processes using wastewater sludge as a raw material. J. Ind. Microbiol. Biotechnol. 31, 545-552. https://doi.org/10.1007/s10295-004-0176-z
  26. Zhu, H., F. Qu, and L.H. Zhu. 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Res. 21, 5279-5280. https://doi.org/10.1093/nar/21.22.5279