The Korean Journal of Pesticide Science (농약과학회지)

The Korean Society of Pesticide Science (한국농약과학회)
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Insecticidal Activity and Molecular Characteristics of Bacillus thuringiensis CAB530 Isolated from Anomala albopilosa (Rutelidae: Coleoptera)

청동풍뎅이에서 분리한 Bacillus thuringiensis CAB530 균주의 살충활성 및 분자학적 특성

  • Beom, Jong-Il (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Seo, Mi-Ja (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • You, Joo (Sulloccha Research Institute) ;
  • Youn, Young-Nam (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Yu, Yong-Man (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University)
  • 범종일 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 서미자 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 유주 (설록차연구소) ;
  • 윤영남 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 유용만 (충남대학교 농업생명과학대학 응용생물학과)
  • Received : 2011.05.31
  • Accepted : 2011.06.13
  • Published : 2011.06.30
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Abstract

Bacillus thuringiensis CAB530 was isolated from dead Anomata albopilosa (Rutelidae: Coleoptera) and soil of green tea field, and confirmed its insecticidal activities. CAB530 isolate showed a high insecticidal activity against the beet armyworm among the many lepidopteran insects that are difficult to control. $LC_{50}$ value of CAB530 isolate against the second larva of Spodoptera exigua was $1.49{times}10^4$ spore concentration (cfu/$m{\ell}$). SDS-PAGE result of insecticidal toxin protein of CAB530 isolate showed a band at 130 kDa that is similar pattern with B. thuringiensis subsp. kurstaki that took insecticidal activity against S. exigua. Otherwise, the crystal protein of the CAB530 isolate was conformed at 65 kDa level after 30 minute of incubation in S. exigua midgut juice. Six crystal genes (cry1Aa, cry1Ab, cry1C, cry1D, cry1F and cry1I) were identified by PCR. It different from genes of B. thuringiensis subsp. kurstaki. Crystal shape and pattern of toxin protein was similar with B. thuringiensis subsp. kurstaki, however, insecticidal activity and PCR result of CAB530 isolate was similar with B. thuringiensis subsp. aizawai.

제주도의 녹차 밭에 서식하는 딱정벌레목인 청동풍뎅이 (Anomala albopilosa)의 사체와 녹차 밭 토양에서 분리한 Bacillus thuringiensis CAB530 균주의 생물효과를 검토하였다. 이 균주는 몇 종류의 해충에 대한 살충활성에서 난방제 농업해충 가운데 하나인 파밤나방에 높은 효과를 나타냈다. 파밤나방 2령 유충에 대한 살충활성 검정에서 CAB530 균주는 $LC_{50}$값이 $1.49{\times}10^4$(cfu/$m{\ell}$)으로 고활성을 보였다. 이 균주가 생산하는 살충성 독소단백질의 SDS-PAGE에서는 파밤나방에 살충활성이 있는 기존의 B. thuringiensis subsp. kurstaki와 비슷한 130kDa의 밴드를 나타내었다. 또한 파밤나방 중장액으로 반응을 시킨 후에 약 65kDa의 활성 독성단백질을 확인할 수 있었다 PCR수행에서 CAB530 균주는 cry1Aa, cry1Ab, cry1C, cry1D, cry1F 그리고 cry1I등 6 개의 유전자가 존재하는 것으로 밝혀졌으며, B. thuringiensis subsp. kurstala기준 균주와 차이가 있었다. 딱정벌레목에서 분리 선발한 B. thuringiensis CAB530균주는 crystal의 형태와 SDS-PAGE의 결과는 B. thuringiensis subsp. kurstaki와 유사하게 나타났지만, 살충활성 검정과 PCR product 전기영동 결과는 B thuringiensis subsp. aizawai와 유사하게 나타났다.

Keywords

References

  1. Agrawal N., P. Malhotra and R. K. Bhatnagar (2002) Interaction of gene-cloned and insect cell-expressed aminopeptidase N of Spodoptera litura with insecticidal crystal protein cry1C. Appl. Environ. Microbiol, 68:4583-4592. https://doi.org/10.1128/AEM.68.9.4583-4592.2002
  2. Agrawal, N., P. Malhotra and R. K. Bhatnagar (2004) siRNA-directed silencing of trans gene expressed in cultured insect cell. Biochem. Biophys. 320:428-434. https://doi.org/10.1016/j.bbrc.2004.05.184
  3. Ahn S. B., I. S. Kim, W. S. Cho, M. H. Lee and K. M. Choi (1989) The occurrence of the crop insect pests from Korea in 1988. Korean J. Appl. Entomol. 28(4):246-253.
  4. Armengol, G., M. C. Escobar, M. E. Maldonado and S. Orduz (2007) Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects. J. Appl. Microbiol. 102:77-88. https://doi.org/10.1111/j.1365-2672.2006.03063.x
  5. Aronson, A. I., E. S. Han, W. McGaughey and D. Johnson (1991) The solubility of inclusion protein from Bacillus thuringiensis is defendant upon protoxin composition and is a factor in toxicity to insect. Appl. Environ. Microbiol. 57:981-986.
  6. Bechtel, D. B. and Jr. L. A. Bulla (1976) Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis. J. Bacteriol. 127:1472-1781.
  7. Ben-Dov, E., A. Zaritsky, E. Dahan, Z. Barak, R. Sinal, R. Manasherob, A. Khamraev, E. Troitskaya, A. Dubitsky, N. Berezina and Y. Margalith (1997) Extended screening by PCR for seven cry-groups genes from field-collected strains of Bacillus thuringiensis. Appl. Environ. Microbiol. 63:4883-4890.
  8. Bernhard, K., P. Jarrett, M. Meadows, J. Butt, 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 insect pests. J. Invert. Pathol. 70:59-68. https://doi.org/10.1006/jipa.1997.4669
  9. Burton, S. L., D. J. Ellar, J. Li and D. J. Derbyshire (1999) N-Acetylgalactosamine on the putative insect receptor aminopeptidase N is recognized by a site on the domain III lectinlike fold of a Bacillus thuringiensis insecticidal toxin. J. Mol. Biol. 287:1011-1022. https://doi.org/10.1006/jmbi.1999.2649
  10. Ceron, J., A. Ortiz, R. Quintero, L. Guereca and A. Bravo (1995) Specific PCR primers directed to identify cry I and cry III genes within a Bacillus thuringiensis strain collection. Appl. and Environ. Microbiol. 61:3826-3831.
  11. Crickmore, N., D. R. Zeigler, J. Fcitelson, E. Schnepf, J. V. Rie, D. Lereclus, J. Baum and D. H. Dean (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbio. Mol. Biol. Rev. 62:807-813.
  12. Cummings, C. E., G. Armstrong, T. C. Hodgeman and D. J. Ellar (1994) Structural and functional studies of a synthetic peptide mimicking a proposed membrane inserting region of a Bacillus thuringiensis $\delta$-endotoxin. Mol. Membr. Biol. 11:87-92. https://doi.org/10.3109/09687689409162225
  13. Dai, S., Gao, Li. X. M and R. Li (1996) Distribution of Bacillus thuringiensis in soils of north and south of China. Acta Microbiol. Sin. 36:295-302.
  14. De Maagd, R. A., M. S. G. Kwa, H. Van der Klei, T. Yamamoto, B. Schipper, J. M. Vlak, W. J. Stiekema and D. Bosch (1996) Domain III substitution in Bacillus thuringiensis delta-endotoxin Cry1Aa (b) result in superior toxicity for Spodoptera exigua and altered membrane protein recognition. Appl. Environ. Microbiol. 62:1537-1543.
  15. 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.
  16. Edwards, D. L., J. Payne and G. G. Soares (1990) Novel isolates of Bacillus thuringiensis having activity against nematodes. U. S. Patent 4:948-734.
  17. Feitelson, J. S., J. Payne and L. Kim (1992) Bacillus thuringiensis: insects and beyond. Bio/Technol. 10:271-275. https://doi.org/10.1038/nbt0392-271
  18. Gazit, E. and Y. Shai (1995) The assembly and organization of the a5 and a7 helices from the pore-forming domain of Bacillus thuringiensis $\delta$-endotoxin. J. Biol, Chem. 270:2571-2578. https://doi.org/10.1074/jbc.270.6.2571
  19. Goh H. G., J. D. Park, Y. M. Choi and I. S. Park (1991) The host plants of beet armyworm, Spodoptera exigua (Hubner), (Lepidoptera: Noctuidae) and its occurrence. Korea J. Appl, Entomol. 30(2):111-116.
  20. Goh, H. G., J. S. Choi, K. B. Uhm, K. N. Choi and J. W. Kim (1993a) Spatial distribution pattern of beet armyworm, Spodoptera exigua (Hubner), larvae in the welsh onion field. Korean J. Appl. Entomol. 32(2):134-138.
  21. Goh, H. G., J. S. Choi, K. B. Uhm, K. N. Choi and J. W. Kim (1993b) Seasonal fluctuation of beet armyworm, Spodoptera exigua (Hubner), adult and larva. Korean J. Appl. Entomol. 32(4):389-394.
  22. Gonzalez, J. M. and B. C. Carlton (1980) Patterns of plasmid DNA in crystalliferous and acrystalliferous strains of Bacillus thuringiensis, Plasmid 3:92-98. https://doi.org/10.1016/S0147-619X(80)90038-4
  23. Grochulski, P., L. Masson, S. Borisova, M. Pusztai-Carey, J. L. Schwartz, R. Brousseau and M. Cygler (1995) Bacillus thuringiensis Cry1A(a) insecticidal toxin: Crystal structure and channel formation. J. Mol. Biol. 254:447-464. https://doi.org/10.1006/jmbi.1995.0630
  24. Herrnstadt, C., G. G. Soares, E. R. Wilcox and D. L. Edwards (1986) A new strain of Bacillus thuringiensis with activity against coleopteran insects. Bio/Techno. 4:305-308. https://doi.org/10.1038/nbt0486-305
  25. Hofte, H. and H. R. Whiteley (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53:242-255.
  26. 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. Bacteriol. 165:527-533. https://doi.org/10.1128/jb.165.2.527-533.1986
  27. Ichimatsu, T., E. Mizuki, K. Nishimura, T. Akao, H. Saitoh, K. Higuchi and M. Ohba (2000) Occurrence of Bacillus thuringiensis in fresh water of Japan. Curr. Microbiol, 40:212-217.
  28. Ishiwata, S. (1901) On a kind of severe flachrie (sotto disease). Dainihon sanshi Kaiho. 114:1-5.
  29. Jung, S. Y. (2010) Molecular genetics studies of Bacillus thuringiensis subsp. kurstaki KB099 of insecticidal activity of Spodopetra litura. Chungnam University MS Thesis.
  30. Jensen, S., L. Cavarec, M. P. Gassama and T. Heidmann (1995) Defective I elements introduced into Drosophila as trans genes can regulate reactivity and prevent I-R hybrid digenesis. Europ. Dros. Res. Conf. 14:198.
  31. Kim H. S., D. W. Lee, H. W. Park, Y. M. Yu, J. I. Kim and S. K. Kang (1995a) Distributional characterization of Bacillus thuringiensis isolated from soils of sericultural farms in Korea. Korean J. Seric. Sci. 31(1):57-61.
  32. Kim, H. S., H. W. Park, D. W. Lee, Y. M. Yu and S. K. Kang (1995b) Characterization of Bacillus thuringiensis isolated in granary dust. Korea J. Appl. Entomol. 34(3):243-248.
  33. Lereclus, D., M. M. Lecadet, J. Ribier and R. Dedonder (1982) Molecular relationships among plasmids of Bacillus thuringiensis: conserved sequences through 11 crystalliferous strains, Mol. Gen. Genet. 186:391-398. https://doi.org/10.1007/BF00729459
  34. Laemmli, U. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 277:680-685.
  35. Li, R., S. Dai, X. Li, X. Li, C. Luo, Z. Sheng and M. Sun (1990) Survey of Bacillus thuringiensis and Bacillus sphaericus from soils of four provinces of China and their principal biological properties. Acta Microbiol. Sin. 30:380-388.
  36. Li, J., J. Carroll and D. J. Ellar (1991) Crystal structure of insecticidal $\delta$-endotoxin from Bacillus thuringiensis at $2.5\AA$ resolution. Nature 353:815-821. https://doi.org/10.1038/353815a0
  37. Liburd, O. E., J. E. Funderburk and S. M. Olson (2000) Effect of biological and chemical insecticides on Spodoptera species (Lep, Noctuidae) and marketable yields of tomatoes. J. Appl. Entomol. 124:19-25. https://doi.org/10.1046/j.1439-0418.2000.00418.x
  38. Loeza-Laraa, P. D., G. Benintendeb, J. Cozzib, A. Ochoa-Zarzosaa, V. M. Baizabal-Aguirrea, J. J. Valdez-Alarcona and J. E. Lopez-Mezaa (2005) The plasmid pBMBt1 from Bacillus thuringiensis subsp. darmstadiensis (INTA Mo14-4) replicates by the rolling-circle mechanism and encodes a novel insecticidal crystal protein-like gene. Plasmid. 25:229-240.
  39. Lu, H., F. Rajamohan and D. H. Dean (1994) Identification of amino acid residues of Bacillus thuringiensis $\delta$-endotoxin Cry1A (a) associated with membrane binding and toxicity to Bombix mori. J. Bacteriol. 176:5554-5559. https://doi.org/10.1128/jb.176.17.5554-5559.1994
  40. Martin, P. A. W. and R. S. Travers (1989) Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl. Environ. Microbiol. 55:2437-2442.
  41. McDowel, D. G. and N. H. Mann (1991) Characterization and sequence analysis of a small plasmid from Bacillus thuringiensis var. kurstaki HD1-DIPEL. Plasmid. 25:113-120. https://doi.org/10.1016/0147-619X(91)90022-O
  42. Moar, W. J., L. Masson, R. Brousseau and J. T. Trumble (1990) Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp, kurstaki HD-1 and NRD-12. Appl. Environ. Microbiol. 56:2480-2483.
  43. Ohba, M. and K. Aizawa (1978) Serological identification of Bacillus thuringiensis and related bacteria isolated in Japan. J. Invertebr. Pathol. 32:303-309. https://doi.org/10.1016/0022-2011(78)90193-3
  44. Ohba, M., N. Wasano and E. Mizuki (2000) Bacillus thuringiensis soil populations naturally occurring in the Ryukyus, a subtropic region of Japan. Microbiol. Res. 155:17-22. https://doi.org/10.1016/S0944-5013(00)80017-8
  45. Park, J. D., H G. Goh, J. H Lee, W. J. Lee and K. J. Kim (1991) Flight activity and injury characteristics of beet armyworm, Spodoptera exigua (Hubner), (Lepidoptera: Noctuidae) in southern region of Korea. Korean J. Appl. Entomol. 30(2):124-129.
  46. Porcar, M., J. Irarte, V. Cosmao 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. 8:640-648.
  47. Rajagopal, R., S. Sivakumar, N. Agrawal, P. Malhotra, V. Bhatnaga and K. Raj (2002) Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor. J. Biol. Chem. 277:46849-46851. https://doi.org/10.1074/jbc.C200523200
  48. Sanchis, V., D. Lereclus, G. Menou, J. Chaufaux, S. Gou and M. M. Lecafet (1989) Mucleotide sequence and analysis of the n-terminal coding region of the Spodoptera-active $\delta$-endotoxin gene of Bacillus thuringiensis aizawai. Mol. Microbiol. 3:229-238. https://doi.org/10.1111/j.1365-2958.1989.tb01812.x
  49. Schnepf, H. E., K. Tomczak, J. P. Ortega and H. R. Whiteley (1990) Specificity-determining regions of lepidopteran-specific insecticidal proteins produced by Bacillus thuringiensis. J. Biol. Chem. 265:20923-20930.
  50. 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 insecticidal proteins. Microbiol, Mol. Biol. Rev. 62:774-806.
  51. Shelton, A. M., J. L. Robertson, H. D. Tang, C. J. Perez, S. D. Eigenbrode, H. K. Preisler, W. T. Wilsey and R. J. Cooley (1993) Resistance of diamondback moth (Lepidoptera, Plutellidae) to Bacillus thuringiensis subspecies in the field. J. Econ, Entomol. 86:697-705. https://doi.org/10.1093/jee/86.3.697
  52. Tabashnik, B. E., N. L. Cushing, N. Inson and M. W. Johnson (1990) Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera, Plutellidae). J. Econ. Entomol. 83:1671-1676. https://doi.org/10.1093/jee/83.5.1671
  53. Uribe, D., W. Martinez and N. J. Cero (2003) Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia. J. Invert. Pathol. 82:119-127. https://doi.org/10.1016/S0022-2011(02)00195-7
  54. Vadlamudi, R. K., E. Weber, I. Ji, T. H. Ji and Jr. L. A. Bulla (1995) Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. J. Biol. Chem. 270:6783-6788.
  55. Vilas-Boas, G. T and M. V. F. Lemos (2004) Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil. Can. J. Microbiol. 50:605-613.
  56. Visser, B., E. Munsterman, A. Stocker and W. G. Dirkse (1990) A novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crystal protein. J. Bacteriol. 172:6783-6788. https://doi.org/10.1128/jb.172.12.6783-6788.1990
  57. 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. https://doi.org/10.1016/0006-291X(81)90468-X