Protein Patterns and Larvicide Activity of Crystalline Inclusions of Bacillus thuringiensis ssp. kumamotoensis DSM 6070

Authors
J. Kutasi 1
R. Kovacs 1
I. Puspan 1
J. Makk 2
K. Takacs 3
B. Erdelyi 3
C. Imre 1
E. Karpati 4
1 BioFil Ltd., Budapest, Hungary.
2 Department of Microbiology, Eötvös Loránd University, Budapest, Hungary.
3 Fermentia Ltd., Budapest, Hungary.
4 Saniplant Ltd., Budapest, Hungary.
Abstract
Morphological and electrophoretic analysis of the crystalline inclusions (parasporal crystals) of sporulated cultures of B. thuringiensis ssp. kumamotoensis DSM 6070 (Bt 6070) was conducted via phase contrast and scanning electron microscopy and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The activity of the spore-crystal suspensions against house fly (Musca domestica) larvae was also assessed. Bipyramidal and smaller, irregular shaped crystals were observed in the sporulated cultures. 130, 75 and 25 kDa bands were detected in the protein pattern. The presence of 25 kda proteins in Bt6070 has not been reported earlier. The spore-crystal suspension showed significant larvicide activity against housefly larvae. Larvicide activity of B. thuringiensis ssp. kumamotoensis against any dipteran species has not been detected yet. Further studies are needed on identifying the dipteran- active fraction.

Keywords

1. Ben-Dov, E. 2014. Bacillus thuringiensis subsp. israelensis and Its Dipteran-specific Toxins. Toxins, 6: 1222–1243.
2. Cannon, R. J. C. 1995. Bacillus thuringiensis in Pest Control. In: “Biological Control: Benefits and Risks”, (Eds.): Hokkanen H. M. T. and Lync, J. M. Cambridge University Press, PP. 190–197.
3. Chen, J., Dai, L. Y., Wang, X. P., Tian, Y. C. and Lu, M. Z. 2005. The Cry3Aa Gene of Bacillus thuringiensis Bt886 Encodes a Toxin against Long-horned Beetle. Appl. Environ. Microbiol., 67: 351–356.
4. Crickmore, N., Zeigler, D. R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J. and Dean, D. H. 1998. Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins. Microbiol. Mol. Biol. Rev., 62: 807–813.
5. deMaagd, R. A. 2014. Bacillus thuringiensis-based Products for Insect Pest Control. In: “Principles of Plant-microbe Interactions”, (Ed.): Lugtenberg, B. Springer, PP. 185–192.
6. Donovan, W. P., Rupar, M. J., Slaney, A. C., Malvar, T., Gawron-Burke M. C. and Johnson, T. B. Appl. 1992. Characterization of Two Genes Encoding Bacillus thuringiensis Insecticidal Crystal Proteins Toxic to Coleoptera Species. Appl Environ. Microbiol., 58: 3921-3927.
7. Federici, B. A. 1999. Bacillus thuringiensis in Biological Control. In: “Handbook of Biological Pest Control”, (Eds.): Bellows, T. S., Fisher, T. W., Caltagirone, L. E., Dahlsten, D. L., Gordh, G. and Huffaker, C. B. Elsevier Inc., PP. 575–593.
8. Federici, B. A., Park, H. W. and Bideshi, D. K. 2010. Overview of the Basic Biology of Bacillus thuringiensis with Emphasis on Genetic Engineering of Bacterial Larvicides for Mosquito Control. Open Toxinol. J., 3: 83–100.
9. Heins, S. D., Jimenez, D. R., Manker, D. C., Marrone, P. G., Mccoy, R. J. and Orjala, J. E. 2000. Compositions and Methods for Controlling Plant Pests. US Patent WO2000029426 A1.
10. Hernández-Rodríguez, C. S. and Ferré, J. 2005. Ecological Distribution and Characterization of Four Collections of Bacillus thuringiensis Strains. J. Basic. Microbiol., 49: 152–157.
11. Höfte, H. and Whiteley H. R. 1989. Insecticidal Crystal Proteins of Bacillus thuringiensis. Microbiol. Rev., 53: 242-255.
12. Ibarra, J. E., del Rincon, C. M., Ordúz, S., Noriega, D., Benintende, G., Monerrat, R., Regis, L., de Oliveira, C. M. F., Lanz, H., Rodrguez, M. H., Sanchez, J., Pena, G. and Bravo, A. 2003. Diversity of Bacillus thuringiensis Strains from Latin America with Insecticidal Activity against Different Mosquito Species. Appl. Environ. Microbiol., 69: 5269–5274.
13. Insell, J. P. and Fitz-James, P. C. 1985. Composition and Toxicity of the Inclusion of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol., 50: 56–62.
14. Kati, H., Sezen, K., Nalcacioglu, R. and Demirbag, Z. 2007. A Highly Pathogenic Strain of Bacillus thuringiensis serovar kurstaki in Pepidopteran Pests. J. Microbiol., 45: 553–557.
15. Merdan, B. A. 2012. Bacillus thuringiensis as a Feed Additive to Control Musca domestica Associated with Poultry Houses. J. Basic Appl. Zool., 65: 83–87.
16. Mwamburi, L. A., Laing, M. D. and Miller, R. 2011. Laboratory and Field Evaluation of Formulated Bacillus thuringiensis var. israelensis as a Feed Additive and Using Topical Applications for Control of Musca domestica (Diptera: Muscidae) Larvae in Caged-poultry Manure. Environ. Entomol., 40: 52–58.
17. Ohba, M., Ono, K., Aizawa, K. and Iwanami, S. 1981. Two New Subspecies of Bacillus thuringiensis Isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (Serotype18) and Bacillus thuringiensis subsp. tochigiensis (Serotype19). J. Invertebr. Pathol., 38: 184–190.
18. Palma, L., Munoz, D., Berry, C., Murillo, J. and Caballero, P. 2014. Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity. Toxins, 6: 3296–3325.
19. Payne, J. M. and Fu, J. M. 1994. Coleopteran-active Bacillus thuringiensis Isolates and Genes Encoding Coleopteran-active Toxins. Patent Number US5286486.
20. Rupar, M. J., Donovan, W. P., Groat, R. G., Slaney, A. C., Mattison, J. W., Johnson, T. B., Charles, J. F., Cosmao Dumanoir, V. and de Barjac, H. 1991. Two Novel Strains of Bacillus thuringiensis Toxic to Coleopterans. Appl. Environ. Microbiol., 57: 3337–3344.
21. Schnepf, H. E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R. and Dean, D. H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775–780.
22. Singh, G. J., Schouest, L. P. and Gill, S. S. 1986. Action of Bacillus thuringiensis subsp. israelensis Delta-endotoxin on the Ultrastructure of the House Fly Larva Neuromuscular System In vitro. J. Invertebr. Pathol., 47: 155–166.
23. Takács, K., Kárpáti, É., Puspán, I., Kutasi, J., Kovács, R., Kalinák, Cs., Erdélyi, B. 2010. Optimization of the fermentation conditions for toxin production of Bacillus thuringiensis sp. In: Abstract book of the Conference of Hungarian Society for Microbiology, Keszthely, Hungary, pp. 85-86.
24. Takács, K. 2010. Optimization of the Fermentation Conditions for Toxin Production of Bacillus thuringiensis sp. In: “Abstract Book of the Conference of Hungarian Society for Microbiology”, Keszthely, PP. 85–86.
25. Takebe, S., Morinaga, S., Mizuhashi, A., Komano, T. 2005 Improved technique for refining the crystal of Bacillus thuringiensis by NaBr gradient centrifugation. In: Proceedings of 6th Pacific Conference on the Biotechnology of Bacillus thuringiensis and its environmental impact, Victoria, BC pp. 111-112.
26. van Frankenhuyzen, K. 2009. Insecticidal Activity of Bacillus thuringiensis Crystal Proteins. J. Invertebr. Pathol., 101: 1–16.
Journal of Agricultural Science and Technology
Volume 18, Issue 7
November and December 2016
Pages 1945-1951