Journal of Biological Control

Open Access Open Access  Restricted Access Subscription Access

Molecular characterization of an indigenous lepidopteran toxic Bacillus thuringiensis strain T532


Affiliations
1 Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore – 635110, Tamil Nadu, India
 

Bacillus thuringiensis Berliner (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, four indigenous Bt isolates (T489, T491, T495 and T532) were taken and investigated for their properties such as crystal morphology, cry gene(s) content and insecticidal activity. Bipyramidal, cuboidal and spherical crystals were observed. Among the four isolates only one isolate was found positive for lepidopteran specific cry genes. The isolate T532 which showed the presence of cry1, cry2Aa and cry2Ab genes was selected for protein profiling and bioassay. The isolate showed the presence of two proteins of molecular weights at ~135 and ~65 kDa sizes. Artificial diet based bioassay resulted in 100% mortality of the neonate larvae of H. armigera and S. litura for the isolate T532 which was comparable to that of the positive control (HD1). The full length cry2A gene was amplified and cloned into E. coli DH5α strain. The recombinant plasmids were sequenced and the sequence was deposited in GenBank nucleotide database. The accession number of the gene is MH475905. The gene was named by the Bacillus thuringiensis delta endotoxin nomenclature committee as cry2Aa23.

Keywords

Bacillus Thuringiensis, Cloning, Cry2Aa, PCR.
User
Notifications

  • Ali S, Zafar Y, Ali GM, Nazir F. 2010. Bacillus thuringiensis and its application in agriculture. Afr J Biotechnol. 9(14): 2022-2031.

  • Asokan R, Puttaswamy. 2007. Isolation and characterization of Bacillus thuringiensis Berliner from soil, leaf, seed dust and insect cadaver. J Biol Control. 21(1): 83-90.

  • Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamaraev A, Baizhanov M, Glupov V, Margalith Y. 1999. Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol. 65(8): 3714-3716. PMid:10427071 PMCid:PMC91556

  • Ben-Dov E, Zaritsky A, Dahan E, Barak ZE, Sinai R, Manasherob R, Khamraev A, Troitskaya E, Dubitsky A, Berezina N. (1997). Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis. Appl Environ Microbiol. 63(12): 4883-4890. PMid:9406409 PMCid:PMC168816

  • Bernhard K, Jarrett P, Meadows M, Butt J, Ellis DJ, Roberts GM, Pauli S, Rodgers P, Burges HD. 1997. Natural isolates of Bacillus thuringiensis worldwide distribution, characterization and activity against insect pests. J Invertebr Pathol. 70: 59-68 https://doi.org/10.1006/ jipa.1997.4669

  • Crickmore N, Baum J, Bravo A, Lereclus D, Narva K, Sampson K, Schnepf E, Sun M, Zeigler DR. 2016. Bacillus thuringiensis toxin nomenclature. Available from: http://www.btnomenclature.info

  • Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean DH 1998. Revision of nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev. 62: 807-813. PMid:9729610 PMCid:PMC98935

  • Darsi S, Prakash GD, Udayasuriyan V. 2010. Cloning and characterization of truncated cry1Ab gene from a new indigenous isolate of Bacillus thuringiensis. Biotechnol Lett. 32(9): 1311-1315. https://doi.org/10.1007/s10529010-0301-1 PMid:20480206

  • Feitelson JS, Payne J, Kim L. 1992. Bacillus thuringiensis: insects and beyond. Nature Biotechnol. 10: 271-275. https://doi.org/10.1038/nbt0392-271

  • Jain D, Sunda SD, Sanadhya S, Nath DJ, Khandelwal SK. 2017. Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains. 3 Biotech 7(1): 4. https://doi.org/10.1007/s13205-016-0583-7 PMid:28391468 PMCid:PMC5385171

  • Jain D, Udayasuriyan V, Arulselvi PI, Dev SS, Sangeetha P. 2006. Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1. Appl Biochem Biotechnol. 128(3): 185-194. https://doi.org/10.1385/ABAB:128:3:185

  • Kalman S, Kiehne KL, Libs JL, Yamamoto T. 1993. Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae. Appl Environ Microbiol. 59(4): 1131-1137. PMid:8476286 PMCid:PMC202250

  • Konecka E, Baranek J, Hrycak A, Kaznowski A. 2012. Insecticidal activity of Bacillus thuringiensis strains isolated from soil and water. Sci World J. Article ID: 710501. doi:10.1100/2012/710501. https://doi.org/10.1100/2012/710501

  • Kumar S, Singh A. 2015. Biopesticides: Present status and the future prospects. J Fertil Pestic. 6(2): 1-2. https:// doi.org/10.4172/2471-2728.1000e129

  • Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685. https://doi.org/10.1038/227680a0 PMid:5432063

  • Manikandan R, Ramalakshmi A, Balasubramani V, Udayasuriyan V. 2015. Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis. Mol. Bio. 49(4): 520-526. https://doi.org/10.1134/S0026893315040111

  • Martin PA, Travers RS. 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl Environ Microbiol. 55(10): 2437-2442. PMid:16348022 PMCid:PMC203101

  • Milne R, Kaplan H. 1993. Purification and characterization of a trypsin-like digestive enzyme from spruce budworm (Choristoneura fumiferana) responsible for the activation of δ- endotoxin from Bacillus thuringiensis. Insect Biochem Molec Biol. 23: 663-673. https://doi.org/10.1016/0965-1748(93)90040-Y

  • Mizuki E, Ichimatsu T, Hwang SH, Park Y, Saitoh H, Higuchi K, Ohba M. 1999. Ubiquity of Bacillus thuringiensis on phylloplanes of arboreous and herbaceous plants in Japan. J Appl Microbiol. 86(6): 979-984. https://doi.org/10.1046/j.1365-2672.1999.00778.x

  • Nagarkatti S, Prakash S. 1974. Rearing of Heliothis armigera (Hub.) on artificial diet. Tech Bull Commonwealth Inst Biol Control 17: 169-173.

  • Palma L, Mu-oz D, Berry C, Murillo J, Caballero P. 2014. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 6(12): 3296-3325. https://doi.org/10.3390/ toxins6123296 PMid:25514092 PMCid:PMC4280536

  • Pan Z, Xu L, Zhu Y, Shi H, Chen Z, Chen M, Chen Q, Liu B. 2014. Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L. World J Microbiol Biotechnol. 30(10): 2655-2662. https://doi.org/10.1007/s11274014-1689-x PMid:24943249

  • Patel KD, Ingle SS. 2012. Molecular characterization of novel serovars of Bacillus thuringiensis isolates from India. Indian J Microbiol. 52(3): 332-336. https:// doi.org/10.1007/s12088-011-0240-0 PMid:23997321 PMCid:PMC3460129

  • Ramalakshmi A, Udayasuriyan V. 2010. Diversity of Bacillus thuringiensis isolated from Western Ghats of Tamil Nadu state, India. Curr Microbiol. 61(1): 13-18. https:// doi.org/10.1007/s00284-009-9569-6 PMid:20033169

  • Reyaz AL, Arulselvi PI. 2016. Cloning, characteriztion and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain SWK1, native to Himalayan valley Kashmir. J Invertebr Pathol. 136: 1-6. https://doi.org/10.1016/j.jip.2016.02.005 PMid:26906447

  • Reyaz AL, Gunapriya L, Arulselvi PI. 2017. Molecular characterization of indigenous Bacillus thuringiensis strains isolated from Kashmir valley. 3 Biotech 7(2): 143. https:// doi.org/10.1007/s13205-017-0756-z PMid:28597156 PMCid:PMC5465046

  • Sasaki J, Asano S, Hashimoto N, Lay BW, Hastowo S, Bando H, Iizuka T. 1997. Characterization of a cry2A gene cloned from an isolate of Bacillus thuringiensis serovar sotto. Curr Microbiol. 35(1): 1-8. https://doi.org/10.1007/s002849900201 PMid:9175551

  • Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler D, Dean D. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev. 62(3): 775-806. PMid:9729609 PMCid:PMC98934

  • Schnepf HE, Whiteley H. 1981. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci. 78(5): 2893-2897. https://doi.org/10.1073/pnas.78.5.2893

  • Shishir A, Roy A, Islam N, Rahman A, Khan S, Hoq MM. 2014. Abundance and diversity of Bacillus thuringiensis in Bangladesh and their cry genes profile. Front Environ Sci. 2: 1-10. https://doi.org/10.3389/fenvs.2014.00020

  • Unalmis S, Ayvaz A, Yilmaz S, Azizoglu U. 2015. Molecular screening and bioactivity of native Bacillus thuringiensis isolates. J Agric Sci Technol. 17(5): 1197-1207.

  • Wasano N, Saitoh H, Maeda M, Ohgushi A, Mizuki E, Ohba M. 2005. Cloning and characterization of a novel gene cry9Ec1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain. Can J Microbiol. 51(11): 988-995. https://doi.org/10.1139/w05-084 PMid:16333339

  • Yilmaz S, Azizoglu U, Ayvaz A, Temizgul R, Atciyurt ZB, Karaborklu S. 2017. Cloning and expression of cry2Aa from native Bacillus thuringiensis strain SY49-1 and its insecticidal activity against Culex pipiens (Diptera: Culicidae). Microb Pathogenesis 105: 81-85. https://doi.org/10.1016/j.micpath.2017.02.016 PMid:28215855

  • Zheng A, Zhu J, Tan F, Guan P, Yu X, Wang S, Deng Q, Li S, Liu H, Li P. 2010. Characterisation and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain JF19-2. Ann Microbiol. 60: 129–134. https://doi.org/10.1007/s13213-009-0011-x


Abstract Views: 292

PDF Views: 112




  • Molecular characterization of an indigenous lepidopteran toxic Bacillus thuringiensis strain T532

Abstract Views: 292  |  PDF Views: 112

Authors

K. V. Nayan Ganesh
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore – 635110, Tamil Nadu, India
A. L. Reyaz
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore – 635110, Tamil Nadu, India
N. Balakrishnan
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore – 635110, Tamil Nadu, India

Abstract


Bacillus thuringiensis Berliner (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, four indigenous Bt isolates (T489, T491, T495 and T532) were taken and investigated for their properties such as crystal morphology, cry gene(s) content and insecticidal activity. Bipyramidal, cuboidal and spherical crystals were observed. Among the four isolates only one isolate was found positive for lepidopteran specific cry genes. The isolate T532 which showed the presence of cry1, cry2Aa and cry2Ab genes was selected for protein profiling and bioassay. The isolate showed the presence of two proteins of molecular weights at ~135 and ~65 kDa sizes. Artificial diet based bioassay resulted in 100% mortality of the neonate larvae of H. armigera and S. litura for the isolate T532 which was comparable to that of the positive control (HD1). The full length cry2A gene was amplified and cloned into E. coli DH5α strain. The recombinant plasmids were sequenced and the sequence was deposited in GenBank nucleotide database. The accession number of the gene is MH475905. The gene was named by the Bacillus thuringiensis delta endotoxin nomenclature committee as cry2Aa23.

Keywords


Bacillus Thuringiensis, Cloning, Cry2Aa, PCR.

References





DOI: https://doi.org/10.18311/jbc%2F2018%2F21604