Abstract
The huge family status and diversified agricultural applications of Bacillus thuringiesnsis (Bt) Berliner made the bacterium, a highly specialized bio-insecticide with worldwide commercial usage. Novel strains and cry genes always expand potential usage of the bacterium. In the present investigation, the authors isolated 45 candidate strains of Bt from a unique ecological niche of Indian subcontinent, i.e., Uttarakhand state belongs to North Western Himalayas of India. All the isolates were characterized for light microscopy, PCR, SDS-PAGE and bioassays against major insect pests. The diversity in collection is manifested by existence of two major crystal shapes (Bipyramidal and spherical) and 21 different cry gene profiles with predominance of cry1 (cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1I) family genes followed by cry2 (cry2A, cry2B) and cry26. Interestingly, coleopteran specific genes were governed by cry8 not the cry3. The sequencing of major cry genes showed preferential divergence of cry2A genes and led to partial identification of novel cry1D gene from UKBt13. Majority of the isolates showed existence of 68 and 130 kDa crystal toxins. The bioefficacy studies presented here led us to identify putative novel strains with potential applications in pest management programs and associated toxicological diversity of Bt in Uttarakhand Himalayas.
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Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P (2011) Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol J 9:283–300. doi:10.1111/j.1467-7652.2011.00595.x
Meadows MP (1993) Bacillus thuringiensis in the environment: ecology and risk assessment. In: Entwistle P.F. et al. (ed) Bacillus thuringiensis, an environmental biopesticide: theory and practice John Wiley and Sons, New York. pp. 193–220
Nazarian A, Jahangiri R, Jouzani GS, Seifinejad A, Soheilivand S, Bagheri O, Keshavarzi M, Alamisaeid K (2009) Coleopteran-specific and putative novel cry genes in Iranian native Bacillus thuringiensis collection. J Invertebr Pathol 102(2):101–109
Crickmore N, Baum J, Bravo A, Lereclus D, Narva K, Sampson K, Schnepf E, Sun M, Zeigler DR (2016). “Bacillus thuringiensis toxin nomenclature” http://www.btnomenclature.info/ (assessed on 16th May, 2016)
Uribe D, Martinez W, Ceron J (2003) Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia. J Invertebr Pathol 82:119–127
Hastowo S, Lay BW, Ohba M (1992) Naturally occurring Bacillus thuringiensis in Indonesia. J Appl Bacteriol 73:108–113
Martin PA, Travers RS (1989) Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appli Environ Microbiol 55(10):2437–2442
Patel KD, Chudasama CJ, Ingle SS (2012) Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India. J Basic Microbiol 52:437–445
Das J Dangar TK (2007) Diversity of Bacillus thuringiensis in the rice field soils of different ecologies in India. Indian J Microbiol 47(4):364–368
Swamy HM, Asokan R, Nagesha SN, Arora DK, Birah A, Mahmood R (2011) Cloning, characterization and diversity of insecticidal crystal protein genes of Bacillus thuringiensis native isolates from soils of Andaman and Nicobar Islands. Curr Microbiol 63(5):420–425
Jain D, Kachhwaha S, Jain R, Kothari SL (2012) PCR based detection of cry genes in indigenous strains of Bacillus thuringiensis isolated from the soils of Rajasthan. Indian J Biotechnol 11(4):491–494
Kaur S, Singh A (2000) Natural occurrence of Bacillus thuringiensis in leguminous phylloplanes in the New Delhi region of India. World J Microbiol Biotechnol 16(7):679–682
Swamy HM, Asokan R, Mahmood R, Nagesha SN (2013) Molecular characterization and genetic diversity of insecticidal crystal protein genes in native Bacillus thuringiensis isolates. Curr Microbiol 66(4):323–330
Ramalakshmi A, Udayasuriyan V (2010) Diversity of Bacillus thuringiensis isolated from western ghats of Tamil Nadu state. India Curr Microbiol 61(1):13–18
Kumar K (2013) Molecular characterization of Lepidopteran specific Bacillus thuringiensis strains isolated from Hilly Zone Soils of Karnataka. India African J Biotechnol 12(20):2924
Swamy HM, Nagesha SN, Gowda TKS, Asokan R, Mahmood R (2012) Isolation, characterization and detection of cry gene contents from Bacillus thuringiensis native isolates in southern states of India. Plant Cell Biotechnol Molec Bio 13(1-4)
Shankar S, Jaiswal L, Kaur S (2010) Diversity of Bacillus thuringiensis isolated from diverse habitats of North India for the presence of cry gene profiles. J Appl Biosci 36:124–129
Tripathi M, Kumar A, Kalia V, Saxena AK, Gujar G (2016) Isolation and characterization of Lepidoptera specific Bacillus thuringiensis strains predominantly from North-Eastern states of India
Travers RS, Martin PAW, Reichelderfer CF (1987) Selective process for efficient isolation of soil Bacillus spp. Appl Environ Microbiol 56:1263–1266
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Finney DJ (1971) Probit Analysis, 3rd edn. Cambridge University Press, Cambridge
Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18(2):265–267
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Porcar M, JuaRez-PeRez VM (2003) PCR-based identification of Bacillus thuringiensis pesticidal crystal genes. FEMS Microbiol Rev 26(5):419–432
Bravo A, Sarabia S, Lopez L, Ontiveros H, Abarca C, Ortiz A, Ortiz M, Lina L, Villalobos FJ, Pena G, Valdez MEN, Soberon M, Quintero R (1998) Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl Environ Microbiol 64(12):4965–4972
Seifinejad A, Jouzani GS, Hosseinzadeh A, Abdmishani C (2008) Characterization of Lepidoptera-active cry and vip genes in Iranian Bacillus thuringiensis strain collection. Biol Cont 44(2):216–226
Wang JA, Boets J, VanRie G (2003) Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China. J Invertebr Pathol 82:63–71
Armengol G, Escobar MC, Maldonado ME, Orduz S (2007) Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects. J. Appli Microbiol 102(1):77–88
Ferrandis MD, Juarez-Pe´rez VM, Frutos R, Bel Y, Ferre J (1999) Distribution of cryI, cryII and cryV genes within Bacillus thuringiensis isolates from Spain. Syst Appli Microbiol 22:179–185
Sanchis VD, Lereclus G, Menou J, Chaufaux MM, Lecadet (1988) Multiplicity of δ-endotoxin genes with different specificities in Bacillus thuringiensis aizawai. Mol Microbiol 2:393–404
Bravo A, Likitvivatanavong S, Gill SS, Soberón M (2011) Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mole Biol 41(7):423–431
Devassy B, Chubicka T, Devak G, Nair H, Padikala J (2009) Novel Cytotoxic Proteins of Bacillus thuringiensis Isolated from Peninsular India. Can Res 69(24):3143
Walters FS, deFontes ChM, Hart H, Warren GW, Chen JS (2010) Lepidopteran active variable-region sequence imparts coleopteran activity in Cry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein. Appl Environ Microbiol 76:3082–3088
Mohan M, Gujar GT (2000) Susceptibility pattern and development of resistance in the diamondback moth, Plutella xylostella L, to Bacillus thuringiensis Berl var kurstaki in India. Pest Manag Sci 56(2):189–194
Estela A, Escriche B, Ferré J (2004) Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: noctuidae). Appli Environ Microbiol 70:1378–1384
Acknowledgements
This work was supported by the Indian Council of Agricultural Research (ICAR) and College of Agriculture, GBPUA and T, Pantnagar. The present work is a part of Ph.D thesis submitted to GBPUA and T, Pantnagar.
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The study reports isolation of 45 Bt strains from a novel ecological niche, the Uttarakhand Himalayas. The entomopathogenicity is manifested by novel cry gene combinations and toxicity against insect pests. The study also led to identification of four isolates likely to be biocontrol agents with evolutionary divergent cry2 genes.
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Subbanna, A.R.N.S., Khan, M.S., Stanley, J. et al. Diversity of Bacillus thuringiensis Isolates Native to Uttarakhand Himalayas, India and Their Bioefficacy Against Selected Insect Pests. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 88, 1489–1498 (2018). https://doi.org/10.1007/s40011-017-0892-6
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DOI: https://doi.org/10.1007/s40011-017-0892-6