A Comparative Study on Isolation and Identification of Bacillus thuringiensis from Different Localities of Gujranwala City
Author's: Fakhar-un-Nisa Yunus, Faiza Kanwal, Farzana Rashid, Asfa Ashraf, Muhammad Naeem Iqbal, Shiwei Xiao
Corresponding Author: Muhammad Naeem Iqbal
Corresponding Author Email: email@example.com
Article Type: Research Article
Published Online: May. 14, 2016
This was a comparative study on isolation and identification of Bacillus thuringiensis isolates from different localities of city Gujranwala. Isolates of B. thuringiensis were identified using microscopic characters, colony morphology and biochemical characters. Then SDS-PAGE was performed to obtain a high protein profile on the basis of Cry proteins. A total of 74 out of 100 samples were found positive with B. thuringiensis. The maximum diversity of isolates was present in the soil (25%) as compared to other samples like dust (23.8%), cow dung (17.5%), donkey dung (17.5%) and bird droppings (16.2%). The average value of bacterial load was highest in soil (1 ± 0.192 CFU/ml) and lowest in bird droppings (0.650 ± 0.131 CFU/ml). The different sizes of bands with representative nine isolates from all sample sources on SDS-PAGE represent characteristics of crystal proteins having different target order specificity. It was concluded that B. thuringiensis is prevalent in various localities of the city. The need of the hour is to characterize these isolates and use them for pesticidal activity.
Bacillus thuringiensis, Cry proteins, SDS-PAGE, biopesticide.
Agriculture and forests are important resources which prolong the social, economical and ecological systems. The main concern is to protect these resources against the pests, but due to undesirable effects of chemical insecticides, biopesticides have been used greatly (Marrone, 1999). Most commonly, all animal species are being regulated by other living organisms known as antagonists that are present naturally and this phenomenon is known as Natural Biological Control (NBC).
Bacillus thuringiensis is a type of bacteria which is Gram positive and produces spores during sporulation. It has rod shape and it is about 1µm in width and 5 µm in length (Sakai et al., 2007). The genome size of B. thuringiensis ranges from a minimum of 2.4 to a maximum of 5.7 million bp. Many isolates contain numerous extra chromosomal parts which contain both globular as well as linear shapes (Carlson et al., 1994). There are almost 34 subspecies (also called serotypes or varieties) of B. thuringiensis and possibly over 800 strains or isolates (Lambert and Peferoen, 1992). Biopesticides based on the B. thuringiensis are extremely important and their percentage in the world’s biopesticide market is about ninety seven percent (Cannon, 1993).
Depending upon the composition of protoxin, crystals have numerous shapes such as Cry1 having bipyramidal, Cry2 having cuboidal, Cry3A having flat rectangular, Cry3B having irregular, Cry4A and Cry4B having spherical and Cry11A having rhomboidal shapes (Schnepf et al., 1998). Any particular bioactivity by B. thuringiensis is controlled via insecticidal crystalline proteins (ICPs) which are being codified in the specific genes known as cry genes so they are vulnerable towards specific insects like Coleopterans, Dipterans and Lepidopterans. Some other insect orders such as Homoptera, Hymenoptera, Dictyoptera and Mallophaga are also controlled by B. thuringiensis. In addition, several crystal toxins are also active against non-insect species such as roundworms, Acarid mites, Platyhelminthes along with protozoans (Marroquin et al., 2000). Both spores and ICPs produced by B. thuringiensis have been used to control insect pests since the 1920s (Peggy, 2008). Each proteolytically activated ICP molecule having insecticidal activity has two terminal domains. One of the domains is C-terminal that is unpredictable and specific for the identification of receptor of their hosts and other is an N- terminal domain that contains preserved sequences so brings toxicity by causing minute openings in the gut of insects (Li et al., 1991).
Since the first cry gene was cloned from B. thuringiensis then sequenced and expressed in Escherichia coli in 1981, more than 335 different genes have been described (Lecadet et al., 1999). From an ecological point of view natural habitat of B. thuringiensis is not exactly known. Historically, it has been found in soil, sick or deceased pest and stored food like tobacco, grain and flour (Hongyu et al., 2000) although it is discovered in dust, in deferral, on plant leaves, in food, animal skin and in the aquatic environment (Meadows et al., 1992; Akhurst et al., 1997; Bernhard et al., 1997; Smith and Barry, 1998; Maeda et al., 2000).
The present work was conducted to isolate and identify B. thuringiensis from different localities of city Gujranwala in order to describe the vast diversity of B. thuringiensis.
Akhurst, R.J., Lyess, E.W., Zhang, Q.Y., Cooper, D.J., Pinnock, D.E., 1997. A 16S r RNA gene oligonucleotide probe for identification of Bacillus thuringiensis isolates from sheep fleece. J. Invert. Pathol., 69: 24-31.
Bel, Y., Granero, F., Alberola, T.M., Sebastian, M.J., Ferre, J., 1997. Distribution, frequency and diversity of Bacillus thuringiensis in olive tree environments in Spain System. J. Appl. Microbiol., 20: 652-658.
Bergey, S.A., 1984. Bergey, Manual of Determinative Bacteriology, 9th edition, Williams & Wilkins., Philadelphia.
Bernhard, K., Jarret, P., Meadows, M., Butt, J., Ellis, D.J., Roberts, G.M., Pauli, S., Rodgers, P., Burges, H.D., 1997. Natural isolates of Bacillus thuringiensis: worldwide distribution, characterization and activity against insect pests. J. Invert. Pathol., 70(1): 59-68.
Buentello-Wong, S., Galan-Wong, L., Arevalo-Nino, K., Almaguer-Cantu, V., Rojas-Verde, G., 2015. Characterization of Cry Proteins in Native Strains of Bacillus thuringiensis and Activity against Anastrepha ludens. Southwest. Entomol., 40(1):15-24.
Cannon, R.J.C., 1993. Prospects and progress for Bacillus thuringiensis based pesticides. Pest. Sci., 37(4): 331-335.
Carlson, C.R., Caugant, D.A., Kolsto, A.B., 1994. Genotypic diversity among Bacillus cereus and Bacillus thuringiensis strains. Appl. Environ. Microbiol., 60(6): 1719-1725.
El-Didamony, G., 2014. Occurrence of Bacillus thuringiensis and their phages in Yemen soil. Virus dis., 25(1): 107-113.
GRAM, H. C 1884. Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten (in German). Fortschr. Med. 2: 185-189.
Hongyu, Z., Ziniu, Y., Wangxi, D., 2000. Composition and ecological distribution of Cry proteins and their genotypes of Bacillus thuringiensis isolates from warehouses in China. J. Invert. Pathol., 76: 191-197.
Iqbal, M.N., Anjum, A.A., Ali, M.A., Hussain, F., ALI, S., Muhammad, A., Irfan, M., Ahmad, A., Irfan, M. and Shabbir, A., 2015. Assessment of microbial load of un-pasteurized fruit juices and in vitro antibacterial potential of honey against bacterial isolates. Open Microbiol. J., 9: 26-32. DOI: 10.2174/1874285820150601E001.
Karim, S., Riazuddin, S., 1999. Rice insect pest of Pakistan and their control. A lesson from past for sustainable future integrated pest management. Pak. J. Biol. Sci., 2(2): 261-276.
Karnataka, J., 2009. Distribution of Bacillus thuringiensis Berliner strains in the soils of different habitats and their activity against white grubs. J. Agri. Sci., 22(3): 628-630.
Laemmli, U.K., 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature., 227(5259): 680-683.
Lambert, B., Peferoen, M., 1992. Insecticidal promise of Bacillus thuringiensis. Biosci., 42(2): 112-122.
Lecadet, M.M., Frachon, E., Dumanoir, V.C., Ripouteau, H., Hamon, S., Laurent, P., THIERY, I., 1999. Updating the H-antigen classification of Bacillus thuringiensis. J. Appl. Microbiol., 86(4): 660-672.
Li, J., Carrol, J., Ellar, D.J., 1991. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 Å resolutions. Nature., 353(6347): 815-821.
Maeda, M., Mizuki, E., Nakamura, Y., Hatano, T., Ohba, M., 2000. Recovery of Bacillus thuringiensis from marine sediments of Japan. Curr. Microbiol., 40(6): 418-422.
Makhdoom, R., 1997. Cloning and sequencing of the delta endotoxin gene from locally isolated Bacillus thuringiensis toxic against spotted bollworm. Ph.D. thesis. University of the Punjab, Lahore. 20-25.
Marrone, P.G., 1999. Microbial pesticides and natural products as alternatives. Agri. Outlook., 28(3): 149-154.
Marroquin, L.D., Elyassnia, D., Griffitts, J.S., Feitelson, J.S., Aroian, R.V., 2000. Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Genet. 155(4): 1693-1699.
Martin, P.A., Traverse, R.S., 1989. World-wide abundance and distribution of Bacillus thuirngiensis isolates. Appl. Environ. Microbiol., 55(10): 2437-2442.
Meadows, M.P., Ellis, D.J., Butt, J., Jarrett, P., Bruges, H.D., 1992. Distribution, frequency and diversity of Bacillus thuringiensis in an animal feed mill. Appl. Environ. Microbiol., 58(4): 1344-1350.
Moraga, E.Q., Tovar, E.G., Garcia, P.V., Alvarez, C., 2004. Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain. Res. Microbiol. 159(1): 59-71.
Peggy, L., 2008. Genetically Engineered Plants and Foods: A Scientist’s Analysis of the Issues (Part I). Annu. Rev. Plant Biol., 59: 771-812.
Sakai, H., Howlader, M.T.H., Ishida, Y., Nakaguchi, A., Oka, K., Ohbayashi, K., Yamagiwa, M., Hayakawa, T., 2007. Flexibility and strictness in functional replacement of domain III of Cry insecticidal proteins from Bacillus thuringiensis. J. Biosci. Bioeng., 103(4): 381-383.
Schnepf, E., Crickmore, N., Rie, J.V., Lereclus, D.J., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its Pesticidal Crystal Proteins. Microbiol. Mol. Biol. Rev. 62(3): 775-806.
Shapiro, J.A., 1995. The significances of bacterial colony patterns. BioEssays., 17: 597-607.
Shishir, A., Akter, A., Hassan, M.H., Kibria, G., Ilias, M., Khan, N.S., Hoq, M.M., 2012. Characterization of locally isolated Bacillus thuringiensis for the development of Eco-friendly Biopesticides in Bangladesh. J. Biopest., 5 (Supplementary): 216-222.
Smith, R.A., Barry, J.W., 1998. Environmental persistence of Bacillus thuringiensis spores following aerial application. J. Invert. Pathol., 71: 263-267.
Wakano, J.Y., Maenosono, S., Komoto, A., Eiha, N., Ymaguchi, Y., 2003. Self-organized pattern formation of a bacteria colony modeled by a reaction .diffusion system and nucleation theory. Phys. Rev. Lett., 90(25): 258102.
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