AV Gomashe et al.

, IJSID, 2012, 2 (2), 304-309

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article

An International peer Review Journal for Science

Available online through www.ijsidonline.info A. V. Gomashe*, A. R. Rewatkar, P. G. Dharmik,

ANTAGONISTIC ACTION OF BACILLUS THURINGINESIS AGAINST PATHOGENIC BACTERIA Shri Shivaji Science College, Congress Nagar, Nagpur-440012, Maharashtra, India ABSTRACT characteristics protein i.e. crystal protein (cry protein). Bt cry proteins are diverse group of proteins has insecticidal activity against various insects, nematodes and other pathogenic microbes. Therefore Bt has been used to control the effect of entomopathogen on vertebrates. Their primary action is to lyses midgut epithelial cells by inserting into target membrane and forming pore and finally there is death of pathogenic microbes. In the given study, 96 hrs old Bt culture shows
Address: Name: Dr. Ashok V. Gomashe Place: Shivaji Science College, Congress Nagar, Nagpur Maharastra, India E-mail: drgomashe@rediffmail.com

Received: 19.02.2012 Accepted: 30.04.2012

*Corresponding Author

Bacillus thuringiensis (Bt) is Gram positive bacterium that form

highest antagonist activity against Escherichia coli, Staphylococcus aureus, Proteus spp. So therefore pathogenic microbes can be controlled by using 96 hrs old Bt culture. From this investigation, due to insecticidal activity, Bt is used in control of

INTRODUCTION agricultural insect pest and insect vector of human disease.

activity, pathogenic microbes, and sensitivity test.

Keywords- Antagonist action, Bacillus thuringiensis, cry proteins, insecticidal

INTRODUCTION

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AV Gomashe et al., IJSID, 2012, 2 (2), 304-309 alternatively, the Cry toxin may be extracted and used as a pesticide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on the dark surfaces of plants. (1) toxin and exotoxin production. (2) Bacillus thuringiensis (Bt) is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide; INTRODUCTION Bacillus thuringiensis bacterium is now widely known as Bt. some of the strains are pathogenic to snail, cockroaches

protozoan. Due to a wide range of host killing Bt occupies tremendous significance in agricultural and other field due to endo

organisms differ mainly in their plasmids. Like other members of the genus, all three are aerobes capable of producing the plasmid.[4][5][6] B. thuringiensis-based insecticides are often applied as liquid sprays on crop plants, where the insecticide must be

endospores.[1] Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal -endotoxins (called crystal proteins or Cry proteins), which are encoded by cry genes.[3] In most strains of B. thuringiensis, the cry genes are located on ingested to be effective. The solubilized toxins are thought to form pores in the midgut epithelium of susceptible larvae. Recent research has suggested the midgut bacteria of susceptible larvae are required for B. thuringiensis insecticidal activity.[7] target insect, are innocuous to humans, vertebrates and plants, and are completely biodegradable. Therefore, Bt is a viable specifically toxic to the insect while Cyt toxins are mostly found in Bt strains active against Diptera. (9) Mode of action Bt produce insecticidal proteins during the sporulation phase as parasporal crystals. These crystals are predominantly

B. thuringiensis is closely related to B.cereus, a soil bacterium, and B.anthracis, the cause of anthrax: the three

comprised of one or more proteins (Cry and Cyt toxins), also called -endotoxins. These toxins are highly specific to their alternative for the control of insect pests in agriculture and of important human disease vector (8). Cry proteins are The mode of action of Cry toxins has been characterized principally in insects. It is widely accepted that the primary

action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming pores in the apical microvilli membrane of

the cells.Cry proteins pass from crystal inclusion protoxins into membrane-inserted oligomers that cause ion leakage and cell lysis. The crystal inclusions ingested by susceptible larvae dissolve in the alkaline environment of the gut, and the solubilized case of the long Cry protoxins. The activated toxin then binds to specific receptors on the brush border membrane of the providing spores a germinating medium leading to a severe septicemia and insect death (10) Bt subspecies Kurstaki (Bt K) controls various types of lepidopterous insects. Bt subspecies isralensis (Bt i) effective against mosquitoes, blackflies and some midges Bt subspecies japonensis (Bt j) effective against many species of scarabid beetles Bt subspecies aizawai (Bt a) effective against wax moth larvae in honeycombs. (11) Bt subspecies are classified on the basis of serotype supplemented by morphological and biochemical criteria.

inactive protoxins are cleaved by midgut proteases yielding 6070 kDa protease resistant proteins. Toxin activation involves midgut epithelium columnar cells before inserting into the membrane. Toxin insertion leads to the formation of lytic pores in microvilli of apical membranes. Subsequently cell lysis and disruption of the midgut epithelium releases the cell contents Bt subspecies tenebrions (Bt te) effective against certain beetle, species and the boll weevil,

the proteolytic removal of an N-terminal peptide and approximately half of the remaining protein from the C-terminus in the

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AV Gomashe et al., IJSID, 2012, 2 (2), 304-309 Natural occurrence microbial insecticide. Many include vegetables, cotton, tobacco, tree crops, forest crops, landscaping. Bt has been found isolated from dead and drying insect. (12) Uses Bacillus thuringiensis (Bt) occurs naturally in the soil and on plants. It is, a species that has been developed as a

extensively in the phylloplane that is isolated from coniferous trees, deciduous trees, also spores persist in soil. It is also first pesticide-producing crop to be approved in the USA] By 1996, Bt maize, Bt potato and Bt cotton were being grown by 115900 km of Bt cotton). This was equivalent to 11.1% and 33.6%, respectively, of global plantings of corn and cotton in 2006.(13) There are several advantages in expressing Bt toxins in transgenic Bt crops that are the level of toxin expression can In 1995, potato plants producing Bt toxin were approved safe by the Environmental Protection Agency, making it the

farmers in the USA. Bt crops (in corn and cotton) were planted on 281,500 km in 2006 (165,600 km of Bt corn and

be very high, thus delivering sufficient dosage to the pest, the toxin expression is contained within the plant system, hence only replaces the use of synthetic pesticides in the environment. The latter observation has been well documented worldwide. (13) Health and safety Overall, Bt-modified crops appear to be environmentally safe. Bt toxins are a potential alternative to broad-spectrum

those insects that feed on the crop perish, the toxin expression can be modulated by using tissue-specific promoters, and insecticides. It is nontoxic to vertebrates and many beneficial arthropods, because Bt works by binding to the appropriate

receptor on the surface of midgut epithelial cells. Any organism that lacks the appropriate receptors in its gut cannot be

affected by Bt. (14) B. thuringiensis-based insecticides are often applied as liquid sprays on crop plants, where the insecticide like Escherichia coli, Staphylococcus aureus, Proteus species, and Pseudomonas species. MATERIALS AND METHOD Materials 1) Bacillus thuringiensis 3) Mueller Hinton Agar, Method 4) Nutrient broth. Bt produced several toxins. The given experiment was carried out antagonist action of Bt against pathogenic bacteria

must be ingested to be effective. Bacillus thuringiensis serovar israelensis, a strain of B. thuringiensis is widely used as a

larvicide against mosquito larvae, where it is also considered an environmentally friendly method of mosquito control.(15)

2) Test organisms (Escherichia coli, Staphylococcus aureus, Proteus species, and Pseudomonas species) The test organisms used were provided by Government Medical College Nagpur. Sub- culturing was done on nutrient

agar slants and was maintained in refrigerator at 9 0c. Given test organisms Escherichia coli, Staphylococcus aureus, Proteus shows antagonist action against 4 test cultures by Kirby-Bauer test. Broth test culture (0.1ml) i.e. 24 hrs old culture inoculated International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

species, and Pseudomonas species inoculated in nutrient broth and incubated at 37 0c for 6-8 hrs, and for 24 hrs. 4 nutrient in the Mueller Hinton Agar plates. Wells are prepared with the help of autoclaved cork borer in Mueller Hinton Agar plates.

broths of Bacillus thuringiensis is also inoculated in nutrient broth and incubated at 370c for 24hrs, 48hrs, and 96hrs. Bt culture Broth cultures of Bt (24 hrs old culture) poured in the well with the help of micropipette. Incubate the plate at 37 0c for 24hrs.

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After incubation the plates are observed for zone of inhibition. Same method is follow for 6-8 hr old test organisms with 24 hrs, 48 hrs, 96 hrs Bt cultures. RESULTS AND DISCUSSIONS Staphylococcus aureus, Proteus species, but not against Pseudomonas species. The first combination of 24 hrs Bt didnt show any result. 96 hrs old Bt culture shows highest antagonist action against E. coli, S. aureus, Proteus spp. and Pseudomonas species are resistant to Bt.

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inhibition was observed, while Staphylococcus aureus, Proteus species shows 16mm and 11 mm zone of inhibition respectively Lycopersici depending upon the host strains of the bacterium. (2) B. thuringiensis was rediscovered in Germany by Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars. In 1976, Robert A. Zakharyan reported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore and crystal formation [16][17]. Cry proteins are defined as a parasporal inclusion protein from Bt that exhibits toxic effects to a target organism, or any protein that has obvious sequence similarity to a known Cry protein Cyt toxins are included in this definition but it was agreed that proteins that are structurally related to Cyt toxins retain the mnemonic Cyt (9) Broderick et.al., 2006 showed that Bt is an opportunistic pathogen and have insecticidal activity against Enterobacteriaceae inhibitory effect on all bacteria. (20) According to Cahon et al 2008 reported that Cyt 1Aa is endotoxin produced by Bt subsp. are activated against B. thuringiensis is the most important bacilli reported to kill a wide range of insects like moths, beetle, mosquitoes,

While from figure 2, maximum antagonistic action shown by Bt was towards Escherichia coli i.e. 26mm zone of

Figure 1 showed that 96 hrs Old Culture of Bacillus thuringiensis has antagonistic action against Escherichia coli,

flies, aphides, insect, ants, fomites, midges, butterflies, even some pathogenic fungi such as pythium, ultium, Fusarium species,

pathogenic strain E.coli and S. aureus. These protieins found to be bactericidal for E.coli and bactriostatic for S. aureus.(18) spp., E.coli and S. aureus (19). According to Sarker et.al., 2009, Bt is used as a pesticide. The inhibitor effect of Bt against The present study is an attempt to find out the antagonist activity of Bt against pathogenic bacteria. It is evident that Bacillus subtillis, Pseudomonas aeruginosa was observed by disc diffusion method. It was found that Bt strain showed an The Bt is very well known for the production of crystal proteins having the insecticidal activity and also for its importance in fisheries and agriculture. Cry proteins show toxicity to the pathogenic bacteria. In the current study, cry proteins in Bt plate. Table 1- Antagonist action of Bt culture against various test organisms Hrs old broth culture Escherichia Staphylococcus Proteus Pseudomonas combination coli aureus species species Pathogenic Bacillus bacteria thuringiensis 24 hrs 24 hrs 6-8 hrs 24 hrs 6-8 hrs 48 hrs 6-8 hrs 96 hrs + + + Legend - = No zone of inhibition, + = zone of inhibition in mm International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

the Bt has antibacterial activity. This activity may be produced the production of bacteriocin or the endotoxin produced by Bt. organism shows lethal effect on Escherichia coli, Staphylococcus aureus, Proteus species by inhibition zone on Mueller Hinton

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AV Gomashe et al., IJSID, 2012, 2 (2), 304-309 Sr no. 1 2 3 4 Table 2- Antagonistic action of 96 hrs old Bt culture against 4 test organisms Test organisms Escherichia coli Staphylococcus aureus Proteus species Pseudomonas species Old Culture of Bt Zone of inhibition 96 hrs +ve 96 hrs +ve 96 hrs +ve 96 hrs -ve Legend +ve = positive, -ve = negative CONCLUSION Zone in diameter 26mm 16mm 11mm 0mm

related to mass production. Bt act as biopesticide in USA and now hundreds of Bt strains are commercially available. The endotoxin is protein and prevalent to midgut. Bt produces several toxins but these are essentially nontoxic to human being, research which may help in development of a new type of product which can be used in agriculture and medicinal field. 1. 2. 3. 4. 5. 6. 7. 8. 9. Madigan M, Martinko J (editors) Brock Biology of Microorganisms (11th ed.) (2005).. Prentice Hall. REFERENCES 708 Circkmore N. "Bacillus thuringiensis toxin nomenclature".

pests, and wildlife. Bt product are available in different forms and genetically engineered microorganisms. The current Staphylococcus aureus, Proteus species are very sensitive to 96 Hrs Bt culture. So the exact mechanism and observation needs HIROETSU WABIKO, GARY A. HELD,t AND LEE A. BULLA, JR. Protoxin Gene of Bacillus thuringiensis subsp. berliner 1715 http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html. Retrieved 2008-11-23. Prospects Engineering.". Biotechnol. Genet. Eng. Rev. (1984). 84: 34163. ISSN 0264-8725. thuringiensis doi:10.4039/Ent124587-4. berliner research and development". Canadian Entomologist

Bacillus thuringiensis occupies tremendous significance in agriculture due to exo and endo toxin production and

experiment concluded that 96 hrs old Bt culture shows antagonist activity against pathogenic bacteria. In that Escherichia coli,

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Broderick NA, Raffa KF, Handelsman J "Midgut bacteria required for Bacillus thuringiensis insecticidal activity". Proc. Natl. Acad. Sci. U.S.A. (October 2006). 103 (41): 151969. doi:10.1073/pnas.0604865103. PMC 1622799. PMID 17005725. Bravo A, Gill SS, Sobern M. Comprehensive Molecular Insect Science. Elsevier BV; 2005. Bacillus thuringiensis Mechanisms Crickmore, N.; Zeigler, DR.; Feitelson, J.; Schnepf, E.; van Rie, J.; Lereclus, D.; Baum, J.; Dean, DH. Revision of the 813.http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html Trends in Genet 2001;17:193199. [PubMed: 11275324]

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