Antagonistic action of bacillus thuringinesis against pathogenic bacteria

AV Gomashe et al., IJSID, 2012, 2 (2), 304-309

International Journal of Science Innovations and Discoveries, Volume 2, Issue 2, March-April 2012

304

ANTAGONISTIC ACTION OF BACILLUS THURINGINESIS AGAINST PATHOGENIC BACTERIA

A. V. Gomashe*, A. R. Rewatkar, P. G. Dharmik,Shri Shivaji Science College, Congress Nagar, Nagpur-440012, Maharashtra, India

INTRODUCTION

INTRODUCTION

ISSN:2249-5347IJSID

International Journal of Science Innovations and Discoveries An International peerReview Journal for Science

Research Article Available online through www.ijsidonline.info

Received: 19.02.2012

Accepted: 30.04.2012

*Corresponding Author

Address:

Name:Dr. Ashok V. Gomashe

Place:

Shivaji Science College,

Congress Nagar, Nagpur

Maharastra, IndiaE-mail:[email protected]

ABSTRACT

Bacillus thuringiensis (Bt) is Gram positive bacterium that formcharacteristics protein i.e. crystal protein (cry protein). Bt cry proteins are diversegroup of proteins has insecticidal activity against various insects, nematodes andother pathogenic microbes. Therefore Bt has been used to control the effect ofentomopathogen on vertebrates. Their primary action is to lyses midgut epithelialcells by inserting into target membrane and forming pore and finally there isdeath of pathogenic microbes. In the given study, 96 hrs old Bt culture showshighest antagonist activity against Escherichia coli, Staphylococcus aureus, Proteus

spp. So therefore pathogenic microbes can be controlled by using 96 hrs old Btculture. From this investigation, due to insecticidal activity, Bt is used in control ofagricultural insect pest and insect vector of human disease.Keywords- Antagonist action, Bacillus thuringiensis, cry proteins, insecticidal

activity, pathogenic microbes, and sensitivity test.



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INTRODUCTION

Bacillus thuringiensis (Bt) is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide;alternatively, the Cry toxin may be extracted and used as a pesticide. B. thuringiensis also occurs naturally in the gut ofcaterpillars of various types of moths and butterflies, as well as on the dark surfaces of plants. (1)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 endotoxin and exotoxin production. (2)B. thuringiensis is closely related to B.cereus, a soil bacterium, and B.anthracis, the cause of anthrax: the threeorganisms differ mainly in their plasmids. Like other members of the genus, all three are aerobes capable of producingendospores.[1] Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal δ-endotoxins (called crystalproteins or Cry proteins), which are encoded by cry genes.[3] In most strains of B. thuringiensis, the cry genes are located onthe plasmid.[4][5][6]B. thuringiensis-based insecticides are often applied as liquid sprays on crop plants, where the insecticide must beingested to be effective. The solubilized toxins are thought to form pores in the midgut epithelium of susceptible larvae. Recentresearch has suggested the midgut bacteria of susceptible larvae are required for B. thuringiensis insecticidal activity.[7]Bt produce insecticidal proteins during the sporulation phase as parasporal crystals. These crystals are predominantlycomprised of one or more proteins (Cry and Cyt toxins), also called δ-endotoxins. These toxins are highly specific to theirtarget insect, are innocuous to humans, vertebrates and plants, and are completely biodegradable. Therefore, Bt is a viablealternative for the control of insect pests in agriculture and of important human disease vector (8). Cry proteins arespecifically toxic to the insect while Cyt toxins are mostly found in Bt strains active against Diptera. (9)

Mode of actionThe mode of action of Cry toxins has been characterized principally in insects. It is widely accepted that the primaryaction of Cry toxins is to lyse midgut epithelial cells in the target insect by forming pores in the apical microvilli membrane ofthe cells.Cry proteins pass from crystal inclusion protoxins into membrane-inserted oligomers that cause ion leakage and celllysis. The crystal inclusions ingested by susceptible larvae dissolve in the alkaline environment of the gut, and the solubilizedinactive protoxins are cleaved by midgut proteases yielding 60–70 kDa protease resistant proteins. Toxin activation involvesthe proteolytic removal of an N-terminal peptide and approximately half of the remaining protein from the C-terminus in thecase of the long Cry protoxins. The activated toxin then binds to specific receptors on the brush border membrane of themidgut epithelium columnar cells before inserting into the membrane. Toxin insertion leads to the formation of lytic pores inmicrovilli of apical membranes. Subsequently cell lysis and disruption of the midgut epithelium releases the cell contentsproviding spores a germinating medium leading to a severe septicemia and insect death (10)Bt subspecies are classified on the basis of serotype supplemented by morphological and biochemical criteria. Bt subspecies Kurstaki (Bt K) controls various types of lepidopterous insects. Bt subspecies tenebrions (Bt te) effective against certain beetle, species and the boll weevil, 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)



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Natural occurrence

Bacillus thuringiensis (Bt) occurs naturally in the soil and on plants. It is, a species that has been developed as amicrobial insecticide. Many include vegetables, cotton, tobacco, tree crops, forest crops, landscaping. Bt has been foundextensively in the phylloplane that is isolated from coniferous trees, deciduous trees, also spores persist in soil. It is alsoisolated from dead and drying insect. (12)Uses In 1995, potato plants producing Bt toxin were approved safe by the Environmental Protection Agency, making it thefirst pesticide-producing crop to be approved in the USA] By 1996, Bt maize, Bt potato and Bt cotton were being grown byfarmers in the USA. Bt crops (in corn and cotton) were planted on 281,500 km² in 2006 (165,600 km² of Bt corn and115900 km² of Bt cotton). This was equivalent to 11.1% and 33.6%, respectively, of global plantings of corn and cotton in2006.(13)There are several advantages in expressing Bt toxins in transgenic Bt crops that are the level of toxin expression canbe very high, thus delivering sufficient dosage to the pest, the toxin expression is contained within the plant system, hence onlythose insects that feed on the crop perish, the toxin expression can be modulated by using tissue-specific promoters, andreplaces the use of synthetic pesticides in the environment. The latter observation has been well documented worldwide. (13)Health and safetyOverall, Bt-modified crops appear to be environmentally safe. Bt toxins are a potential alternative to broad-spectruminsecticides. It is nontoxic to vertebrates and many beneficial arthropods, because Bt works by binding to the appropriatereceptor on the surface of midgut epithelial cells. Any organism that lacks the appropriate receptors in its gut cannot beaffected by Bt. (14) B. thuringiensis-based insecticides are often applied as liquid sprays on crop plants, where the insecticidemust be ingested to be effective. Bacillus thuringiensis serovar israelensis, a strain of B. thuringiensis is widely used as alarvicide against mosquito larvae, where it is also considered an environmentally friendly method of mosquito control.(15)Bt produced several toxins. The given experiment was carried out antagonist action of Bt against pathogenic bacterialike Escherichia coli, Staphylococcus aureus, Proteus species, and Pseudomonas species.

MATERIALS AND METHOD

Materials1) Bacillus thuringiensis2) Test organisms (Escherichia coli, Staphylococcus aureus, Proteus species, and Pseudomonas species)3) Mueller Hinton Agar,4) Nutrient broth.Method The test organisms used were provided by Government Medical College Nagpur. Sub- culturing was done on nutrientagar slants and was maintained in refrigerator at 90c. Given test organisms Escherichia coli, Staphylococcus aureus, Proteus

species, and Pseudomonas species inoculated in nutrient broth and incubated at 370c for 6-8 hrs, and for 24 hrs. 4 nutrientbroths of Bacillus thuringiensis is also inoculated in nutrient broth and incubated at 370c for 24hrs, 48hrs, and 96hrs. Bt cultureshows antagonist action against 4 test cultures by Kirby-Bauer test. Broth test culture (0.1ml) i.e. 24 hrs old culture inoculatedin the Mueller Hinton Agar plates. Wells are prepared with the help of autoclaved cork borer in Mueller Hinton Agar plates.Broth cultures of Bt (24 hrs old culture) poured in the well with the help of micropipette. Incubate the plate at 370c 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 24hrs, 48 hrs, 96 hrs Bt cultures.RESULTS AND DISCUSSIONSFigure 1 showed that 96 hrs Old Culture of Bacillus thuringiensis has antagonistic action against Escherichia coli,

Staphylococcus aureus, Proteus species, but not against Pseudomonas species. The first combination of 24 hrs Bt didn’t show anyresult. 96 hrs old Bt culture shows highest antagonist action against E. coli, S. aureus, Proteus spp.While from figure 2, maximum antagonistic action shown by Bt was towards Escherichia coli i.e. 26mm zone ofinhibition was observed, while Staphylococcus aureus, Proteus species shows 16mm and 11 mm zone of inhibition respectivelyand Pseudomonas species are resistant to Bt.B. thuringiensis is the most important bacilli reported to kill a wide range of insects like moths, beetle, mosquitoes,flies, aphides, insect, ants, fomites, midges, butterflies, even some pathogenic fungi such as pythium, ultium, Fusarium species,Lycopersici depending upon the host strains of the bacterium. (2) B. thuringiensis was rediscovered in Germany by ErnstBerliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars. In 1976, Robert A. Zakharyanreported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore andcrystal formation [16][17]. Cry proteins are defined as a parasporal inclusion protein from Bt that exhibits toxic effects to atarget organism, or any protein that has obvious sequence similarity to a known Cry protein Cyt toxins are included in thisdefinition but it was agreed that proteins that are structurally related to Cyt toxins retain the mnemonic Cyt (9)According to Cahon et al 2008 reported that Cyt 1Aa is endotoxin produced by Bt subsp. are activated againstpathogenic strain E.coli and S. aureus. These protieins found to be bactericidal for E.coli and bactriostatic for S. aureus.(18)Broderick et.al., 2006 showed that Bt is an opportunistic pathogen and have insecticidal activity against Enterobacteriaceaespp., E.coli and S. aureus (19). According to Sarker et.al., 2009, Bt is used as a pesticide. The inhibitor effect of Bt against

Bacillus subtillis, Pseudomonas aeruginosa was observed by disc diffusion method. It was found that Bt strain showed aninhibitory effect on all bacteria. (20)The present study is an attempt to find out the antagonist activity of Bt against pathogenic bacteria. It is evident thatthe Bt has antibacterial activity. This activity may be produced the production of bacteriocin or the endotoxin produced by Bt.The Bt is very well known for the production of crystal proteins having the insecticidal activity and also for its importance infisheries and agriculture. Cry proteins show toxicity to the pathogenic bacteria. In the current study, cry proteins in Btorganism shows lethal effect on Escherichia coli, Staphylococcus aureus, Proteus species by inhibition zone on Mueller Hintonplate.Table 1- Antagonist action of Bt culture against various test organisms

Hrs old broth culturecombination

Escherichiacoli

Staphylococcusaureus

Proteusspecies

PseudomonasspeciesPathogenicbacteria Bacillus

thuringiensis24 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



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Table 2- Antagonistic action of 96 hrs old Bt culture against 4 test organisms

Sr no. Test organisms Old Culture of Bt Zone of inhibition Zone in diameter1 Escherichia coli 96 hrs +ve 26mm2 Staphylococcus aureus 96 hrs +ve 16mm3 Proteus species 96 hrs +ve 11mm4 Pseudomonas species 96 hrs -ve 0mmLegend +ve = positive, -ve = negativeCONCLUSION

Bacillus thuringiensis occupies tremendous significance in agriculture due to exo and endo toxin production andrelated to mass production. Bt act as biopesticide in USA and now hundreds of Bt strains are commercially available. Theendotoxin is protein and prevalent to midgut. Bt produces several toxins but these are essentially nontoxic to human being,pests, and wildlife. Bt product are available in different forms and genetically engineered microorganisms. The currentexperiment concluded that 96 hrs old Bt culture shows antagonist activity against pathogenic bacteria. In that Escherichia coli,

Staphylococcus aureus, Proteus species are very sensitive to 96 Hrs Bt culture. So the exact mechanism and observation needsresearch which may help in development of a new type of product which can be used in agriculture and medicinal field.REFERENCES1. Madigan M, Martinko J (editors) Brock Biology of Microorganisms (11th ed.) (2005).. Prentice Hall.2. HIROETSU WABIKO, GARY A. HELD,t AND LEE A. BULLA, JR. “Protoxin Gene of Bacillus thuringiensis subsp. berliner 1715Is Necessary for Insecticidal Activity” APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1985, Vol. 49, No. 3p. 706-7083. Circkmore N. "Bacillus thuringiensis toxin nomenclature".http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html. Retrieved 2008-11-23.4. Stahly D.P "Biochemical Genetics of the Bacterial Insect-Control Agent Bacillus thuringiensis: Basic Principles andProspects Engineering.". Biotechnol. Genet. Eng. Rev. (1984). 84: 341–63. ISSN 0264-8725.5. Clayton C. Beegle and Takhashi Yamamoto "INVITATION PAPER ( C. P. ALEXANDER FUND) : History of Bacillusthuringiensis berliner research and development". Canadian Entomologist (1992). 124 (4): 587–616.doi:10.4039/Ent124587-4.6. XU Jian, LIU Qin, YIN Xiang-dong and ZHU Shu-de "A review of recent development of Bacillus thuringiensis ICP geneticallyengineered microbes". Entomological Journal of East China (2006). 15 (1): 53–58.7. 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): 15196–9. doi:10.1073/pnas.0604865103. PMC 1622799. PMID 17005725.http://www.pnas.org/cgi/pmidlookup?view=long&pmid=17005725.8. Bravo A, Gill SS, Soberón M. Comprehensive Molecular Insect Science. Elsevier BV; 2005. Bacillus thuringiensis Mechanismsand Use; pp. 175–206.9. Crickmore, N.; Zeigler, DR.; Feitelson, J.; Schnepf, E.; van Rie, J.; Lereclus, D.; Baum, J.; Dean, DH. Revision of thenomenclature for the Bacillus thuringiensis pesticidal crystal proteins; Microbiol MolBiol Rev. 1998. p. 807-813.http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html10. De Maagd RA, Bravo A, Crickmore N. “How Bacillus thuringiensis has evolved specific toxins to colonize the insect world”.Trends in Genet 2001;17:193–199. [PubMed: 11275324]



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11. Enhanced Toxicity of Bacillus thuringiensis Subspecies kurstaki and aizawai to Black Cutworm Larvae (Lepidoptera:Noctuidae) with Bacillus sp. NFD2 and Pseudomonas sp. FNFD1, Journal of Economic Entomology 104(1):41-46. Dec 2011 :Volume 104 Issue 6)12. Assaeedi, A. S. A.,1 Osman, G. E. H.1,2 and Abulreesh, H. H.”The occurrence and insecticidal activity of Bacillus thuringiensisin the arid environments” Australian journal of Crop Science, AJCS (2011)5(10):1185-119013. Brookes G, Barfoot P "GM crops: the first ten years - global socio-economic andenvironmentaimpacts"(PDF).http://www.pgeconomics.co.uk/pdf/global_impactstudy_2006_v1_finalPGEconomics.pdf.Retrieved 2008-11-23.14. Knowles BH "Mechanism of action of Bacillus thuringiensis insecticidal delta-endotoxins". Advances in Insect Physiology(1994). 24: 275–308. doi:10.1016/S0065-2806(08)60085-5.15. Broderick NA, Raffa KF, Handelsman J ("Midgut bacteria required for Bacillus thuringiensis insecticidal activity". Proc.

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Antagonistic action of bacillus thuringinesis against pathogenic bacteria

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