Rolul lui Bacillus turigiensis.pdf

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Indian Journal of Biotechnology Vol 2, July 2003, pp 302-321 Advances in Pest Control: The Role of Bacillus thuringiensis S R Prabagaran, K R Rupesh, S J Nimal, S Sudha Rani and S Jayachandran* Department of Biotechnology, Pondicherry University, Pondicherry 605 014, India Received 9 January 2003; accepted 20 February 2003 Bacillus thuringiensis (Bt), a popularly known biopesticide, has widest scope in skirmishing diverse species of insects. Numerous reports have appeared on the ecology and distribution of Bt around the world. Characterizing Bt strains based on cry gene contents envisages more on its application potential. In the recent past, organizing the cry gene nomenclature based on protein/nucleotide sequence has overcome many ambiguities that persisted for long. This initiative of the decade has paved way to face the challenges of new additions in cry genes and assigning its phylogenetic position. In vitro evidences on insect mortality are most often not reproducible under field conditions. Therefore, numerous formulations have been developed by various entrepreneurs to combat insect pest menace. In malevolence of all advantages, development of resistance is the greatest threat to Bt indnstry and environmentalists as well. Though several alternative methods are practiced, it needs enormous effort to really understand the mode of development of resistance and to combat it accordingly. In agrarian and health sector, development of transgenic organisms is the recent trendsetter deserving attention. However, there is a cause for concern in advocating transgenics since the long-term effects of transgenics on the living organisms is not well understood. After weighing the advantages and disadvantages of application of Bt in the environment, it could be concluded that Bt definitely offers best scope in being biodegradable, non-toxic, target specific and most importantly renewable compared to chemical insecticides for the control of insect-pests. Keywords: Bacillus thuringiensis, cry genes, o-endotoxins, insect resistance, formulations, transgenics. Introduction Bacillus thuringiensis (Bt) is an ubiquitous gram positive, spore forming bacterium that forms parasporal crystal inclusions normally during the stationary phase of its growth cycle. Since the early 1900's Bacillus group has received great attention for its use as a biopesticide against a variety of insect- pests. The proteinaceous inclusions of Bt are called as crystal proteins or o-endotoxins, which are toxic especially to the class Insecta. Hence, preparations of Bt are being used as bioinsecticides for the control of certain insect species belonging to the orders Lepidoptera, Diptera and Coleoptera (Beegle & Yamamoto, 1992). It is also well-documented that the encoded products of cry genes of certain Bt are toxic against other insect orders such as Hymenoptera, Homoptera, Orthoptera and Mallophaga and against certain nemotodes, mites and protozoa (Feitelson et al, 1992). Bt is a well accepted eco-friendly alternative to chemical pesticides used in agriculture, forest management and health care. It has become one *Author for correspondence: Tel.: 91-413-2655715,2655991; Fax: 91-413-2655265 E-mail: [email protected] of the nodal organism, which serves as a gene pool for transgenic crops and microorganisms to confer insect resistance. Bt is widely distributed in various habitats. Besides biochemical and morphological characteristics, the diversity in Bt is studied using flagellar H-antigen, agglutination reactions (Lecadet et al, 1999), polymerase chain reactions (PCR), cry genes (Kalman et al, 1993), ELISAIWestern blotting of total proteins (Prabagaran, 2002), plasmid analysis (Lereclus et al, 1982), restriction patterns (Carlson et al, 1996), Bioassays (Kranthi et al, 2000), etc. Classification of cry genes of Bt has taken various shapes over the decades and the latest one by Crickmore et al (1998) is considered as the acceptable one, being incorporated with latest available resources and grouping methods. This classification not only accepts the newer submission of novel cry genes but is also designed in such a way to position phylogenetically, based on the amino acid homology. Regulation of cry gene(s) expression and post- transcriptional modification of cry proteins have been reviewed by several workers (Rajamohan & Dean, 1996; Schnepf et ai, 1998). The mechanism of action of Bt cry proteins involves solubilization of the

Transcript of Rolul lui Bacillus turigiensis.pdf

Page 1: Rolul lui Bacillus turigiensis.pdf

Indian Journal of BiotechnologyVol 2, July 2003, pp 302-321

Advances in Pest Control: The Role of Bacillus thuringiensis

S R Prabagaran, K R Rupesh, S J Nimal, S Sudha Rani and S Jayachandran*Department of Biotechnology, Pondicherry University, Pondicherry 605 014, India

Received 9 January 2003; accepted 20 February 2003

Bacillus thuringiensis (Bt), a popularly known biopesticide, has widest scope in skirmishing diverse species ofinsects. Numerous reports have appeared on the ecology and distribution of Bt around the world. Characterizing Btstrains based on cry gene contents envisages more on its application potential. In the recent past, organizing the crygene nomenclature based on protein/nucleotide sequence has overcome many ambiguities that persisted for long.This initiative of the decade has paved way to face the challenges of new additions in cry genes and assigning itsphylogenetic position. In vitro evidences on insect mortality are most often not reproducible under field conditions.Therefore, numerous formulations have been developed by various entrepreneurs to combat insect pest menace. Inmalevolence of all advantages, development of resistance is the greatest threat to Bt indnstry and environmentalistsas well. Though several alternative methods are practiced, it needs enormous effort to really understand the mode ofdevelopment of resistance and to combat it accordingly. In agrarian and health sector, development of transgenicorganisms is the recent trendsetter deserving attention. However, there is a cause for concern in advocatingtransgenics since the long-term effects of transgenics on the living organisms is not well understood. After weighingthe advantages and disadvantages of application of Bt in the environment, it could be concluded that Bt definitelyoffers best scope in being biodegradable, non-toxic, target specific and most importantly renewable compared tochemical insecticides for the control of insect-pests.

Keywords: Bacillus thuringiensis, cry genes, o-endotoxins, insect resistance, formulations, transgenics.

IntroductionBacillus thuringiensis (Bt) is an ubiquitous gram

positive, spore forming bacterium that formsparasporal crystal inclusions normally during thestationary phase of its growth cycle. Since the early1900's Bacillus group has received great attention forits use as a biopesticide against a variety of insect-pests. The proteinaceous inclusions of Bt are called ascrystal proteins or o-endotoxins, which are toxicespecially to the class Insecta. Hence, preparations ofBt are being used as bioinsecticides for the control ofcertain insect species belonging to the ordersLepidoptera, Diptera and Coleoptera (Beegle &Yamamoto, 1992). It is also well-documented that theencoded products of cry genes of certain Bt are toxicagainst other insect orders such as Hymenoptera,Homoptera, Orthoptera and Mallophaga and againstcertain nemotodes, mites and protozoa (Feitelson etal, 1992). Bt is a well accepted eco-friendlyalternative to chemical pesticides used in agriculture,forest management and health care. It has become one

*Author for correspondence:Tel.: 91-413-2655715,2655991; Fax: 91-413-2655265E-mail: [email protected]

of the nodal organism, which serves as a gene pool fortransgenic crops and microorganisms to confer insectresistance.

Bt is widely distributed in various habitats. Besidesbiochemical and morphological characteristics, thediversity in Bt is studied using flagellar H-antigen,agglutination reactions (Lecadet et al, 1999),polymerase chain reactions (PCR), cry genes (Kalmanet al, 1993), ELISAIWestern blotting of total proteins(Prabagaran, 2002), plasmid analysis (Lereclus et al,1982), restriction patterns (Carlson et al, 1996),Bioassays (Kranthi et al, 2000), etc.

Classification of cry genes of Bt has taken variousshapes over the decades and the latest one byCrickmore et al (1998) is considered as the acceptableone, being incorporated with latest available resourcesand grouping methods. This classification not onlyaccepts the newer submission of novel cry genes butis also designed in such a way to positionphylogenetically, based on the amino acid homology.Regulation of cry gene(s) expression and post-transcriptional modification of cry proteins have beenreviewed by several workers (Rajamohan & Dean,1996; Schnepf et ai, 1998). The mechanism of actionof Bt cry proteins involves solubilization of the

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crystal in the insect midgut, proteolytic processing ofthe protoxins by midgut proteases, binding of crytoxin to midgut receptors, and insertions of the toxininto the apical membrane to create ion channels orpores (Schnepf et aI, 1998). Inspite of all advantagesthat Bt offers, the greatest backlog is the developmentof resistance by insect species (Tabashnik et aI, 1994;Escriche et al, 1995). Several such incidences areencountered while managing several pests in differentcountries which underscore a critical need forincreased emphasis to concentrate in this area ofresearch to manage resurgence.

Expression of cry genes in alternate hosts is of lastdecade's trend by the molecular biologists(Merryweather et al, 1990; Crickmore et al, 1998). Inorder to increase the toxicidal spectrum, Bt strainshave been manipulated to harbour combination ofgenes by conjugation and electroporation. Attemptswere made to over-express such cry genes inalternative hosts like Pseudomonas sp., Rhizobioumsp., Bacillus sp., Azospirillum sp., Deinococcus sp.,etc. Several firms were ambitious to introduce crygene(s) in major crops like maize, brassiccas,soybean, cotton, etc. so as to evade insect-pestsespecially internal feeders in crop protection.

Based on the current level of utilization theworldwide sales of Agrochemicals in 2006 areforecasted as $ 28,000 million of which, insecticidesalone will contribute $ 7611 million. The market forBt based biopesticides is still less than 1% of theworld's crop protection market (Navon, 2000). Thebig constraint in this minimal acceptance by thefarmers and health agencies is its production cost.Inspite of the substantial information on developmentof Bt formulation, several problems still remainunresolved in reducing the loss against UV light, rain,etc. While, health workers encourage encapsulation inbiopolymers for slow release in aquatic habitats,additives like wetting agents, stickers, sunscreens,synergists, and phago stimulants are recommended forformulation in the agrarian sector (Navon, 2000).Greater emphasis is given nowadays to reduce theproduction cost by employing cost-effectivebiological resources, which are mostly by-products ofone industry. Several fermentation systems are alsodeveloped to compete with the emerging needs for theproduction of Bt products. In this review the authorshave attempted to give comprehensive up-to-dateinformation on all aspects of Bt and the readers aresuggested to refer to the articles of Schnepf et al

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(1998), Yang and Wang (1998) and Sharma et al(2000) for details other than those covered in thisreview.

Ecology and OccurrenceVarious workers have reported isolation of Bt from

soil (Carozzi et al, 1991; Hastowo et aI, 1992; Landenet aI, 1994; Bernhard et al, 1997; Hossain et al, 1997;Theunis et aI, 1998), water (Ichimatsu et aI, 2000),saw dust (Bravo et al, 1998; Helgason et al, 1998;Hongyu et al, 2000) grain dusts (Jung et al, 1997),dead insects (Chilcott & Wigley, 1993; Bernhard etaI, 1997), dried tobacco leaves (Kaelin & Gadani,2000), warehouses (Hongyu et al, 2000), storedproducts (Bernhard et al, 1997; Chaufaux et aI, 1997),phylloplane (Bernhard et al, 1997; Bora et al, 1994),compost (Bernhard et aI, 1997), mammal faeces(Theunis et al, 1998), etc. Out of the 2362 samplescollected from stored products, soil, insect residues,plant material and other unusual materials, 5303crystal forming isolates were obtained (Bernhard etal, 1997). Bravo et al (1998) who had isolated Btstrains in 456 of the 503 Mexican soils also suggestedthat such collection has greatest value, since samplescollected were from different climatic regions with ahigh diversity of insects. Recently, Kaelin and Gadani(2000) reported that 9% of 132 samples of curedtobacco leaves of different types and originscontained Bt. The compiled data on the distribution ofBt isolates from different ecological horizons indifferent countries in the world are given in Table 1.

Numerous Bt subspecies have been isolated fromcadavers (dead) or moribund (dying insect) larvae andin most cases the isolate has toxic activity to theinsect from which it was isolated (Paily et al, 1987;Hansen et aI, 1996). These organisms have a narrowhost range in the orders Coleoptera, Diptera andLepidoptera and can proliferate within the bodies oftheir host insects. When the infected insect larva dies,the dead insect carcass usually contains relativelylarge quantities of spores and crystals that may bereleased into the environment. Following the earlyisolation of Bt from dead insect larvae, Bt was foundubiquitously by using a novel enrichment techniquethat exploits unique germination properties of thespores (Martin & Travers, 1989). One of the mostimportant aspects about establishing a Bt collection isto have a methodology with which one can rapidlyand accurately characterize the strain, the toxinprotein and the gene. This is especially important if

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Table I--Distribution of Bt in different environments

Origin Source Bt, Isolates Methodology used Referenceobtained

Bangladesh Soil (17) 650 Bioassay, Crystal Morphology Hossain et al, 1997Soil 650 Bioassay Hossain et al, 1997

Eygpt Bioassay, Electron micrcorcopy Hameed et al, 1990China Soil Bioassay, Serotyping Li et al, 1991

Forest soil (384) Bioassay Oai et al, 1994Soil (1491) 221 Bioassay, Serotyping Oai et al, 1996Warehouses PCR, SOS-PAGE, Bioassay, Hongyu et al, 2000

SerotypingChina/World Soil (486) 85 Serotyping , Bioassay Hongyu et al, 2000Columbia Phylloplane (35) 256 Bioassay, Crystal, PCR Maduell et al, 2002

Mud (64) 264 Crystal morphology, Larvicidalactivity, Biochemical characteristics,Antibiotic sensitivity, Bioassay,

Greece Soil 11.2% of SOS-PAGE, Crystal morphology Aptosoglou et al, 1997total

India Soil (4) 4 Bioassay Shakoori et al, 1999Soil Check Crystal morphology, SOS-PAGE, PCR Prabagaran, 2002Soil 1 Bioassay, SOS-PAGE Manonmani and Balaraman,

2001Soil, insect (18) 18/583 Crystal morphology, SOS-PAGE, PCR, Prabagaran, 2002.

Plasmid pattern, Western blotting,Bioassay

Israel Soil (126) 38 PCR Ben-Dov et al, 1997Japan Water sources (107) 195 Serotyping, Bioassay, Crystal Ichimatsu et al, 2000

morphologyJordan Different habitats 80 Crystal morphology, SOS-PAGE, Al-Momani et al, 2002

Plasmid, SerotypingKorea Granery (411) 163 PCR, Plasmids, Serotyping Kim et al, 1998

Oust 4 SOS-PAGE, PCR, Crystal morphology, Jung et al, 1997ONA hybridization, Bioassay

Farm soil, Granery 45 Bioassay, Serotyping Kim, 2000Soil 58 Serotyping, Crystal morphology, PCR, Kim, 2000

Plasmid, BioassayMexico soil (503) 496 PCR, serotyping Bravo et al, 1998New Zealand Soil, insect habitats 6909 Bioassays, SOS-PAGE, Crystal Chilcott & Wigley, 1993

and larvae (455) morphologyNigeria Soil 6 Serotyping, PCR Ogunjimi et al, 2000Malaysia 1 Bioassay, SOS-PAGE, Plasmid, Kawalek et al, 1995

Western, Southern blotMexico Larvae Crystal morphology, SOS-PAGE, Lopex-Meza & Ibarra, 1996

BioassaySoil (503) 496 PCR, Bioassay Bravo et al, 1998(5) 2 AFLP, 16s rRNA Ticknor et al, 2001

Norway Soil 154 Bioassay, Transposons, SOS-PAGE, Helgason et al, 1998Serotyping

Philippines Miscellaneous (801) 3950 ELSIA, PCR, Bioassay Theunis et al, 1998

Soil, dust, larvae, 3950 Bioassay, PCR, SOS-PAGE, Theunis et al, 1998faeces, compost, , Immunoblotting(457)

Sweden Soil (12) 35 Biochemical, Crystal morphology, Landen, 1994secretion of InA, Resistance topenicillin G, Plasmid pattern

Spain Soil, dust, insects 1401 SOS-PAGE, Serotyping, Bioassay Iriarte et al, 1998(301)

Contd.

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Source

Table I-Distribution of Bt in different environments-Contd.

ReferenceOrigin Bt. Isolatesobtained

Methodology used

Terresterial andaquatic habitatsDead larva

22 serovars Serotyping, PCR, Bioassay,

Porcar & Caballero, 2000

Martinez & Caballero, 2002

Taiwan 225501477

Crystal morphology, Plasmid, SDS-PAGE, PCR, BioassayPCRSDS-PAGE, Bioassay, PCRBioassay, Crystal morphology

Chak et al, 1994Chak et al, 1994Meadows et al, 1992

SerologyFatty acid compositionPCR, Sugar fermentation, Antibioticsensitivity, GrowthBioassayBioassay, PCRBioassays, Crystal morphology, SDS-PAGE, Western blotCrystal morphology,. Bioassay

Soil

UnitedKingdomUnited states

Stored product (36)

Soil (115)ContainersSoil

25027.9%95

Worlwide Soil, (785)Soil (148)Tobacco leaves andinsects (20)Stored products,soils, insects, plantmaterials, etc, (2363)Tobacco leaves (133)

891665178

5303

24 PCR, Crystal morphology Kaelin & Gadani, 2000

the differences among endotoxin genes, carried by acertain strain, are critical for its specificity andtoxicity. The bioassay analysis is an exhaustive andtime-consuming process because it is necessary toscreen all the isolates in all the target insects. Variousmethodologies, viz. Southern blot analysis in searchof homologous genes (Kronstad & Whiteley, 1986),reactivity to different monoclonal antibodies (Hofte &Whiteley, 1989), electrophoretic analysis of PCRproducts (Perez et al, 1997), phospholipid and fattyacid analysis (Siegel et al, 1995; Hathout et al, 2000),16S rRNA sequence comparison (Giffel et ai, 1997;Joung et al, 2001), amplified length polymorphism(Keirn et ai, 1997; Ticknor et ai, 2001), genomicrestriction digests (Schnepf et al, 1998), SDS-PAGEanalysis of total proteins (Helgason et al, 1998), etc.have been described to simplify this process. Amongall the approaches, SDS-PAGE and PCR areconsidered to be the best choice because both themethods are highly sensitive, relatively fast and canbe used routinely (Kumar et al, 1996).

Soil being the repository of all living forms of life,it supports maximum number of Bt isolates. Based onthe present data available, it is well-documented thatBt are target specific. The target specificity of the crytoxin proteins is mostly because of a) specificity oftoxin receptor binding in the gut and b) the three-dimensional configuration of protein that enables thistoxin to bind to the receptor (Schnepf et al, 1998). In

DeLucca et al, 1981Siegel et al, 2001Ejiofor & Johnson, 2002

Martin and Travers, 19R9Ceron et al, 1994Kaelin et al, 1994

recent times many workers have established andidentified a variety of cry genes of different molecularweight (Chilcott & Wigley, 1993), expresseddifferentially (Helgason et al, 1998) and virulent tovarying levels to different insects (Frankenhuysen etal, 1992). Greater emphasis is given nowadays toidentify cry genes which are novel exhibiting eitherwider spectrum of activity for different insect pests orgreater virulence per se to any specific insect inquestion (Crickmore et al, 1998; Schnepf et al, 1998).In this direction Prabagaran et al (2002) have recentlyshown the presence of different cry gene families in avariety of Bt isolates obtained from the Indian soils.The greater divergence of different cry genes withrespect to molecular weight, distribution andpreponderance, supports the general view that the crygenes are too diverse in nature and the presence ofdifferent permutations and combinations of cry genesis a rule of thumb in the ecosystem.In the light of significance attached to evolution,

the remarkable diversity of Bt strains and toxins isdue, at least in part, to a high degree of geneticplasticity. It is fascinating that Bt has evolved an arrayof molecular mechanisms to produce large amounts ofpesticidal toxins after cessation of growth(Rajamohan & Dean, 1996). The significance of Btusing several cry gene expression systems is notknown. Co-expression of multiple toxins, no doubt,will increase the virulence of the toxin besides

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increasing the host range of a given strain or of apopulation exchanging toxin genes. In a naturalpopulation of Bt inhabiting the soil, cry genecontaining plasmid transfer between Bt strains is anestablished phenomenon observed by many workers(Thomas et al, 2001).

Though several authors have studied and identifiedmany functional (Jenson et al, 1995; Gonzalez-Cabrera et al, 2001) as well as cryptic cry genes(Lereclus et al, 1983) in diverse Bt isolates obtainedfrom divergent samples, differential expression of crygenes offers evolutionary significance over otherbacteria. Masson et al (1998) and Theunis et al (1998)studied such expressions and concluded that not allcry genes are expressed. Prabagaran et al (2002) alsoclaimed that amongst three of their isolates studied,two strains showed elevated levels of expression ofcry genes. Based on in vitro methods of testing, astrain can be claimed as toxic only if its toxic activityis exhibited in laboratory/field testing. Hence,priority-wise one can conclude that crystalmorphology, SDS-PAGE, PCR, western blotting,bioassay and field experimentation are essential forsuch studies on Bt.

Most of the workers have not taken sibling strainsinto their consideration. Bravo et al (1998) haveemphasized such grouping on siblings. ThoughPrabagaran et al (2002) have isolated 583 strains fromdifferent agro-climatic regions in India, based oncrystal morphology and SDS-PAGE profile of crytoxin proteins, only 18 strains were subjected todetailed investigations based on PCR product ofamplified cry genes. Several workers (Bernhard et al,1997; Hongyu et al, 2000) have grouped such hugepopulations based on one or other methods. Moreoften it is considered worthwhile to undertake in-depth studies on a limited Bt population that holdsgreater promise for the control of insect-pests ratherthan to take a large population with no provenpotential. Statistical analysis with thousands of Btstrains regarding potency generally does not have anypractical value. On the contrary, studies with selectedelite strains with novel gene or high potency will havehigh commercial value under field conditions. Hence,workers can restrict their studies to handle limitednumber of strains after grouping from a bigpopulation in either of the methods and explore thepossibilities of such superior strains with highapplication potential.

Insecticidal Crystal ProteinsDuring sporulation, Bt synthesizes a protoxin

crystal, known as 8-endotoxins (or Cry protein), asinclusion bodies against Lepidopteran, Dipteran andColeopteran insect-pests (Li et al, 1991). Cry proteinshave a range of about 130-140 kDa, depending on thesubtypes. Over 100 cry genes have been sequencedand a few have been expressed in E. coli after cloning(Rajamohan & Dean, 1996). Cry proteins wereoriginally classified by several workers with arbitrarydesignation from the working groups as icp(McLinden et al, 1985), cry (Donovan et al, 1988;Ward & Ellar, 1987), kurhdl (Geiser et al, 1986), Bt a(Sanchis et al, 1989), Btl, Bt2, etc (Hofte et al, 1986),type B and type C (Hofte et al, 1988), protein size(Kronstad & Whiteley, 1986) and vip (Estruch et al,1996). However, the classification proposed by Hofteand Whitely (1989) was widely accepted by thescientific community over a long period. Accordinglycry genes were classified into four major classesbased upon their protein toxicity towards insects andprimary reactivity with corresponding genes viz. cry](lepidopteran specific), cryll (lepidopteran anddipteran specific), cryIll (coleopteran specific) andcryIV (dipteran specific).

However, with spurt in scientific knowledge,several working groups (Crickmore et al, 1998)reported Bt toxins with a range of toxicity and withcross-reacting groups. This posed problem whileclassifying the organism based on the classificationsuggested by Hofte and Whiteley (1989). Toovercome the chaotic and confounding confusionsthat existed in the classification suggested by Hofteand Whiteley (1989), Crickmore et al (1998) deviseda new nomenclature system, which provided a usefulframework for classifying the expanding cry genes.Thus, the inconsistencies that existed in the previousclassification system to accommodate genes that werehighly homologous to known genes but did notencode a toxin with a similar insecticidal spectrumwas also solved. With the wealth of data produced bygenomic sequencing efforts, a new nomenclaturalparadigm emerged by assigning names to members ofgene superfamilies according to their degree ofevolutionary divergence as estimated by phylogenetictree algorithms. Such nomenclature format is designedto convey rich informational content about theserelationships, which allows appending of new crygenes along with details on phylogenetic divergencewith already reported genes. Thus, sequence-based

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nomenclature allows closely related toxins to begrouped together and minimized bioassays of eachnew protein against a growing series of organismsbefore assigning a valid name. Based on phylogeneticrelationships and ranking, the cry genes are nowassigned with an Arabic number (primary level of45% identity), followed by an uppercase letter(secondary: 75% relatedness), lowercase letter(tertiary: 95% identity) and Arabic number(Quaternary with further relatedness). With thesequence information of the coding region of a crygene suspected, one can easily locate its phylogeneticposition and claim to be novel after such analysis(Crickmore et al, 1998). Besides, an advisorycommittee on cry gene nomenclature has been set-up,which is responsible for analyzing andaccommodating new cry genes. An official websitewith other relevant information is also set-up with thefollowing URL http://www.biols.susx.ac.uk/Home/Neil jCrickmore/B, thuringiensis/ for thispurpose. The cyt genes, which have different mode ofaction, and newly added vip genes are grouped inseparate linkages. The phylogenetic tree depicting thenumber of cry gene submissions at the time ofpreparation of this review is given in Fig. 1.

Basically, the structure of the cry toxins revealsfive conserved amino acid blocks, concentratedmainly in the centre of the domain or at the junctionbetween domains (Rajamohan & Dean, 1996). It isknown that the Cry toxin consists of 3 domains, eachwith a specific function (Fig. 2). Domain I is made upof seven a helices, which is responsible for insertinginto the gut membrane and creating a pore (Steve,1995, Park & Federici, 2000). Domain II appears as atriangular column of 3 anti-parallel p-sheets, which issimilar to antigen-binding regions ofimmunoglobulins, which implies that it is responsiblefor binding to the receptors on the epithelial linings ofthe insect midgut. Domain III contains antiparallel Pstrand in a P sandwich form (Li et al, 1991; Schnepfet al, 1998). Though the structure of domain III hasbeen elucidated, its function remains obscure. Somesuggest it might serve to protect the Cry toxin frominappropriate cleavage by gut proteases (Li et al,1991), others suggest it may be involved with ionchannel formation, receptor binding and insectspecificity (Rajamohan et al, 1998). It is suspectedthat many cases of insect resistance to specific cryproteins may be due to altered receptor-binding. Justas antibodies contain conserved and variable domains,

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so are toxin genes and proteins made up of alternatingconserved and variable regions. The N-terrninal partof the toxin protein is responsible for its toxicity andspecificity and contains five conserved regions. TheC-terminal part is usually highly conserved andprobably responsible for crystal formation. For moredetailed information on the structure-functionrelationship of Cry proteins the readers can refer torecent articles of Gazit et al (1997), Butko et al(1997) and Schnepf et al (1998).

Mode of Action of Cry ProteinsWhen delivered as parasporal crystal form, the

mechanism of action of the Bt cry proteins involvesthree major steps: (1) solubilization and activation ofthe crystal in the insect midgut, (2) binding of theactivated toxins to receptors on the epithelial liningsin the midgut, and (3) insertion of the activated toxininto the midgut apical membrane to create ionchannels or pores (Hill & Pinnock, 1998). Uponingestion of the endotoxin, crystalline inclusions aredissolved in alkaline pH of the gut juices to yield theprotoxin. Subsequently upon proteolytic activation,considerable stretch of C terminal is removed leavingthe functional toxic domain at the trimmed N terminal(Tojo & Aizawa, 1983). The major proteases of thelepidopteran insect midgut are trypsin-like (Milne &Kaplan, 1993) or chymotrypsin-like (Johnston et al,1995; Peterson et al, 1995; Novillo et at, 1997).

The activated toxin binds readily to specificreceptors on the apical brush border of the midgutmicrovillae of susceptible insects. Binding is a two-stage process involving reversible (Hofmann & Luthy,1986; Hofmann, et al, 1988) and irreversible (Van Rieet at, 1989; Ihara et al, 1993; Rajamohan et al, 1995)steps. The latter steps may involve a tight bindingbetween the toxin and receptor, insertion of the toxininto the apical membrane, or both. It has beengenerally assumed that irreversible binding isexclusively associated with membrane insertion (Iharaet al, 1993; Rajamohan et al, 1995). Insertion into theapical membrane of the columnar epithelial cellsfollows the initial receptor-mediated binding,rendering the toxin insensitive to proteases andmonoclonal antibodies (Wolfersberger, 1990) andinducing ion channels or non-specific pores in thetarget membrane. In vitro electrophysiological studiesof voltage clamping of lipid bilayers (Slatin et al,1990; Schwartz et al, 1991) and sections of wholeinsect midguts (Rajamohan et al, 1995) support the

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308 INDIAN J BIOTECHNOL, JULY 2003

functional role of the toxin in pore or ion channelformation. The nature of the ion channel or pore-forming activity of cry toxins in the insect is stillcontroversial. The pores are K+ selective (Sacchi etal, 1986), permeable to cations (Wolfersberger,

1~

1989), anions (Hendrickx et al, 1989), and solutessuch as sucrose, irrespective of the charge (Schwartzet al, 1991). Besides all conditions, cessation of K+pump leads to swelling of columnar cells and osmoticlysis (Knowles & Dow, 1993). The disruption of gut

4" ... IM.~,~. <".;t'I~

~.Jl~I MAl

V1~i ry1

'y... ~. ~l

•...

~....--t

rylJ(--!' .~,-L

roo- l ,lib

--•... ..

'--- .~yeca

~~.7ftI"

...- '---I 11lb{I---

~'All

I- --J

~- ··It---' eIry I - .1:.,.

fill1MJlM

Fig. I-Updated phylogenetic tree showing the amino acid sequence identity of cry proteins (Reproduced with kind permission fromCrickmore et al, 1998).

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••Domain I Domain II Domain 1Il

Fig. 2-Position of conserved block of a representative cryprotein. Sequence blocks are shown as dark grey, which showhigh degree of homology to the conserved block. Generalstructural features of proteins as deduced from amino acidsequences. Regions marked 1 to 5 are highly conserved blocks invarious cry genes (Schnepf et al, 1998).

integrity leads to death of the insect throughstarvation or septicemia (Sharma et al, 2000).

Insecticidal crystal proteins of Bt bind to receptorsin the midgut of susceptible insects leading to poreformation and death of the insect. The identity of thereceptors has not been clearly established. Recently, adirect interaction between a cloned andheterologously expressed amino peptidase (slapn)from Spodoptera litura and the CrylC protein wasdemonstrated (Rajagopal et al, 2002) byimmunofluorescence and in vitro ligand blotinteraction where it reduced its expression. The genesilencing was retained during the insect's moultingand development and transmitted to the subsequentgeneration albeit with a reduced effect. These resultsdirectly implicate larval midgut amino peptidase N asreceptor for Bt insecticidal proteins.

Other major group of proteins excluding cry andcyt proteins is the vegetative insecticidal proteins(VIP1, VIP2 and VIP3). Vegetative cell supernatantsof Bt and B. cerus have been shown to be toxic tovarious pests (Estruch et al, 1996, 1997). Thoughthese were assumed to be similar to cry proteins, theyinduce gut paralysis, followed by complete lysis ofthe gut epithelium cells resulting in larval mortality(Sharma et al, 2000).

Bioassay and Field EvaluationThere is a need of new ways for protecting plant

crops against predators and pathogens while avoidingthe use of environmentally aggressive chemicals tomeet the demands for food for the expanding worldpopulation (Carlini & Grossi-de-Sa, 2002). In thiscontext several biopesticides are in market of whichBt stands high in its usage and performance.Bioassays are routinely used to detect the potency ofcommercial and experimental Bt preparations. They

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are tested in vitro by allowing reared larvae or in vivoby exposing to field conditions (Dulmage et al, 1970).

Martin and Travers (1989) found 40.3% of theirisolates to be active against lepidoptera and 22.7%against mosquitoes. Chilcott and Wigley (1993)observed that the percentage of strains obtained fromthe soil with toxicity against the lepidopteran larvaealone ranged from 37-88%, 43-100% against thedipteran, 0-6% against the coleopteran, 45-77% bothto lepidopteran and dipteran and 20-60% with noactivity against lepidopteran, dipteran or coleopteran.Bernhard et al (1997) tested single-dose assays of5303 Bt isolates against Agrotis ipsilon, 5136 againstH. virescens, 3077 against Peris brassicae, and 3028against S. littoralis.

The biopesticides, viz. Centari, Delfin and Dipel8C were found to cause cent percent mortality within48 hrs after treatment (Shenmar & Brar, 1997).Vaidya (1997) recorded 80% larval mortality after48 hrs of exposure to Bt formulation Centari 3G (B.thuringiensis var. aizawaii at 0.1% concentration.Similarly, Frankenhuyzen et al (1993) reported thetoxicity of crylC against Spodoptera spp., crylDagainst S. littoralis and cry 1E against S. exigua. Bai etal (1993) observed minimal sensitivity of crylAb,crylB, crylD, crylAa and crylAc to S. exempta.Frankenhuyzen et al (1993) also suggested thatcrylAa, crylAb, crylAc, crylB and crylC were toxicto P. xylostella. Of the crylAa, crylAb, crylAc,crylC, crylD and crylAa genes present in Bt activeagainst P. xylostella, crylAb was significantly toxic(Monnerat et al, 1999). The LTso values of H.armigera were less than 24 hrs for Bt at a PCPconcentration of 10 ug/g of synthetic diet (Ingle et al,1997). Natarajan and Srinivasan (1999) recorded100%, 85% and 20% mortality in first, second andthird instar larvae of H. armigera, respectively at aPCP concentration of 20 ug/ml.

Employing Principle Component Analysis (PCA)Leong et al (1980) reported that pathogenicity is thecombined effect of multiple environmental factorslike sunlight, leaf temperature and vapour pressuredeficit. Navon (2000) based on his studies hasindicated that insecticidal power of Bt is: a) ins tardependent; b) solar sensitive; c) dependent onmicrobial load and d) presence of allelochemicals onthe phylloplane. S. litura is an important polyphagouspest of cultivated crops primarily in the tropical andsubtropical regions. Insecticides are often usedpreventively to suppress Spodoptera populations from

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reaching the economic threshold level (Moar et al,1990). Such decreasing population in A. janata, S.litura, Sylepta sp. were observed in field byVimaladevi et al (1996), Kalia et al (1997) andPrabagaran et al (2002), respectively.

B. sphaericus and Bt var. israelensis were studiedfor their toxicity and resistance development byPoopathi et al (2000) who suggested that tolerancelevel did not differ significantly in all the lethalconcentrations they have studied. Paily et al (1987)had demonstrated the efficacy of the freeze-driedpowder preparation of a B. sphaericus mutant againstCulex tritaeniorhynchus. Mittal et al (2001) revealedthat among the two Bt formulations they employedfor their study, Vectobac was more toxic to A. aegyptiand A. stephensi. Most granule formulations ofmicrobial agents such as Bt. var. israelensis yieldedsignificant control of immature mosquitoes for up to7-9 days (Seleena et al, 1999; Nguyen et al, 1999;Nayar et al, 1999).

Puntambekar et al (1997) screened different Btstrains against lepidopteran pests on pigeon pea underlaboratory and field conditions and concluded thatthere was substantial increase in pod yields whencompared to untreated control. A semi field methodwas conducted in tubs to evaluate the efficacy andresidual activity of granular formulations of Bt var.isralensis by Ali et al (1994), who have reportedappreciable larval mortalities up to 9 clays post-treatment.

The objectives of most of the above mentioned invitro bioassay experiments and field evaluations areultimately to explore the potential of Bt for the solepurpose of controlling pests that infect cultivatedcrops. Though extensi ve work has been carried out onthe molecular mechanisms underlying structure of crygenes and the expression of cry toxin proteins in Bt,very meagre information is available on the fieldexperiments to test the efficacy of Bt in controllinginsect-pests infecting multitudes of crops. It is to beremembered that the final test to prove the efficacy ofa Bt strain lies in its supremacy in controlling insect-pest(s).

Insect Resistance to B. thuringiensisIn spite of earlier view that insects would not,

develop resistance to microbial insecticides, it nowappears that resistance in insect population against Btevolves rapidly under extreme selection pressure inlaboratory or field. Resistances in various insects viz.

Plutella xylostella, Ploidia interpunctella andHeliothis virescens were very well demonstrated(Raymond, 1993).

Previous genetic and biochemical analyses of insectstrains with resistance to Bt toxins indicate (Gould etal, 1992) that (i) resistance is restricted to singlegroups of related Bt toxins, (ii) decreased toxinsensitivity is associated with changes in Bt-toxinbinding to sites in brush-border membrane vesicles ofthe larval midgut, and (iii) resistance is inherited as apartially or fully recessive trait. If these threecharacteristics were common to all resistant insects,specific crop-variety deployment strategies couldsignificantly diminish problems associated withresistance in field populations of pests. Gould et al(1992) suggested that Bt toxin resistance in H.virescens, where the resistance is not accompanied bysignificant changes in toxin binding, and resistance isinherited as an additive trait when larvae are treatedwith high doses of CryIA(c) toxin. Despite successfuland extensive use of these toxins in transgenic crops,little is known about toxicity and resistance pathwaysin target insects since these organisms are not idealfor molecular genetic studies (Marroquin et al, 2000).

Poopathi et al (2000) showed that, even whenrecessive, resistant mutants could rapidly increase infrequency, providing some interactions that protectCulex pipiens from disappearance. Grifftts et al(2001) reported the cloning of a Bt toxin resistancegene, bre-5, in Caenorhabditis elegans whichencoded a putative ~-1,3-galactosyltransferase. Lackof bre-5 in the intestine led to resistance to the Bttoxin crySB.

Field-collected colony of the diamondback moth,P. xylostella, which showed 31-fold resistance toCrylC protoxin of Bt was studied by Zhao et al(2000) where after 24 generations of selection withCrylC protoxin in transgenic broccoli expressing aCry 1C protein, the resistance that developed was highenough that neonates of the resistant strain couldcomplete their entire life cycle on transgenic broccoliexpressing high levels of Cry 1C.

Ferre and Van Rie (2002) have reviewed thecurrent knowledge on the biochemical mechanismsand genetics of resistance to Bt products andinsecticidal crystal proteins. The understanding of thebiochemical and genetic basis of resistance to Bt canhelp design appropriate management tactics to delayor reduce the evolution of resistance in insectpopulations.

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Liu et al (2001) suggested that slower developmentof resistant insects on Bt cotton could increase theprobability of mating between resistant adults andaccelerate resistance. Further, negative effects of Btcotton on the survival and development of resistantlarvae could delay evolution of resistance. Carriere etal (2001) reported simulation models that suggestmanipulation of planting date and implementation ofother control cultural methods to reduce the rate ofapplication of insecticides and delay the evolution ofresistance to Bt in the pink bollworm. Gonzalez-Cabrera et al (2001) reported for the first time thatPlutella xylostella showed more than one geneconferring resistance to the same Cry toxin.

Dominance has been assessed in different ways ininsecticide resistance studies, based on threephenotypic traits: (1) the insecticide concentrationrequired to give a particular mortality (DLC); (2)mortality at a particular insecticide dose (DML); and(3) fitness in treated areas (DWT). Bourguet et al(2000) declared that DWT, rather than DML, isrelevant to the resistance management strategy, whichthey suggest to focus on fitness dominance levels inthe presence and absence of insecticide. Geneticanalysis revealed that B. sphaericus resistance wasinherited as a recessive trait and controlled by a singlemajor locus (Wirth et al, 2000). Liu et al (2000)found that resistance in Pectinophora gossypiella wascodominant at a low concentration of CrylAc,partially recessive at an intermediate concentration,and completely recessive at a high concentration.

Transgenic Microorganisms and PlantsWith the advent of recombinant DNA technology,

development of transgenic microbes containing crytoxin genes from Bt and Bt var. israelensis hasbecome possible and easier. Transgenicmicroorganisms are usually applied either as foliarsprays or powder/granules in the soil. While B.megaterium (Bora et al, 1994), Pseudomonas sp.(Skot et al, 1990; Obukowicz et al, 1986 a; b) andClavibacter xyli (Lampel et al, 1994) are applied assprays, Azospirillum sp. (Udayasuriyan et al, 1995),Rhizobium leguminosarum and Pseudomonas cepacia(Skot et al, 1990) and P. fluorescens (Stone et al,1989) are applied as soil formulations for the controlof multiple crop pests that infect different cultivatedcrops. In the aquatic system efficient delivery foreffective control of insect-pests was achieved byapplying transgenic B. sphaericus (Bar et al, 1991;

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Poncet et al, 1994), Caulobacter crescentus(Thanabalu et al, 1992), Anabaena sp.(Manasherob etal, 1998), Synechococcus sp. (Soltes-Rak et al, 1993)and Agmanellum quadruplicatum (Stevens et al,1994) containing various cry toxin genes. Theinherent problems viz., narrow specificity, short shelf-life, low potency, lack of systemic activity, presenceof viable spores, etc., that are associated with wildtype Bt and Bt var. israelensis, which were the majorconstraints and limitations in the large-scale use of Btwere greatly overcome by the transgenic microbeapproach (Lambert & Peferoen, 1992).

The location of cry gene(s) in plasmids in Bt hasenabled construction of transgenic microbes bysimpler techniques such as conjugal transfer, plasmidcuring, etc. (Wiwat et al, 1996) in early stages. Usingsuch approach, Ecogen Corporation has come outwith transgenic strains for commercial exploitation(Gawron-Burke & Baum, 1991). Bora et al (1994)have demonstrated that the transconjucant, B.megaterium, isolated from the cotton phyllospherecontained the cry toxin gene from Bt var. kurstaki andwas effective in controlling cotton bollworms. Similarsuch studies were carried out using transgenicPseudomonas against Helicoverpa in cotton. Anefficient alternative method was designed by Lerecluset al (1992), where insertion sequence IS232 wasengaged for expression of cryIIIA gene into anotherisolate, which produced crylAc protein. In yetanother strategy, non-sporulating mutants of Bt,which overexpressed crystal proteins, were developedfor application in silkworm rearing regimes (Lerecluset al, 1995).

An elegant and perhaps the most effective deliverysystem for Bt toxins is the transgenic plant (Stewart etal, 1997). Since, it has become possible to expressexotic genes into the plant genome that conferresistance to insects, such transgenic plants provideprotection independent of weather conditions anduntil the end of crop. This approach offers effectivecontrol of insects, which feed from interior part ofplants where it is difficult to applyinsecticide/biopesticide to control all the stages ofinsect development. With the advent of moleculartechniques like genetic engineering, almosttransformation of all the dicotyledonous crops is nowfeasible ranging from Agrobacterium approach toparticle bombardment protocol. Several crops havebeen genetically modified to express Bt toxins tomanage major insect-pests. In tobacco, the transgenic

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technology favoured remarkable results from 1987,where cloning of Bt crystal protein genes in aT-DNAvector was expressed using Agrobacteriumtumefaciens (Barton et al, 1987; Fischhoff et al, 1987;Vaeck et al, 1987). However, transgenic crops ofcotton, potato, brassicas and com were allowed tocommercial level only from 1996 (McLaren, 1998).Since then several vectors with the entire codingsequence for the CrylAa, CrylAb or CrylAcprotoxins were available for use in crop scienceengineering (Barton et al, 1987; Vaeck et al, 1987).Several promoters were available in plant systembased on the place of delivery of crystal protein intransgenic plants. The level of expression withchimeric crystal protein genes was typically lowerthan those obtained with other chimeric trans genes inplants which indicated that the coding region of crygenes inhibits the efficiency of expression in plantsystem (Koziel et al, 1993). Several truncatedexpression of Cry proteins in crops also conferredgood resistance in tomato and cotton plants (Fischhoffet al, 1987), which proved to resist infection by H.virescens and Helicoverpa zea larvae. With atruncated CrylAb gene under the control of a wound-stimulated promoter of potato, several potato varietieswere developed to resist potato tuber moth larvae

(Phthorimaea opercuiella) (Peferoen et al, 1990).Transgenic tobacco plants (Warren et al, 1992)expressing truncated crystal protein genes alsoshowed considerable level of insecticidal activity.However, truncated gene expression and use ofdifferent promoters with enhancer sequences andfusion proteins conferred only limited levels in cropimprovement and its pest management (Barton et al,1987; Vaeck et al, 1987; Carozzi et al, 1992). A listof transgenic microbes and plants containing singleand combinations of cry genes is given in Table 2.

FermentationBecause of the proven potential of Bt in controlling

a variety of insect-pests of agricultural and publichealth importance, concerted efforts have been madein recent times for large-scale multiplication of Bt forwide distribution to the agrarian sector as well aspublic health authorities. Of the many factors thatcontrol large-scale production of biopesticides, cost ofproduction is the primary factor. Therefore,optimization of media constituents so as to minimizethe cost of production rules high in the minds offermentation technologists. Many workers, in theirendeavour to develop suitable media for fermentativeproduction of Bt have proposed the use of cheap

CroplMicrobe Cry gene

Table 2-·List of certain transgenic microbes and crops containing cry toxin genes

Reference

MicrobesPseudomonas sp.Agrobacterium radiobacterPseudomonas fluorescensClavibacter xyli subsp cyanodontisClavibater xyliAzospirillum sp.B. sphaericus

CrylAbCrylAb

CrylAcCrylAc

Most genes

E. coli Cry4A, cryllacytlAa andp20MosquitocidalCrylVA

AnabaenaCyanobacteriaCropsTobacco CRY1Aa

Brassica napus CrylAc

Rice CrylAc

Potato CrylllACotton Cry2AbSoybean CrylAcMaize Bt toxin

Method

Transposon tn5Obukowicz et al, 1986Obukowicz et al, 1986

Intergative plasmidTurner et al, 1991Lampel et al, 1994Udayasurian et al, 1995Bourgouin et al, 1990Bar et ai, 1991Khasdan,2oo1

Shuffling

Manasherob et al, 1998Soltes-Rak et al, 1993

CrylA Barton et al, 1987;Fischhoff et al, 1987Vaeck et al, 1987Stewart et al, 1996Agrobacterium. Mediated

transformationAgrobacterium. Mediatedtransformation, biolistic gunTouchdown PCR method

Khanna & Raina, 2002

Mel'nychuk et al, 2002Tabashnik et al, 2002Zeng et al, 2002Reuter et al, 2002

Particle bombardmentBt toxin against Ostrinia nubilalis

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carbon sources like jaggery, com, black strapmolasses, whey, etc. (Pearson & Ward, 1988; Hameedet al, 1990; Ejiofor et al, 1991). The primarydeterminant in the large-scale production of Bt ismost often the carbon source. Great attention has beenpaid in finding out cheap utilizable carbon sources,which at the same time are eco-friendly and non-polluting.

During fermentation, Bt vegetatively multipliesuntil it reaches early stationary phase (Avignone-Rossa et al, 1992). When a critical nutrientcomponent becomes depleted, it commits to sporulateconcomitantly producing crystal inclusion, whichharbours o-endotoxin (Priest, 1992). After completionof sporulation, cells lyse to release spores and crystalproteins in the medium where Bt is grown.

Great strides with regard to optimization of mediafor fermentative production of therapeutic agents(Bongaerts et al, 1997), organic acids (Rychlik et al,2000), beverages (Guerra et al, 2001), etc. haverecently been made. Strangely, not much progressseems to have been achieved on the optimization ofnutrient parameters for mass multiplication of Bt. Thedevelopment of a fermentative process is largelyempirical and must be determined separately for eachBt strain selected. The goal in commercial productionof Bt is to make the maximum amount of activeingredient and final formulated product for theminimum cost (Koziel et al, 1993).

When formulating the nutrient medium, carbon isprovided by mono, di and poly saccharides such asglucose, starch, molasses, etc. If their concentration istoo high, the pH will drop below 5.6 to 5.8 and aciditymay prevent growth. It depends on balancing the levelof saccharide and the source of nitrogen in as much asBt is producing alkaline components from thenitrogen bearing material and these can neutralize theacidic products. Usually the medium is with the initialpH of 6.8 to 7.2, which will drop to 5.8-6.0 and thenwill rise towards 8.0-8.3.

Bulla et al (1980) and Luthy et al (1982)demonstrated that glucose was assimilated throughEMP pathway. Subsequently the acetate formed in theTCA cycle served as energy for growth andsporulation. Because there exists a distinct differencein the utilization of acetate in the presence of glucosein the medium in Bt (Liu et al, 1994) and E. coli(Yang et al, 1992) many workers have recommendedthe use of glucose for fermentative production of Bt.Since most of the industrially useful strains are not

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able to utilize high levels of sugars, such as sucrose,lactose, etc. (Bulla et al, 1980), attempts have beenmade to use industrial by-products for fermentativeproduction of Bt. Prabagaran (2002) employed locallyavailable inexpensive raw materials like fish meal,molasses, sago, paddy straw and whey forfermentative production of Bt and showed that fishmeal and molasses supported maximum growth.

Both inorganic (Avignone-Rossa et al, 1990) aswell as organic nitrogen sources (Ampofo, 1995) havebeen employed for the production of Bt in thelaboratory. Bulla et al (1980) and Areas (1984) havereported that inorganic ammonium compound such asammonium sulfate, did not support the growth of Bt.However, organic nitrogen sources such as meatpreparation, fish meal and soybean flour werereported to support rapid growth (Yang et al, 1998).Though different amino acids were also shown(Avignone-Rossa & Mignone, 1995) to support goodbiomass production and toxin protein synthesis in Bt,their prohibitive cost prevented amino acids frombeing used as the source of nitrogen. On the otherhand, in continuous culture, proteins, instead ofglucose, was found to be a major limiting factor forgrowth of cells (Chang et al, 1993). In addition, thebatch-to-batch uniformity of fermentable substratesmay prove to be critical for development of an easilyreproducible process.

Wakisaka et al (1982) reported that metal ions suchas Ca2+ and Mn2+ were critical in the medium tosupport the growth of Bt in the medium. P04

3. was

found (Ma et al, 1993) to be critical for growth in themedium since glucose uses EMP pathway to enter theTCA cycle.

Bt requires high O2 uptake with simultaneousgeneration of large amount of heat during vegetativegrowth. An O2 uptake rate of as much as 150 mMlhILwas reported by Yang and Wang (1998). Flores et al(1997) in their study reported an O2 formationcoefficient kLa of 270 1Ih by a sulphite oxidationmethod. It is critical to make sure that large-scalecultivation processes for Bt have adequate O2formation capacity and cooling capacity as well.Another approach, other than nutrient optimization toincrease biomass and O-endotoxin production of Bt isto employ genetically modified Bt strain for increasedo-endotoxin expression. While Koziel et al (1993)have employed y-irradiation for developing a mutantBt var. tenebrionis. Other workers (Donovan et al,1992; Gamel & Piot, 1992) have employed genetic

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engineering for developing a Bt strain, which hadincreased potential to produce more 8-endotoxin.They have conclusively shown that 8-endotoxinexpression can be regulated by placing 8-endotoxingenes under the control of promoters for eitherendotoxin or non-endotoxin genes. This kind ofregulatory change together with change in gene copynumber and alteration at the ribosomal binding siteshould make it possible to further enhance 8-endotoxin production. The other possibilities forincreasing 8-endotoxin production are: a) directingmore of the cell's biosynthetic capacity to 8-endotoxin production, b) to prolong the time periodallowed for biosynthesis and c) to synthesizeendotoxin during vegetative phase. In this directionspore negative 8-endotoxin producing mutants of Bt(Wakisaka et al, 1982) deserve attention. Herrnstadtet al (1987) and Koziel et al (1993) have reportedspore negative Bt strain where 8-endotoxinproduction has increased relative to the parental strainto a tune of. 1.5-fold.

Harvesting microorganisms from submergedfermentation is often difficult due to the lowconcentration of the products, their thermolabilenature and in some cases their poor stability. Sporeforming Bt are usually concentrated prior to dryinglike centrifugation or filtration. In a continuouscentrifuge, the product from 2-3% to 15% suspendedsolid can be achieved. Spray drying the centrifugate at175°C (Dulmage, 1981) is advocated. Many patents,such as foam floatation process exist for separating Btsporulation products.

FormulationBiopesticide formulation is the process of

transferring the biopesticides into a product, whichcan be applied by practical methods to permit itseffective, safe and economic use. The aim of theformulation is to avoid practice that might inhibit orharm the pathogen and wherever possible to enhancethe possibility of infection. Thus, not only one mustavoid agents in anyway antimicrobial, but also withBt, any compounds capable of denaturing the 8-endotoxin. The formulation of spore/toxin materialand carrier is devised to present a suitable amount ofcry protein to larvae in an acceptable form foringestion.

For effective reach and ingestion by larvae,wettable powder, disposable granules, dusts, microgranules, aqueous flowable liquid or oil band

flow able liquids have been developed (Navon, 2000).Early formulation was often difficult to apply andtheir performance was poor and unreliable. Advancesin application equipment and improvement informulation technology have largely overcomeapplication difficulties.

The feeding habits of mosquito larvae greatlyinfluence formulation design. Larvae of the species ofCulex and Aedes are filter feeders. Ingestion of thetoxin depends on the rate of feeding, the rate at whichthe toxin falls to the bottom of the pool and becomesinaccessible and competition to ingestion from othersuspended organic materials. Ranjith (2001) hasshown that the rate of settling of Bti in cesspit water isgreatly determined by the BOD and COD of cesspitwater. Mulla (1995) recommended two-fold increasein the rate of application of Bt var. israelensis/B.sphaericus in turbid and polluted water wheremosquitoes breed.

One of the major drawbacks in the use of Bt var.isralensis is its rapid inactivation in the environment(Siegel et al, 2001). Since there is little persistence ofthe toxin and the bacterium does not recycle, furtherapplications are necessary to effect continuouscontrol. Paily et al (1987) have shown that repeatedapplication of Bt var. isralensis was able toeffectively control Culex tritaeniorhynchus breedingin paper factory effluent up to 14 days post-application. The overall strategy for application ofbiopesticide formulations should be substantialsavings in cost and labour.

Many new spreaders and stickers have beendeveloped to help evenly distribute and bind thematerial over the applied surface. Persistence of thesprayed formulation is another important criterionwhich determines the effectiveness and efficiency ofthe sprayed Bt. Though UV light in the sun-rays andhigh pH have been implicated to reduce thepersistence of the sprayed Bt, addition of additives, tohelp prevent loss of the active ingredient orencapsulating the material in such a way that theactive ingredient is protected from inactivation havegreatly helped to enhance the persistence of Bt.

To reduce the cost of application it would be betterif the Bt is compatible with other pesticides,herbicides and additives intended to promote insectfeeding. As suggested by Lacey et al (1984), otherconsiderations are ease of handling, stability, bothduring storage and in the field and cost.

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Future ProspectsThe damage caused to cultivated crops due to

attack by different pests and diseases are enormousand a huge sum of money is spent in using plantprotection chemicals. Similarly from the health pointof view, biological control of vectors poses greatestchallenge to the health workers. The safe mode ofaction by Bt, because of its specificity to target pests,makes it attractive for field applications. Besides, itsbroad spectrum of activity among insect species andnarrow range of target specific insect orders is anadvantage in terms of safety. Its long-term persistenceenvisaged due to sporulation behaviour aids recyclingpotential. Since the genetics, physiology andbiochemistry of Bt is well understood, developmentof transgenics using alternate bacterial systems andcrop plants is well standardized. Increasingcompetition among the pesticide producingcompanies to enter into the venture of introducingsuch biopesticides in their catalogue is highlylaudable. In this context the expanding number offormulations released with long keeping quality (up to3 years) and decreasing cost of Bt production is anencouraging attribute. Nowadays, several seed-producing firms make attempt in developingtransgenic crops, which confer resistance to majorinsect pests of selected crops.

Inspite of all the above facts, still the share of Bt ininsect control programmes is minimal and deserves tobe encouraged to eliminate the development ofresistance in insects to chemical insecticides and toincrease the ecological impact of biological controlprogrammes. Thus, it is inevitable to include Bt basedmicrobial pest management with novel formulation,improved application technologies, andbiotechnological methods to increase Bt activitieswith broad host range (Navon, 2000). The productionof Bt on commercial scale is thus an economicallyimportant aspect as it is necessary to consideravailable human resources, technology for producingequipments and controls, adequate and cheap rawmaterials, adapted microorganisms and bioassayfacilities (Alves & Lemos, 2000). Such proceduresmight generate a lower production cost andconsequently an increase in such productconsumption either by farmers or health programauthorities.

Insect resistance is not limited to syntheticinsecticides alone, but also includes a wide range of'natural products' including pathogens and insect

315

growth regulators. It is unfortunate to know that Bt isnot included under this topic. Resistance managementhas a wider potential to work in the field for the firstgeneration of insecticidal plants. Present trend underthis context includes Bt expression modes that subjectinsects to impose selection pressure for specifiedperiods of time, particularly, plant parts by usinginducible and/or tissue specific promoters. Suchtechniques allow the research worker for exploringsusceptible alleles both within the field and within aregion while at the same time minimizing crop loss.Alternatively, pyramiding two dissimilar toxin genesin the same plant has been devised which is expectedto delay the onset of resistance much more effectivelythan single-toxin plants released spatially ortemporally and may require smaller refuges (Roush,1994). Though the development and implementationof engineered insecticidal plants is considered asinfancy but it has started providing substantialbenefits for seed industries, crop growers and theenvironment. It is important that industry, public-sector scientists and farmers work together to developa second generation of technology and implementstrategies to ensure insects not to develop resistanceto Bt crops.

AcknowledgementThe authors thank Department of Biotechnology,

New Delhi and All India Council for TechnicalEducation, New Delhi, India for providing funds tocarry out research on Bt.

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