Molecular characterization of indigenous Bacillus thuringiensis strains isolated from Kashmir valley.

Bacillus thuringiensis (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, new indigenous B. thuringiensis strains were isolated and investigated so that these could be used as an alternative and/or support the current commercial strains/cry proteins in use. For this, 159 samples including soil, leaf and spider webs were collected from ten districts of Kashmir valley (India). Of 1447 bacterial strains screened, 68 Bt strains were identified with 4 types of crystalline inclusions. Crystal morphology ranking among the Bt strains was spherical (69.11%) > spore attached (8.82%) > rod (5.88%) = bipyramidal (5.88%) > spherical plus rod (4.41%) > spherical plus bipyramidal (2.94%) = cuboidal (2.94%). SDS-PAGE investigation of the spore-crystal mixture demonstrated Bt strains contained proteins of various molecular weights ranging from 150 to 28 kDa. Insecticidal activity of the 68 indigenous Bt strains against Spodoptera litura neonates showed that Bt strain SWK1 strain had the highest mortality. Lepidopteron active genes (cry1, cry2Ab, cry2Ab) were present in six Bt strains. Further, analysis of a full-length cry2A gene (~1.9 kb) by PCR-RFLP in strain SWK1 revealed that it was a new cry2A gene in Bt strain SWK1 and was named as cry2Al1 (GenBank Accession No. KJ149819.1) using the Bt toxin nomenclature ( http://www.btnomenclature.info ). Insect bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 is toxic to S. litura with LC50 2.448 µg/ml and H. armigera with LC50 3.374 µg/ml, respectively. However, it did not produce any mortality in third instar larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi larvae/pupae insects (100 µg/ml) at 28 ± 2 °C and 75 to 85% relative humidity under a photoperiod of 14L:10D.


Introduction
Bacillus thuringiensis is ubiquitous, Gram-positive, sporeforming bacterium that is characterized by the production of insecticidal crystal proteins known as d-endotoxin (Shishir et al. 2014). These have a great potential to control a number of insect pests belonging to Lepidoptera, Diptera and Coleoptera and are benign to the environment Zhong et al. 2000;Vidyarthi et al. 2002;Valicente et al. 2010). There are reports of Bt strains active against livestock ectoparasites (Gough et al. 2002) and nematodes (Wei et al. 2003). Recently several reports of Bt and its toxicity against various insect pests have been documented (Ozturk et al. 2008;de Escudero et al. 2014;Neethu et al. 2015). Cry toxins' primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores (Bravo et al. 2007). Commercial Bt-based bioinsecticides used worldwide are applied at 10-50 g/acre or about 10 20 molecules/acre, while chemical pesticides such as organophosphates and pyrethroids are applied about 8 9 10 24 and 3 9 10 22 molecules/acre, respectively. Thus, the molecular potency of these toxins is 80,000 times better than organophosphates and 300 times greater than synthetic pyrethroids (Feitelson et al. 1992). Ever since the cloning of first cry gene (cry1Aa) from B. thuringiensis spp. kurstaki HD-1 (Schnepf and Whiteley 1981), 304 such cry holotype protein genes have been reported (Crickmore et al. 2016;25th April 2016, http:// www.btnomenclature.info/). These insecticidal crystal protein genes are the major source for the development of insect-resistant transgenic plants (Romeis et al. 2006). Genetically engineered crops which produce insecticidal proteins of Bt for the control of pests have been planted on a cumulative total of [560 million hectares worldwide since 1996 (James 2013). However, there have been some tribulations with this approach, for instance narrow insecticidal range and evolution of insect resistance (Sumerford et al. 2013;Pardo-López et al. 2013;Gassmann et al. 2014). An investigation of 24 cases, with each case relating responses of single pest species in one country to an individual Bt toxin, demonstrated that the practical influence of field-evolved resistance can differ from none to severe, based on the magnitude, incidence and spatial distribution of resistance (Tabashnik et al. 2014). Hence, isolation of new Bt strains with novel toxins are of significance for providing alternatives to these problems. In this regard, the present study was carried out to explore diversity of Bt present in samples collected at Kashmir valley (roughly between 32°.15 0 to 37°.05 0 North latitude and 72°.35 0 to 83°.20 0 East longitude) situated at the northern western tip of Himalayan biodiversity hotspot (Mittermeier et al. 2005). This may yield new Bt strains with novel cry gene sequences which could encode crystal proteins with significant difference in the level of toxicity due to variation in their sequences.

Reference strain
Bacillus thuringiensis subsp. kurstaki HD1 was obtained from Bacillus Genetic Stock Center (Columbus, Ohio) which served as positive control.

Sample collection and bacterial strains
A total of 159 samples from different spots in ten different divisions (Anantnag, Kulgam, Pulwama, Shopian, Budgam, Srinagar, Ganderbal, Bandipore, Baramulla and Kupwara) of the Himalayan valley Kashmir, India were used for isolation of Bt (Online resource 1). To our best knowledge, Bt insecticides had not been formerly applied in the sampled areas. All the soil samples (each *5 g) were collected from 2 to 4 cm below the surface after scraping off the surface material with a spatula. Leaf samples were collected from dense forest areas to get the maximum microbial population. Spider webs were collected using sterilized small sticks. All the samples were stored in sterile screw-capped vials.

Isolation and maintenance of Bt strains
Isolation of Bt strains from the samples was performed according to the method described by Ramalakshmi and Udayasuriyan (2010). In the laboratory, one gram of sample was suspended in 10 ml of sterile distilled water (10 -1 ) in a boiling tube. The boiling tube was kept at 65°C for 30 min and allowed to settle. One milliliter of this treated suspension was added to four ml of saline (0.85% NaCl), which gave 5 -1 dilution. Similarly, dilutions were made up to 5 -5 . One ml aliquots of dilutions 10 -1 , 5 -1 -5 -5 were taken in six different Petri plates over which melted T 3 agar medium (Travers et al. 1987) was poured and mixed clockwise and anti-clockwise directions. The plates were incubated (Hasthas Incubator, Hasthas Scientific Instruments, India) at 30°C for 2-3 days. From every soil sample, around 7-10 Bt-like colonies were chosen, sub-cultured as ribbon streak on T 3 agar medium and incubated for 48 h. To check the presence of protein crystals in the strains, culture smears were prepared, heat fixed and stained with the Coomassie Brilliant Blue stain (0.133% Coomassie Brilliant Blue G250 in 50% acetic acid). Then, the smears were washed softly in running tap water, blot dried with blotting paper and observed through bright-field microscope for the existence of crystalline inclusions. The strains which showed the presence of crystalline inclusions were selected as Bt. Single colony purification was performed on T 3 agar medium and stored on T 3 agar medium for further studies.
Isolation of spore-crystal mixture from Bt strains A loopful of Bt culture from the T 3 medium slant was inoculated into 5 ml of T 3 broth and incubated in a shaking incubator (Hasthas Shaking Incubator, Hasthas Scientific Instruments, India) maintained at 30°C and 200 rpm. After overnight growth, 1% inoculum was added to 250-ml flask containing 25 ml of T 3 medium and incubated at 30°C in a shaking incubator maintained at 200 rpm for nearly 48-60 h. The bacterial sporulation was monitored through bright-field microscope. When more than 90% of cells lysed, the sporulated broth culture was transferred to 4°C, at least half an hour before harvesting. The T 3 broth containing spore-crystal mixture was centrifuged at 4°C. The pellet was resuspended in 25 ml of ice-cold Tris-EDTA buffer [Tris 10 mM, EDTA 1 mM, pH 8.0 with 1 mM phenyl methyl sulphonyl fluoride (PMSF)] and washed once with 25 ml of ice-cold 0.5 M NaCl centrifuged for 10 min followed by two washes with 25 ml Tris-EDTA buffer with 0.5 mM PMSF at the same speed and time. Finally, the spore-crystal pellet was suspended in 500 ll of sterile distilled water containing 1 mM PMSF and stored at -20°C. All centrifugation steps were performed at the 5600g speed at 4°C (Superspin R-V/FM Plasto Crafts, Plasto Craft Scientific Pvt. Ltd, Mumbai, India).

Cry protein electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method of Laemmli (1970) using 10% running and 4% stacking gels. The gels were stained with 0.4% Coomassie Blue R250. The molecular mass of proteins was determined using a higher range protein molecular weight marker (myosin rabbit muscle 205 kDa, phosphorylase b 97.4 kDa, bovine serum albumin 65 kDa, ovalbumin 43 kDa and carbonic anhydrase 29 kDa) obtained from GeNei TM , Bengaluru, India.
Insecticidal activity of Bacillus thuringiensis strains against Spodoptera litura A modified diet overlay assay method was employed to test the susceptibility of Spodoptera litura neonates to indigenous Bt strains. In each 1.8-ml cryovial (Tarson Ò ; 1 cm dia.), 1 ml of artificial diet (Nagarkatti and Prakash 1974) was poured and 20 ll of spore-crystal mixture (1:5 dilution) of indigenous Bt strains was smeared over the diet surfaces. Bt 4D1 served as positive control. Cryovials were air dried for 1.5-2 h in laminar airhood before infesting with the neonates of Spodoptera litura. One neonate was transferred into each vial and lids were half closed for aeration. The infested culture vials were kept at 27°C, 50% RH, and a photoperiod of 16-8 (LD) h for 7 days. Mortality was calculated after every 24 h. Next different protein concentrations of spore-crystal mixtures (1-5 lg) of potent Bt strains were used and results noted. The mean 50% lethal concentration was estimated by Probit analysis using statistical parameters.
Distribution of cry genes in selected potential indigenous Bt strains Genomic DNA extracted using Sambrook and Russell's (2001) method from Bt strains was used for the amplification of cry1 and cry2 genes. For each polymerase chain reaction (PCR), 0.1 lg of total genomic DNA and 1 lM of each primer (Juarez-Perez et al. 1997;Ben-Dov et al. 1997) were mixed with 10 ll of 29 PCR Master Mix (GeNei TM , Bengaluru, India) consisting of dNTPs, Taq polymerase and PCR buffer. The final volume was made up to 20 ll with sterile double distilled water. PCR amplification was performed in a thermal cycler (cyber cycler-P series PCR Peltier model p96? USA).

Sample collection and bacterial isolation
A total of 159 Kashmir samples including soil, leaves and spider webs were collected from the Himalayan valley Kashmir. Out of 159 samples of Kashmir valley, 68 Bt strains (Table 1) were obtained based on the presence of protein crystals and T 3 as a selective medium. The number of Bt strains was isolated higher from the soil samples collected from Bandipore with 0.075 Bt index and the lowest number was isolated from Baramulla samples with 0.028 Bt index. The strains were further purified as a single colony and were maintained at 4°C.

Crystal morphologies
In 68 Bt strains, different types of crystal proteins morphologies, viz., bipyramidal crystals, spherical crystals, cuboidal crystals, spore attached crystals, rod-shaped crystals (Online resource 2) were observed under brightfield microscope (Magnus MLXi, Olympus, Japan). A single type of crystal morphology was observed in 73.92% of the strains and more than one type of crystal morphology was present in other 26.08% of the strains. Most of the isolated strains were found to possess spherical crystals. In the present study, crystal morphology ranking was spherical (69.11%) [ spore attached (8.82%) [ rod (5.88%) = bipyramidal (5.88%) [ spherical plus rod (4.41%) [ spherical plus bipyramidal (2.94%) = cuboidal (2.94%) among the strains from Kashmir.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
A total of 23 strains were studied for crystal protein profile(s) by SDS-PAGE. The time for parasporal crystal formation and completion of sporulation was checked always before isolation. Bt 4D1 was used as a positive control. SDS-PAGE investigation of the spore-crystal mixture demonstrated that Bt strains contained proteins of various molecular weights, viz., 150,135,130,105,98,95,91,90,70,65,45,42,34, 28 kDa ( Fig. 1). Among the selected 23 Bt strains, 05 strains (21.74%) were found to produce single protein band only and 18 Bt strains (78.26%) showed more than one protein band with different molecular weights. Most of the indigenous Bt strains (73.92%) produced proteins in the range of 60 to 70 kDa.
The 130 to 135 kDa proteins were next predominant proteins present in the Bt strains (43.47%) from different environments. Five strains (21.74%) were found to have proteins in the *30-35 kDa range. Two strains (8.7%) out of 23 Bt strains produced proteins of *28 kDa. This diversity in proteins indicated that Bt strains may have diverse cry genes and insecticidal activities. Around 135 and 75 kDa proteins suggest the presence of genes related to cry1 or cry4 and cry2 or cry3, respectively.

Insecticidal activity
Primary screening of insecticidal activity of newly isolated 68 indigenous Bt strains with 20 ll of spore crystal-protein mixtures (1:5 dilutions) against the Spodoptera litura revealed only 19 (27.94%) strains were active. When  Table 2. The mortality data fitted the probit model that was confirmed by the Chi-square goodness-of-fit test.
Distribution of lepidopteran-active cry genes in selected indigenous Bt strains PCR for three types of lepidopteran-active genes in 06 potential Bt strains revealed their presence in most of the strains. The most abundant cry gene was cry1-type gene. All the six selected strains (100%) showed the presence of cry1 type genes (*1500 bp) (Fig. 2) and cry2Aa type genes (*498 bp) (Fig. 3). It was followed by cry2Ab. Out of 06 Bt strains, 05 Bt strains (90%) gave positive results for cry2Ab gene with a PCR product of expected size at around 500 bp with specific primers (Fig. 4)

Discussion
Isolation of Bt strains B. thuringiensis strains have been found worldwide from diverse habitats, including the micro flora of soil and aquatic habitats (Martin and Travers 1989;Martinez and Caballero 2002;Uribe et al. 2003, Unalmis et al. 2015, phylloplane (Mizuki et al. 1999), freshwater (Ichimatsu et al. 2000), marine sediments (Maeda et al. 2000), freeliving animals (Swiecicka et al. 2002), bank voles (Swiecicka and De Vos 2003) and other environment (Meadows et al. 1992). This bacterium is widely distributed in five continents (Bernhard et al. 1997), ranging from tropics (Hastowo et al. 1992) to the high altitude (Landen et al. 1994). B. thuringiensis strains were isolated from different agro-climatic regions of India (Prabagaran et al. 2002 (Chilcott and Wigley 1993). In other earlier reports frequency for isolation of Bt from soil samples varied, ranging from 3 to 85% (Wang et al. 2003). Recently Shishir et al. (2014) reported 0.86 Bt index in their samples from Bangladesh. Vilas-Böas and Manoel (2004) suggested the Bt index may be an outcome of the biotic environmental factor, e.g., the vegetal top, the type of insect commonly found in the area, or microorganism in the soil, besides, abiotic factors such as the nutrient availability, texture, pH, temperature and humidity. On these grounds, they circumvent comparing their result with those of other authors, as they considered that the procedures of collection of sample, storage, processing, and identification of the bacteria were influenced by the experience of the working group and this may influence the result.

Protein profiling
Grouping of Bt strains according to crystal protein(s) profile studied by SDS-PAGE gives a preface for the presence of diversity in cry genes. Therefore, analysis of crystal proteins(s) profile could be useful to predict the presence of cry genes. The 130-138 kDa lepidopteran-active Cry proteins are encoded by cry1 genes. The Cry2 and Cry3 proteins are 65 and 70 kDa, respectively. The dipteran active, Cry4 and Cry10 or Cry11 proteins are 135 and 80 kDa, respectively (Chambers et al. 1991 Gough et al. (2005) studied Bacillus thuringiensis strains collected from various Australian soil samples which produced crystals containing 130 and 28 kDa proteins. These strains were highly toxic to feeding larvae of sheep blowfly (Lucilia cuprina). Ibarra et al. (2003) reported Bacillus thuringiensis strains (LBIT315, LBIT320, LBIT348, and IB604) from Latin America which are highly toxic against different mosquito species with major proteins of 130, 70, and 28 kDa similar to B. thuringiensis subsp. israelensis. They also reported Bt strain 147-8906 which had four different protein bands of 100, 75, 65, and 26 kDa. Earlier some investigators have reported a correlation between crystal morphology and type of proteins produced. The Cry1 (130-138) kDa lepidopteran-active proteins formed bipyramidal crystalline inclusions. The dipteran active, Cry4 and Cry10 or Cry11 proteins of 135 and 80 kDa sizes, respectively, formed spherical inclusions (Chambers et al. 1991). But in the present study, we could not establish a correlation between crystal morphology, molecular weight and a number of proteins produced by a Bt strain. Our results are in agreement with Arrieta and Espinoza (2006) who also reported there was no correlation between the morphology of the inclusion body, the molecular weight of the d-endotoxins and the number of Cry proteins produced. They reported a strain from Braulio Carrillo with pleomorphic crystals having proteins of 60, 70 and 100 kDa, while another strain with the same crystal morphology isolated from Santa Rosa contained a single polypeptide of 70 kDa.

Bioinsecticidal activity against Spodoptera litura
Spodoptera litura (Lepidoptera: Noctuidae) has surfaced as a key polyphagous pest in the recent past (Brown and Dewhurst 1975;Holloway 1989). This pest has been reported to attack a broad array of crops (about 40 species of plants) in Indian sub-continent (Chari and Patel 1983). Because of their ability to inflict serious economic damage to the crops in India, this insect pest has been subjected to heavy doses of insecticidal treatments. Indiscriminate and non-judicious use of insecticides has resulted in the development of resistance in this pest to as many as 20 different active ingredients of insecticides (Whalon et al. 2007). It is known that insects could be susceptible to different Cry toxins (Liao et al. 2002;Sauka et al. 2007). Protein profiling of selected indigenous strains confers that our indigenous strains have different Cry toxins which could be susceptible to various types of insects belonging to different orders. In this study, we tested the susceptibility of our Bt strains towards Spodoptera litura using an artificial diet. Modified diet overlay assay method was used by Liu et al. (2009) to test the susceptibility of Helicoverpa armigera and Spodoptera exigua neonates to Bt11 and showed 100% mortality of these two pests at the concentration of 5.9 9 10 7 spores/ml. Thaphan et al. (2008) screened toxicities of various Bt strains primarily on second-instar larvae of Plutella xylostella and on neonate larvae of Spodoptera litura and S. exigua with highly concentrated spore-crystal suspension and observed mortality after 3 days. Preliminary insect bioassay carried out in vitro by Reddy et al. (2013) with crude protein extracted from DOR Bt-1 isolate had shown that the strain was effective against Spodoptera litura, H. armigera and A. janata insect pest. We performed the primary screening of insecticidal activity of newly isolated 68 indigenous Bt strains with diluted 20 ll (1:5) of spore crystal-proteins mixture against the Spodoptera litura pest. Among these, 19 (27.94%) strains were found active with different toxicities levels. Whitlock et al. (1991) have reported a narrow range of pathogenicity of B. thuringiensis products against S. litura. According to Federici (1999), B. thuringiensis products were not always effective in controlling certain noctuid pests, especially the species of Spodoptera, viz., S. litura, S. frugiperda (Smith) and S. littoralis (Boisduval). Prabagaran et al. (2002) reported that out of 18 Bt strains subjected to single-dose assays against second-instar larvae of S. litura, five Bt strains (27.78%) had been found to be effective in killing at least 50% of the laboratory-reared insect population. Hire et al. (2009) reported a cry2Aa14 from an indigenous Bt strain HD-550 toxic to lepidopteran and dipteran insects. The expressed protein had shown toxicity to Spodoptera litura and Culex quinquefasciatus.
The Bt isolate 01-CHI-01 obtained from cadavers of a silkworm, Bombyx mori L. caused 71.3% mortality of Spodoptera litura by Leaf dip method (Manimegalai et al. 2005). In our study, 08 Bt strains have shown above 60% mortality of Spodoptera litura by surface diet method with diluted (1:5) spore-crystal mixture. Similarly, preliminary assays performed with spore-crystal mixture by Patel et al. (2009) revealed that all their 07 Bt strains (100%) tested against second-instar larvae of H. armigera were toxic with varying percentage of mortality ranging from 20 to 80% after 48 h of infestation. Bioassay study conducted by Goudar et al. (2012) with 44 strains against the third instar larvae of Plutella xylostella resulted in the cumulative mortality which ranged from 17 to 100% after 72 h of exposure. The insecticidal toxicity of 56 native B. thuringiensis strains investigated against first instar larva of E. kuehniella showed 80% of the strains had varying degrees of toxicity, whereas 20% of the strains were not toxic. About half of the native strains (44.6%) caused mortality between 10 and 30%. In addition, the mortality range of 13 strains was lower than 10% and that of 6 strains was moderate (30-50%). However, only one strain (85PPb) showed very high toxicity (84% mortality) (Apaydin et al. 2008). Detection of Lepidopteran-active cry genes in new Bt strains by PCR Lepidopteran-specific toxic proteins are produced by cry genes . PCR for three types of lepidopteran-active genes in 06 potential Bt strains revealed their presence in most of the strains. All the six selected strains (100%) showed the presence of cry1 type genes (1500 bp) and cry2Aa type genes (498 bp). It was followed by cry2Ab. Out of 06 Bt strains, 05 Bt strains (90%) gave positive results for cry2Ab gene with a PCR product of expected size at around 500 bp with specific primers. Five Bt strains, viz., SWK1, KS2-3, 2M-6, KS2-6, QZ-19 contained all cry1, cry2Aa and cry2Ab genes like Bt strain 4D1. All these five strains were found to have proteins of 135 and 65 kDa. Patel et al. (2013) reported the presence of cry1, cry2, cry3, cry7, cry8, cry4, cry5, cry12, cry14, cry21, cry11, cry13 and cyt1 genes from Bt strains isolated from various regions of India by PCR amplification. In their investigation, the cry1 gene was also the most abundant gene present. Prabagaran et al. (2002) reported the presence of cry1 genes (cry1Aa1, cry1Ab1, cry1Ac1, cry1Ca1, cry1Da1) in different indigenous B. thuringiensis strains potentially active against Spodoptera litura.
Recently, the abundance of cry genes in the Bt strains from Bangladesh was screened by Shishir et al. (2014) and reported cry1 gene as the most abundant with (30.8%) followed by cry2 (25.5%), cry3 (22.2%), and cry9 (7.2%) genes. Reddy et al. (2013) have reported a novel cry1 gene from Bt-1 DOR isolate effective against Spodoptera litura and other lepidopteran pests. Further, in our studies we found cry2Aa genes more in abundance than cry2Ab. Our results differed from the results of Shu et al. (2013) wherein they reported an abundance of cry2Ab genes more compared to cry2Aa by pooled clone method. This difference or variation in the cry2A sub type's content may be due to the fact that we selected the Spodoptera litura active Bt strains alone for PCR amplification. Five Bt strains, viz., SWK1, KS2-3, 2M-6, KS2-6, QZ-19 contained all cry1, cry2Aa and cry2Ab genes like Bt strain 4D1. All these five strains were found to have proteins of *135 and *65 kDa. Porcar et al. (2000) reported that Cry1C, Cry1D, Cry1E, Cry1F and Cry9C proteins were highly toxic towards Spodoptera species. Earlier Tabashnik et al. (1994) reported that Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1F and Cry2A were highly toxic to P. xylostella. Song et al. (2003) reported that Cry1I was toxic to P. xylostella, Ostrinia furnacalis and Leguminivora glycinivorella. Xue et al. (2008) reported that Cry1Aa was highly toxic to Ostrinia furnacalis, Helicoverpa armigera, Chilo suppressalis, and Plutella xylostella. Shu et al. (2013) developed an improved PCR-Restriction Fragment Length Polymorphism (RFLP) method for the identification of cry1-type genes which could efficiently identify single and clustered cry1-type genes. Among the evaluated cry1-type genes in their novel strain collections of B. thuringiensis, they identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. In our study, PCR-RFLP analysis of fulllength cry2A genes revealed the presence of a novel cry2A gene in Bacillus thuringiensis SWK1 strain which was named as cry2Al1 by Bt toxin nomenclature committee. Expressed Cry2Al1 protein showed promising larvicidal activity against S. litura (LC 50 = 2.448 lg/ml) and H. armigera (LC 50 = 3.374 lg/ml) (Reyaz and Indra Arulselvi 2016). However, it did not show any activity against the epidemiological disease-causing vectors: Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi larvae/pupae at 100 lg/ml. Zheng et al. (2010) reported a novel haplotype cry2A-type gene (Cry2Ag1 protein) by PCR-RFLP method with insecticidal activity against A. aegypti (Diptera), P. xylostella (Lepidoptera), and H. armigera (Lepidoptera). It had shown LC 50 of 2.541 lg/ml against A. aegypti, LC 50 of 9.745 lg/ml against H. armigera and LC 50 of 23.478 lg/ml against P. xylostella. Hire et al. (2009) reported Cry2Aa14 protein highly toxic to S. litura and H. armigera with LC 50 of 694 ng/cm 2 and LC 50 value of 122 ng/cm 3 , respectively. Lenin et al. (2001) reported a Cry2Aa protein which had shown 71.4% mortality and severe (81-99%) inhibition in larval growth of H. armigera on the seventh day at the concentration of 2.3 lg/ ll. Besides, in transgenic cotton plants Cry2Ab/Cry2Ae has been incorporated in addition to the Cry1A as a resistance managing mechanism (Christou et al. 2006; https://www.isaaa.org). In conclusion, even though many Bt Cry toxins have already been isolated and shown to be an important means for insect control, particularly with the development of transgenic plants, the discovery of additional novel Bt strains with novel cry genes continues to harness the further improvements of Cry proteins as competitive and successful biological insecticides. Our results advocate that the cry2Al1 gene is not merely a novel resource in the field of research into insecticidal crystal genes, but it may also act as a prospective alternative toxin to resolve some of the impending problems associated with insect resistance. These results also support that our indigenous Bt strains need to be evaluated for other Cry toxins which could be novel and interesting to Bt technology for crop protection. On the whole, our results are of significance because S. litura is known to be tolerant to the majority of the known d-endotoxins (Singh et al. 2004).