Cry-type Genes Research Articles

Isolation and molecular characterization of Bacillus thuringiensis strains obtained from different habitats in Northwest Ethiopia

Bacillus thuringiensis (Bt.) is ubiquitous, a gram-positive and spore-forming bacterium found in natural habitats everywhere in the world. For this study, Bt. has been isolated and characterized using a variety of techniques. Twenty-one strains showed positive results for the four sets of primers including, cry1, cry2, cry3, and cry9 genes. The PCR amplified results cry1 (33.3%) were most abundant among the tested cry-type genes next to cry9 (25%), cry2 (16.6%), and cry3 (12.5%), respectively. Three strains did not amplify. Twenty-four Bt. isolates were tested for the bioassay with a third-instar diamondback moth. The mortality of this insect was not shown after treatment for 24h. However, after 48 and 72h showed 20-61% and 20-79% mortality, respectively. The four Bt. strains tested against diamondback moth larvae showed no insecticidal activity. Therefore, the isolates in this study were promising for bio-insecticidal properties for diamondback moth and plant pest control programs.

Cloning and Characterization of Cry1 and Cry2 Genes from Indigenous Bacillus thuringiensis Isolates

Insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are toxic to specific insect pests. They are used as alternatives to chemicals for controlling insect pests using transgenic plants and biopesticides. Three strains of Bacillus thuringiensis expressing cry1 and cry2 genes were identified in soil samples collected from different locations in India. The full-length cry1, cry1I, and cry2 type genes (3.5, 2.1, and 1.9 kb, respectively) were cloned and sequenced. The full-length clones of the three genes were sub-cloned in the E. coli expression vector, and expressed proteins were analysed. The expression of cry1, cry1I, and cry2 resulted in 132, 81, and 70 kDa proteins, respectively. The expressed proteins were tested against secondinstar larvae of Helicoverpa armigera. The expressed proteins of cry1 and cry2- type from BRI-86 exhibited the highest toxicity to H. armigera larvae with an LC50 of 2.34 and 0.2 µg/ml diet, respectively. The LC50 exhibited by the test proteins was two and four-fold lower than the reference Btk HD-1 proteins. Thus, the spore preparation or the genes can be used individually or in combination to control insect pests.

Isolation and identification of Bacillus thuringiensis strains native of the Eastern Province of Saudi Arabia

BackgroundBacillus thuringiensis (Bt) produces a group of δ-endotoxin proteins designated as cry toxins. No doubt that Bt isolates are excellent potential candidates for biological control strategies.ResultsThe present study showed that 12 Bt strains were isolated and characterized at morphological, biochemical, and molecular levels. All the tested 12 Bt strains were gram-positive, endospore-forming, and possessing typical Bt crystal structures under the scanning electron microscopy (SEM). Universal primers direct and reverse of five pairs were used to detect five Cry-type genes (Cry1, Cry2, Cry3, Cry4, Cry7, and Cry8) by the PCR sizes produced from the studied Bt strains. The 16S rRNA PCR technique, 16S gene primer, DNA template, dNTPs, and Taq polymerase produced unique and distinguishable restriction patterns used for the molecular characterization of the studied Bt strains. Based on the PCR products, the frequency of Cry-gene distribution among the tested strains was Cry1 100%, Cry4 85%, and Cry3 62%, and Cry2 and Cry7 frequency was 54%. Based on the activity of insecticidal of the tested Bt strains, Bt1, Bt9, Bt10, and Bt11 were extremely pathogenic; their pathogenicity ranged from 93 to 100% against dipteran and lepidopteran larvae, compared to the other Bt isolates. The nucleotide sequences of amplified 1500 bp conserved region of 16S rRNA genes of four strains blasted using NCBI database compared to NCBI database sequences, and they were reported as native strains of Bt showing high homology with the known Bt strains (99–100%). The nucleotide sequences of Bt1, Bt9, Bt10, and Bt11 were placed in the GenBank database under accession numbers MN860017, MN843958, MN843959, and MN843960, respectively.ConclusionThe strategies of enhancing the sustainability of crops and vegetables that are targeted by a large number of pathogenic insects require a great effort of exploring novel species and strains of Bt. Herein, native strains of Bt were documented from the eastern province of Saudi Arabia that displayed bio-insecticidal action on larvae of Diptera and Lepidoptera.

Promising active bioinsecticides produced by Bacillus thuringiensis strain BLB427

Bacillus thuringiensis is gaining worldwide importance in the control of pests in agriculture and public health. Characterization of new isolated strains with original, higher and broader spectrum of activity is an ever developing field. Parasporal inclusions from BLB427, a new B. thuringiensis strain isolated from Tunisian soil, contained 2 major Cry proteins having molecular weights of about 130-135 kDa and 65-70 kDa corresponding to cry1-type and cry2-type genes, respectively. These toxins exhibited high toxicities to Ephestia kuehniella and Spodoptera littoralis larvae, with LC50 values of about 81.91 µg/cm2 (+/- 15.30) and 79.70 µg/cm2 (+/- 36.73), respectively. BLB427 supernatant showed a promising activity against Lepidopteran pests due to the presence of vip3-type gene in this strain. The high toxicity of BLB427 supernatant compared to that of BUPM95, used as control, was due in part to the high copy number of vip3 gene of BLB427 compared to that of vip3Aa16 of BUPM95, as demonstrated by Real-time PCR. This gene was cloned, sequenced and the comparison with the most known vip3 genes in databases, demonstrated that vip3(427) gene and the corresponding protein showed differences that can influence the efficiency of the Vip3(427) toxin against Lepidoptera. After overexpression of Vip3(427) protein in Esherichia coli and its purification using His-Trap column, the toxin showed a promising toxicity against the lepidopteran pest S. littoralis with extremely damages in the larvae midgut traduced by the vacuolization of the apical cells, the damage of microvilli and the disruption of epithelial cells. The results described in the present study proved that B. thuringiensis BLB427 strain could be of a great interest for lepidopteran biocontrol by using its d-endotoxins and/or Vip3 toxins in bioinsecticides formulations.

Isolation, identification, and toxicity of native Bacillus thuringiensis against Spodoptera litura

This study aimed at isolation and selection of Bacillus thuringiensis (Bt) with toxicity against Spodoptera litura in soil samples collected from southeastern region of Vietnam. Thirty nine Bt isolates were obtained from 32.63% of the 95 collected samples, resulting the total Bt index of 0.096. Light microscopy observation revealed the presence of variety morphology of parasporal crystals. Mortality rate of S. litura was maximum in the reference strain, B. thuringiensis var kurstaki (95%), followed by the two Bt isolates TN1 and TN2 (80% and 75%). The isolates showing the mortality rate of 20% to 40% and below 20% constituted, respectively, 38.46% and 34.62%of the assessed isolates. PCR analysis method was used with the universal primers to detect the presence of cry1 and cry2 type genes. Ten isolates showed the presence of the expected amplicons and 90% of the Bt isolates contained cry1 genes. Four Bt isolates harbored both cry1 and cry2 genes, including the two isolates with high toxicity on S. litura. This study contributed to the local Bt collection in Vietnam. The obtained isolates with high toxicity on S. litura could be utilized in applications to control lepidonteran insect pests.

DETERMINATION AND DISTRIBUTION OF CRY1-TYPE GENES IN Bacillus thuringiensis ISOLATED FROM NORTH INDIA

Bacillus thuringiensis (Bt) is a spore-forming bacterium which produces insecticidal crystal protein in the sporulation phase. Polymerase chain reaction (PCR)-based identification of Bacillus thuringiensis toxin genes has become a routine step in most B. thuringiensis isolation and characterization initiatives. In the present study, eighteen native Bt isolates from diverse habitats of North India were taken for the presence of cry1 type genes. The distribution of cry1 gene families in native Bt isolates was examined by PCR amplification of genes with three sets of corresponding PCR primers. In native Bt isolates many variant bands were also observed in addition to expected bands on PCR amplification. Different sets of primers for the same gene give different results due to different sites of primer binding. Maximum number of isolates showed expected bands when primers designed by Ceron were used compared to other primers. However, with other primers, the numbers of variant bands was larger. The isolates, SK-13, 63 and 105 showed the maximum number of cry1-type genes, followed by SK-20, 28, 48, 88, 94, 301, 304 and 307, whereas SK-3 showed the presence of only 3 cry1-type genes. RFLP analysis of 1.6 kb fragment indicated the presence of variant bands from the reference strains. Several promising isolates with predicted toxicity towards lepidoptera have been observed in this study.

Pool deconvolution approach for high-throughput gene mining from Bacillus thuringiensis.

Novel genes from Bacillus thuringiensis (Bt) are required for effective deployment in agriculture, human health, and forestry. In an improvement over conventional PCR-based screening, next generation sequencing (NGS) has been used for identification of new genes of potential interest from Bt strains, but cost becomes a constraint when several isolates are to be sequenced. We demonstrate the potential of a DNA pooling strategy known as pool deconvolution to identify commercially important toxin genes from 36 native Bt isolates. This strategy is divided into three steps: (a) DNA pooling, (b) short read sequence assembly followed by gene mining, and (c) host isolate identification. With this approach, we have identified insecticidal protein (ip) genes including nine three-domain (3D) cry genes, three cyt-type genes, three mtx genes (mosquitocidal toxin), and one bin and vip-type gene each. Three cry-type and three cyt-type genes were cloned, out of which, two cry-type genes, ip11 and ip13, were named as cry4Ca2 and cry52Ca1, respectively by the Bacillus thuringiensis nomenclature committee ( http://www.biols.susx.ac.uk/Home/Neil_Crickmore/BT/ ). Our results show that the pool deconvolution approach is well suited for high-throughput gene mining in bacteria.

Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains

Novel cry genes are potential candidates for resistance management strategies, due to their different structures and modes of action. Therefore, it is desirable to clone and express novel cry genes from several new isolates of Bacillus thuringiensis (Bt). In the present study, 28 Bt strains were characterized at morphological and molecular level. All these strains are Gram positive, endospore forming and had shown different crystal morphologies when viewed under the microscope. The ARDRA (16S rDNA PCR-RFLP technique) with AluI, HaeIII, HinfI and TaqI produced unique and distinguishable restriction patterns used for the molecular characterization of these isolates. Based on UPGMA clustering analysis, Bt strains showed significant molecular diversity and the dendrogram obtained differentiated 28 Bt strains into 1 major cluster at a similarity coefficient 0.56. PCR analysis demonstrated that the Bt strains showed diverse cry gene profiles with several genes per strain. The Bt strain G3C1 showed the presence of maximum cry-type genes by PCR. The toxicological characterization of these cry genes will have huge importance in transgenic technology and will be useful in transgenesis of crop plants for better resistance management.

Prevalence of cry2-type genes in Bacillus thuringiensis isolates recovered from diverse habitats in India and isolation of a novel cry2Af2 gene toxic to Helicoverpa armigera (cotton boll worm).

Insecticidal cry and vip genes from Bacillus thuringiensis (Bt) have been used for control of lepidopteran insects in transgenic crops. However, novel genes are required for gene pyramiding to delay evolution of resistance to the currently deployed genes. Two PCR-based techniques were employed for screening of cry2-type genes in 129 Bt isolates from diverse habitats in India and 27 known Bt strains. cry2Ab-type genes were more prevalent than cry2Aa- and cry2Ac-type genes. Correlation between source of isolates and abundance of cry2-type genes was not observed. Full-length cry2A-type genes were amplified by PCR from 9 Bt isolates and 4 Bt strains. The genes from Bt isolates SK-758 from Sorghum grain dust and SK-793 from Chilli seeds warehouse, Andhra Pradesh, were cloned and sequenced. The gene from SK-758 (NCBI GenBank accession No. GQ866915) was novel, while that from SK-793 (NCBI GenBank accession No. GQ866914) was identical to the cry2Ab1 gene. The Bacillus thuringiensis Nomenclature Committee ( http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/toxins2.html ) named these genes cry2Af2 and cry2Ab16, respectively. The cry2Af2 gene was expressed in Escherichia coli and found to be toxic towards Helicoverpa armigera. The cry2Af2 gene will be useful for pyramiding in transgenic crops.

Improved insecticidal activities of Novel Bacillus thuringiensis Cry1-type genes

Recently, Bacillus thuringiensis (Bt) cry genes encoding insecticidal Cry proteins have been widely applied for the construction of transgenic crops resistant to insect pests. This study aimed to construct novel mutant cry1Ac genes for genetically modified crops with enhanced insecticidal activities. Using multi-site directed mutagenesis, 34 mutant cry1Ac genes were synthesized and converted at 24 amino acid residues, located on domain I (8 residues) and domain II (16 residues). These mutant genes were expressed as a fusion protein with polyhedrin using the baculovirus expression system. The expressed proteins were occluded into polyhedra and activated stably to 65kDa by trypsin. Among these, Mut-N04, N06 and N16 showed high levels of insecticidal activities against larvae of Plutella xylostella, Spodoptera exigua, and Ostrinia furnacalis. Mut-N16, which showed the highest insecticidal activity, is expected to be a desirable cry gene for introduction into transgenic crops. This study could provide useful means to construct mutant cry genes with improved insecticidal activities and expand host spectra for transgenic crops.

Diversity of Bacillus thuringiensis isolates from Egyptian soils as shown by molecular characterization.

Different techniques were adopted for molecular characterization of several indigenous strains of Bacillus thuringiensis (Bt) previously isolated from Egyptian soil samples. These isolates show different toxicity levels against neonate larvae of both insect species; Spodoptera littoralis (Biosduval); and Helicoverpa armigera (Hübner). The parasporal crystals among the most potent isolates contained polypeptides of about 127 and 130 kDa. PCR screening for genes encoding different Cry genes was performed. The Cry 1 gene is the most abundant in these isolates (83.33%) among tested Cry-type genes, followed by Cry 1 gene subfamilies (Cry 1B and Cry 1C) with percentage of 38.88% and 77.77%, respectively. The tested isolates showed the presence of Cry 2A(a,b) gene, but not all of these isolates were positive for Cry 2 gene (55.55%). Only 27.77% and 16.66% of the tested isolates harbor Cry 4 and Cry 3 genes, respectively. All strains were negative in PCR assays for the Vip 3Aa1 gene. Moreover, DNA fingerprinting using RAPD-PCR was performed to detect the genetic similarities and dissimilarities among the different isolates and standard strains. Assessment of Bt diversity based on the combined analysis of their protein and RAPD-PCR banding patterns was performed. This study demonstrates that Bt strains isolated from Egyptian soil samples can be distinguished and identified on the basis of the distribution of Cry-type genes and RAPD fingerprints.

Characterization of cry1Cb3 and cry1Fb7 from Bacillus thuringiensis subsp. galleriae

Abstract Two cry1-type genes encoding insecticidal crystal proteins (ICPs) were detected by PCR-RFLP and cloned from Bacillus thuringiensis subsp. galleriae 87. The nucleotide sequences were deposited in GenBank with accession numbers EU679501 and EU679502, and designated as cry1Fb7 and cry1Cb3 respectively by B. thuringiensis Delta- Endotoxin Nomenclature Committee. cry1Cb3 shared 99% homology with other cry1Cb genes. The existence of two additional stop codons indicated cry1Cb3 was a silent gene. The cry1Cb3 was 3531 bp with 38.98% G+C content and its first open reading frame (ORF) encoded a protein of 213 amino acid residues with a calculated molecular weight of 23.8 kDa and a predicted pI value of 4.63. Five amino acid sequence blocks (block 1, block 2, block 3, block 4 and block 5) were found in Cry1Cb3. Translation of cry1Fb7 revealed an ORF of 3525 bp with 39.12% G+C content and a protein with a calculated molecular weight of 133.2 kDa and a predicted pI value of 5.18. Cry1Fb7 had five amino acid sequence blocks (blocks 1, 2, 3, 4 and 5) and three domains (I, II and III), which consisted of 218 residues (Leu34 to Ala252), 197 residues (Thr257 to Asp454), and 138 residues (Ile464 to Glu600), respectively.

Screening <i>Bacillus thringiensis</i> Isolates from Soil Samples of Wuzhishan Primitive Tropic Rainforest in Hainan and Identifying Novel cry-type Genes

The Wuzhishan, located at the center of Hainan Island, is the largest Primitive Tropic Rainforest in Hainna island. In this study, we collected 234 soil samples from Wuzhishan at the different heights above sea level from 700 m to 1 650 m. Following the approach of selection culture medium with sodium acetate and treatment of high temperature, we o Bt ained 886 Bacillus isolates and further detected 21 Bacillus thuringiensis (Bt) isolates through observation of microscopy, of which the isolating rate reached 2.3%. In order to mine the insecticidal genes from the identified Bt isolates, the PCR strategy with universal primer pairs, combined with DNA sequencing technology was used to detected cry genes. The result showed that the Bt isolates from Wuzhishan include cry1-like , cry4-like , cry39-like , cry40-like , cry31-like , cry46-like genes and so on, presenting that the o Bt ained Bt isolates are abundant in novel cry genes, which may facilitate to clone novel Bt crystal toxin genes.

Screening ofBacillus thuringiensisIsolates Recovered from Diverse Habitats in India for the Presence of Insect and Nematode-activecryGenes

Novel cry genes with different structure and mode of action are potential candidates for resistant management strategies. Isolation of novel genes needs thorough screening of large number of native Bacillus thuringiensis (Bt) isolates collected from diverse habitats and ecotypes. In the present study, 63 Bt isolates recovered from different habitats in India have been investigated for their insecticidal and nematicidal cry gene content. Bt isolates were screened through Polymerase Chain Reaction (PCR) using degenerate primers for the presence of cry1, cry1A and nematode-active cry genes. Among the collection of 63 Bt isolates 21 were found positive for the presence of cry1-type genes, 49 for cry1A-type genes and 20 for nematode-active cry genes. Nine Bt isolates were found positive for all three types of cry genes. The study also show that PCR based screening method is rapid and highly useful for characterization of Bt isolates to reveal their insecticidal spectrum.

An improved PCR-restriction fragment length polymorphism (RFLP) method for the identification of cry1-type genes.

The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.

Characterization of a new highly mosquitocidal isolate of Bacillusthuringiensis – An alternative to Bti?

The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis ( Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes ( cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases.

Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin, China

Sichuan basin, situated in the west of China, is the fourth biggest basin in China. In order to describe a systematic study of the cry2-type genes resources from Bacillus thuringiensis strains of Sichuan basin, a total of 791 Bacillus thuringiensis strains have been screened from 2650 soil samples in different ecological regions. The method of PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the type of cry2 genes. The results showed that 322 Bacillus thuringiensis strains harbored cry2-type genes and four different RFLP patterns were found. The combination of cry2Aa/cry2Ab genes was the most frequent (90.4%), followed by cry2Aa (6.8%) and cry2Ab alone (2.5%), and only one novel type of cry2 gene was cloned from one isolate (JF19-2). The full-length of this novel gene was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR), which was designated as cry2Ag1 (GenBank No. ACH91610) by the Bt Pesticide Crystal Protein Nomenclature Committee. In addition, the result of scanning electron microscopic (SEM) observation showed that these strains had erose, spherical, bipyramidal, and square crystal. And the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these strains harbored about one to three major proteins. These strains exhibited a wide range of insecticidal spectrum toxic to Aedes aegypti (Diptera) and Pieris rapae Linnaeus, 1758 (Lepidoptera). Particularly, JF19-2 contained cry2Ag gene had the highest insecticidal activity. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bacillus thuringiensis strains in Sichuan basin.

Genetic Diversity of cry Gene Sequences of Bacillus thuringiensis Strains Analyzed by Denaturing Gradient Gel Electrophoresis

PCR has been widely used to identify cry-type genes, to determine their distribution, to detect new such genes and to predict insecticidal activities. We describe here a molecular approach to analyze the genetic diversity of B. thuringiensis cry-like genes based on denaturing gradient gel electrophoresis (DGGE). This analysis demonstrated that different B. thuringiensis isolates can be distinguished according to its PCR-DGGE profile of cry-like genes. Identification of the resolvable DNA fragments was easy to accomplish by DNA sequencing, which was confirmed in this work. Importantly, the strategy allowed the identification of unknown B. thuringiensis cry-like sequences present in a single strain that remained cryptic after PCR analysis using degenerate primers. The method developed in this work contributes to the availability of molecular techniques for both B. thuringiensis strains and cry-like genes identification and discovery.

Biological characteristics of Bacillus thuringiensis strain Bt11 and identification of its cry-type genes

A novel strain of Bacillus thuringiensis Bt11, isolated from soil samples in China, was classified and characterized in terms of its crystal proteins, cry genes content. The Bt11 strain showed high toxicity against Spodoptera exigua and Helicoverpa armigera neonates. Bt11 strain shares morphological and biochemical characteristics with the previously described Bacillus thuringiensis subsp. kurstaki. SDS-polyacrylamide gel electrophoresis revealed that crystals were composed of several polypeptides ranging from 20 to 130 kDa, of which the 35, 80, and 130 kDa proteins were the major components. PCR-RFLP with total DNA from strain Bt11 and specific primers for cry1, cry2, cry3, cry4/10, cry7, cry8, cry9, and cry11 genes revealed that cry1Aa, cry1Ab, cry1Ia, and cry9Ea genes were present.

Distribution of Bacillus Thuringiensis in Gunung Jerai Forest (Malaysia)with Insecticidal Activity Against Lepidopteran and Dipteran Insects

Globally there has been a concerted effort to isolate Bt strains from different environments for use as microbial pesticides and in transgenic plants. In our study, 106 soil samples were collected from different altitude in Gunung Jerai forest, an undisturbed rain forest ecosystem, Kedah Darul Aman State, Malaysia. Preliminary screening included temperature selection methods to isolate Bacillus-like colonies, followed by phase contrast microscopy to screen presumptive Bt isolates. Twenty three isolates were selected for PCR analysis with Cry1 general primer (gcry1), PCR to identify the presence of cry1-type genes. Out of 23 isolates, only 17 isolates showed the presence of cry1-type genes with the product size of 558 bp. Subsequently, the 17 Bt isolates which showed the presence of cry1-type genes were subjected to pathotype PCR to predict the insecticidal activity against lepidopteran pests. The results of the pathotype PCR revealed that out of 17 Bt isolates, only 11 isolates showed PCR product indicating their possible anti lepidopteran activity. Plasmid profiling studies indicated the presence of a major plasmid of size 22.5 to 28.5kb.Then the hemolytic positive strains were subjected to one step bioassay against the second instar larvae of Aedes aegypti. One Bt isolate, Bt 02, showed elevated larvicidal activity, which was compared to that of the reference strain Bt.subsp. israelensis. The crystal protein studies of the Bt isolate 02 indicated the presence of cuboidal crystals. SDS-PAGE analysis of the Bt isolate 02 showed a major protein of 101.77 kDa. However, presence of a protein of 32.68 kDa, indicted the possible presence of cytolytic toxin. Finally to study the diversity, two Bt isolates, Bt 22 and Bt 42 were subjected to partial DNA sequencing of the PCR amplicon. The results indicated that these two strains have different nucleotide sequences and each strain showed homology with the reported Bt strains which are also different from each other, thus indicating the presence diverse Bt strains in this environment. The Bt isolates showing anti lepidopteran and anti dipteran activity can be used in integrated pest management to control agricultural pests and to control the selected mosquito vectors.