Biological, Immunological, and Genetic Analysis of Bacillus thuringiensis Isolated from Granary in Korea

To isolate a naturally occurring novel Bacillus thuringiensis strain, we investigated the distribution, toxicity, morphology, H serotype, and gene type of B. thuringiensis from residue samples of granary in Korea. A total of 163 B. thuringiensis isolates out of 411 samples producing spore and crystal were obtained. In toxicity tests, 80% of all isolates were toxic to lepidoptera, and 12% were not toxic to any of tested insects. And dipteran-active and lepidopteran/dipteran-active isolates were rare (2% and 6%, respectively). 152 B. thuringiensis isolates produced typical rhomboidal crystals, and the remainder produced parasporal inclusions with various morphologies. Serological test showed that B. thuringiensis isolates in granary represented 12 H serotypes, indicating varied distribution of B. thuringiensis. Of these, the serotype 3ab predominated, followed by the serotype 7 and 4ac. B. thuringiensis isolates of the serotype 3ab, 4ac, 5ab, 7, 8ab, 9, and 23 were toxic to lepidoptera, and the serotype 8bd, 12, 18, and 20ac were nontoxic, while 14 isolates were untypable by 33 B. thuringiensis H antisera. The frequency of toxicity against lepidoptera and diptera was primarily highly toxic. PCR analysis using cryI gene type-specific primers showed that cryIA(b) genes are frequently found and cryIE gene exists in only one isolate. Analysis of B. thuringiensis crystals and plasmid DNAs indicated a diversity of crystal and gene types. Received: 15 January 1998 / Accepted: 18 February 1998     

Distribution, Serological Identification, and PCR Analysis of Bacillus thuringiensis Isolated from Soils of Korea

A total, 58 strains of Bacillus thuringiensis were isolated from soils of various regions in Korea. Serological tests showed that B. thuringiensis isolates represented 10 H serotypes, indicating a varied flora of B. thuringiensis. But the H serotypes did not have a significantly uneven distribution, ranging from 1 to 11 isolates. In toxicity tests, 35% of all isolates were toxic to lepidoptera, 20% were toxic to diptera, and 9% were non-toxic isolates. Especially, a large number of lepidopteran/dipteran-active isolates (36%) were found. Forty all lepidopteran-active isolates produced typical rhomboidial inclusions, and the remainder, which belong to dipteran-active and non-toxic isolates, were spherical in shape. In addition, lepidopteran/dipteran-active isolates produced rhomboidal or spherical inclusions. PCR analysis using cryI, II, III, IV, and V gene-specific primers showed that the frequency of the cryIC gene (57%) predominated, followed by the cryIA(b) (45%) and cryIIA genes (34%). But, the cryIE, cryIF, cryIII, cryIVC and cryV genes were not reactive. Several isolates had unusual PCR products and multiple insecticidal crystal protein genes. PCR results showed varied distribution of the cry-type gene. Seven isolates were selected for evaluation of novel activity according to the following criteria: flagellar serotypes, parasporal inclusion morphology, SDS-PAGE, plasmid DNA patterns, toxicity, and the cry-type gene in PCR analysis. Two isolates, named S333 (H7) and S225 (H7), among them synthesized PCR products of the cryIC gene, but the S333 isolate producing rhomboidal inclusion was toxic to both Plutella xylostella and Culex pipiens, whereas the S225 isolate having toxicity to only C. pipiens produced spherical inclusion. Received: 13 March 1998 / Accepted: 14 April 1998     

Determination and Distribution of cry-Type Genes of Bacillus thuringiensis Isolates from Taiwan

Using PCR with a set of specific oligonucleotide primers to detect cryI-type genes, we were able to screen the cry-type genes of 225 Bacillus thuringiensis soil isolates from Taiwan without much cost in time or labor. Some combinations of cry genes (the cry-type profile) in a single isolate were unique. We identified five distinct profiles of crystal genes from the B. thuringiensis soil isolates from Taiwan. The cry genes included cryIA(a), cryIA(b), cryIA(c), cryIC, cryID, and cryIV. Interestingly, 501 B. thuringiensis isolates (93.5% of the total number that we identified) were isolated from areas at high altitudes. The profiles of cry-type genes were distinct in all isolation areas. The distribution of cry-type genes of our isolates therefore depended on geography. Using PCR footprinting to detect cryIC-type genes, we identified two distinct cryIC footprints from some of our isolates, indicating that these isolates may contain novel cryIC-type genes. B. thuringiensis isolates containing cryIA(a)-, cryIA(b)-, and cryIA(c)-type genes exhibited much greater activity against Plutella xylostella than did other isolates, indicating that multiple cry-type genes may be used as markers for the prediction of insecticidal activities.     

Insecticidal Activity of the Protein Encoded by the cryV Gene of Bacillus thuringiensis kurstaki INA-02

A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.     

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.     

Characterization of INTA 51-3, a New Atypical Strain of Bacillus thuringiensis from Argentina

Several isolates of Bacillus thuringiensis native to Argentina obtained in a nationwide screening program showed atypical crystal morphology. One of these strains, INTA 51-3, was further characterized in order to determine other features like protein composition of its parasporal crystal, plasmid pattern, identification of cry genes and toxicological properties. B. thuringiensis INTA 51-3 (serovar tohokuensis) had an amorphous inclusion containing a major protein component of ca. 130 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique protease-resistant peptide of ca. 90 kDa. The plasmid pattern from INTA 51-3 resembled that of the standard strain HD-1. However, Southern analysis showed no hybridization to fragments of cry1Aa, cry2Aa, cry3A, and cry11A genes. Degenerate primers were used for identification of the cry1 genes by PCR. Nevertheless, the presence of cry1 type gene(s) in B. thuringiensis INTA 51-3 was confirmed. Highly concentrated crystal suspensions showed to be weakly toxic only to lepidopteran species. Received: 23 May 2000 / Accepted: 26 June 2000     

Characterization of the highly variable <Emphasis Type="Italic">cry</Emphasis> gene regions of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain ly4a3 by PCR-SSCP profiling and sequencing

Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%) and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes.     

Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United States

Gram-positive, endospore-forming Bacillus thuringiensis-like strains were isolated from 95 of 413 samples collected at the 0–5 cm depth of noncultivated soils and stagnant or dried-up ponds as well as from dust from stored grain products in South Central United States. Based on the production of parasporal crystals, 25 isolates were identified as B. thuringiensis after examining 227 B. thuringiensis-like colonies. The greatest proportion of samples yielding B. thuringiensis were from the dust from grain storage. The sodium acetate selective medium, heat processing, and crystal staining used in the initial screening revealed diverse populations of B. thuringiensis, which were categorized into distinct crystal morphological groups. Sugar fermentation, antibiotic sensitivity, growth characteristics and PCR studies showed diversity among the isolates that were distributed among 25 of the 58 known strains. The most frequently isolated strains were kurstaki, aizawai, morrisoni, thuringiensis, sotto and kenyae that together represented more than 90% of the characterized isolates. PCR analysis using 30 family primer pairs for cry and cyt genes showed that the frequency of the cry1 gene (62%) was predominant followed by the cry2 genes (30%), and the rest (8%) were other cry gene types, such as cry3, cry4, cry10, cry11, cry14, cry15, cry20, cry24 and cry26. Both cyt1 and -2 genes were also detected. Several isolates showed PCR products on the gel that were not consistent with the expected sizes of nucleotides targeted by the primers. These were suggestive of nonspecific amplifications and were not used in the characterization process. Journal of Industrial Microbiology & Biotechnology (2002) 28, 284–290 DOI: 10.1038/sj/jim/7000244 Received 30 May 2001/ Accepted in revised form 10 January 2002     

Isolation and Characterization of a Strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">morrisoni</Emphasis> PG-14 Encoding δ-Endotoxin Cry1Ac

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.     

Identification and characterization of a new cry-like gene found in a Bacillus cereus strain

Bacillus thuringiensis is the most successful microbial insecticide against different pests in agriculture and vectors of diseases. Its activity is mostly attributed to the Cry proteins expressed during its sporulation phase. However, these proteins are not exclusive to B. thuringiensis. Some cry genes have been found in other Bacillus species, or even in other genera. In this work, cry genes were searched in 223 acrystalliferous bacillaceous strains. From these strains 13 amplicons were obtained, cloned, and sequenced; however, only 6 amplicons tested positive for cry-like genes, and the 6 isolates showed to be the same strain. We report the characterization of an unusual strain of B. cereus (LBIC-004) which is unable to form protein inclusions during the sporulation phase. LBIC-004 showed a high identity to B. cereus using the sequences of 16S rRNA, gyrB and hag genes; in addition, a unique plasmid pattern of the strain was obtained. A 1953-bp cry gene was identified, coding for a 651 amino acid protein with a molecular weight of 74.9 kDa. This protein showed a predicted three-domain structure, similar to all Cry proteins. However, the amino acid sequence of the protein showed only 41% identity its highest hit: the Cry8Ca1 protein, indicating the uniqueness of this cry-like gene. It was cloned and transferred into a mutant acrystalliferous B. thuringiensis strain which was used in bioassays against Caenorhabditis elegans, Aedes aegypti, Manduca sexta and Phyllophaga sp. The recombinant strain showed no crystal formation and no toxicity to the tested species.

     

Isolation and characterization of a strain of Bacillus thuringiensis ssp. kurstaki containing a new delta-endotoxin gene

A strain of Bacillus thuringiensis that showed significantly high toxicity to Plutella xylostella and Spodoptera exigua was isolated from a Korean soil sample and characterized. The isolate, named B. thuringiensis K1, was determined to belong to ssp. kurstaki (H3a3b3c) type by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1, but the parasporal inclusion protein profile of K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1. To verify the δ-endotoxin gene types of K1, PCR analysis with specific cry gene primers was performed to show that K1 contained a new cry gene in addition to cry1Aa, cry1Ab, cry1Ac, cry1E and cry2 genes. PCR-amplified region of the new cry gene, cryX, showed 79% similarity to cry1Fa1 gene (GenBank Accession No. M63897). In an insect toxicity assay, K1 had higher toxicity against Plutella xylostella and S. exigua than ssp. kurstaki HD-1. Received: 21 December 2001 / Accepted: 28 January 2002     

New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Detection and characterisation of a Bacillus thuringiensis crystal protein with nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus

Nematicidal Bacillus thuringiensis (Bt) strains were isolated from forests in Zhejiang, China for further characterisation. PCR analysis was performed with nine pairs of primers specific for cry1, cry2, cry3, cry4, cry5, cry6, cry9, cry11 and cry13 to characterise and classify cry gene groups from Bt isolates. The isolates from individual cry groups were tested for nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus, which is implicated in pine wilt disease. PCR identified 14 different categories of cry gene combinations, indicating a large diversity of cry genes. The cry1 gene was by far the most abundant in Bt isolates and was found in 68% of samples. The Bt isolates zjfc85 and zjfc392 were from two distinct classes, but shared the same cry5 amplification profile and the same ~130 kDa protein; they had the highest nematicidal activity against pinewood nematode during the 48 h exposure tests, resulting in 90 and 59% mortality (9% of mortality under control conditions), respectively. The ~130 kDa Cry protein from isolate zjfc85 was purified and named as Cry5Ba3. Bioassay results indicated pinewood nematode was highly susceptible to Cry5Ba3 and exhibited profound growth abnormalities after exposure to Cry5Ba3. Our results are a novel finding and provide a potential strategy to manage pine wilt disease caused by B. xylophilus based on a nematicidal Bt.     

Characterization of Bacillus thuringiensis isolates from soil and small mammals that harbour vip3A gene homologues

The insecticidal and psychrotropic potential of 132 new isolates of Bacillus thuringiensis from northeastern Poland (74 from animals and 58 from soil) were determined by screening these for vip and cry genes encoding, respectively, vegetative insecticidal proteins (Vip) and Cry proteins, and cspA that encoded the CspA cold shock protein that confers psychrotropy in Bacillus species. The vip3A gene, encoding Vip3A toxic to lepidopterans, was found in ~5% of the isolates from animals and ~17% the isolates from soil, whereas coleopteran-specific vip1 and vip2 genes were present in 8% of the isolates from soil. Nucleotide sequences of vip3A-specific amplicons were highly conserved, with only a few containing minor differences from vip3A. Despite the high level of vip3A conservation, isolates harbouring the gene demonstrated a high level of heterogeneity based on whole-cell genomic DNA RFLP analysis with pulsed-field gel electrophoresis (PFGE) and plasmid profiling. Eight isolates positive for vip3A contained cry1 and six also harboured the cry2 gene, which encodes an endotoxin toxic to lepidopteran insects. However, none of these isolates contained cry genes coding for proteins toxic to coleopteran or dipteran insects. Due to the known potential for synergistic interactions between Vip and Cry proteins, the isolates positive for vip3A and cry genes may be used in resistance management strategies directed against lepidopteran larvae. Finally, all of the B. thuringiensis vip3A-positive isolates harboured the cspA gene, but only two were confirmed to be psychrotrophs.     

Distribution and diversity of Dipteran-specific cry and cyt genes in native Bacillus thuringiensis strains obtained from different ecosystems of Iran

One hundred and twenty-eight Bacillus thuringiensis isolates from fields of different ecological regions of Iran were collected to study the distribution and diversity of Dipteran-specific cry and cyt genes. The percentage of samples with Bt showed significant differences between different regions and also between different fields. The most Bt frequency was observed in the soil samples collected from Caspianic zone (7%) and soils of cotton (17%). Characterization of isolates was based on morphological characteristics of crystals, plasmid profiles and protein band patterns as well as PCR analysis using general and specific primers for 22 different cry and cyt genes encoding proteins active against mosquitoes. Thirty-eight different cry gene profiles were detected in this collection. Several of them were found to be different from all previously published profiles and none of the previous researches reported these numbers of profiles. Strains containing cry2-type genes were the most abundant and represent 57.1% of the isolates. Strains harboring cry24 and cry10 genes were also highly abundant (38.7 and 32.8%, respectively). cry11, cry4, cry17, cry19, cry21, cry29, cyt1, and cry9 genes were less abundant, found in 25.7, 14.3, 11.4, 1.4, 4.3, 1.4, and 10% of the strains, respectively. Among the cry2 gene containing isolates, 37.5% strains harbored cry2Aa, 55% cry2Ab, 2.5% cry2Ac, and 5% other or novel cry2-type genes. Among the cry4 gene containing isolates, 0% strains harbored cry4A, 60% cry4B, and 40% cry4C, cry4D or novel cry4 type genes. Finally, based on crystal morphology, protein patterns and PCR, 21 strains were selected as potentially high Dipteran-active for bioassays. Also our results showed that some of the isolates may harbor minimum a putative novel cry gene.     

Occurrence of Bacillus thuringiensis on Cured Tobacco Leaves

A worldwide survey was conducted to evaluate the frequency and distribution of Bacillus thuringiensis populations on cured tobacco leaves during post-harvest storage. In total, 133 tobacco samples of different types and origins were analyzed. Nine percent of the samples showed the presence of B. thuringiensis, and 24 B. thuringiensis strains were isolated and characterized. The majority of the isolates produced bipyramidal crystals, and three fourths of them showed a second type of crystal protein (cuboidal or heterogeneous crystals). Only three isolates showed the rhomboidal crystal morphology characteristic of the anti-coleopteran B. thuringiensis subsp. tenebrionis. PCR analysis with primers specific for cry1 and cry3 genes revealed eight distinct cry gene profiles. The results of this study indicate that B. thuringiensis is naturally present at low frequency on the phylloplane of cured tobacco leaves and that its distribution is worldwide. Received: 26 August 1999 / Accepted: 5 October 1999     

Cloning,Characterization and Diversity of Insecticidal Crystal Protein Genes of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Native Isolates from Soils of Andaman and Nicobar Islands

Bt strains were isolated from soils of Andaman and Nicobar Islands and characterized by microscopic and molecular methods. Diversity was observed both in protein and cry gene profiles, where majority of the isolates showed presence of 65 kDa protein band on SDS-PAGE while rest of them showed 130, 72, 44, and 29 kDa bands. PCR analysis revealed predominance of cry1I and cry7, 8 genes in these isolates. The PCR screening strategy presented here led us to identify putative novel cry genes which could be active against Coleoptera insects. Variation in the nucleotide sequences of cry genes from the isolates suggests that the genetic diversity of Bt isolates results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats in the soils of Andaman and Nicobar islands. The implications of our studies are important from the point of view of identifying novel cry genes that could be toxic to insects other than lepidoptera.     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

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.     

PCR-based method for the detection of cry genes in local isolates of Bacillus thuringiensis from Thailand

A total of 134 isolates of Bacillus thuringiensis obtained from different geographical and ecological origins in Thailand were analyzed to determine the distribution and diversity of cry1, cry2 and cry9 genes encoding for Cry proteins toxic to lepidopteran insects. Strains containing cry1-type genes (109/134 or 81.3%) were found at the same frequency as strains harboring cry2 gene (108/134 or 80.6%) whereas only 50 strains contained cry9 gene (50/134 or 37.3%). Seventeen percent (23/134) of the B. thuringiensis isolates did not harbor any cry1, cry2 or cry9 genes. Among cry1 containing isolates, cry1A (49.3%), cry1B (50.0%), cry1G (48.5%), cry1I (49.3%), cry1J (35.1%) and cry1L (47.0%) were considered abundant. The cry2 gene was distributed with high frequency (>70%) in every region of the country. The study of cry gene combinations revealed 14 cry gene profiles.