对二点委夜蛾高毒力苏云金芽胞杆菌的筛选及其基因型分析

【目的】获得对二点委夜蛾Athetis lepigone(M(o|¨)schler)高毒力的苏云金芽胞杆菌(Bt)菌株,寻找对该虫具有特异杀虫活性的蛋白毒素,探索Bt菌株或其杀虫基因应用于二点委夜蛾防治的可行性。【方法】通过生物测定方法比较了36株苏云金芽胞杆菌和一株恶臭假单胞工程菌PHB-cry1Ab对二点委夜蛾幼虫的杀虫活性,同时利用PCR-RFLP方法对这些菌株的基因型进行了分析。【结果】不同Bt菌株对二点委夜蛾幼虫的杀虫活性差别很大,杀虫活性高的菌株都含有cry1Ac基因。饲毒72 h后含单基因的BtHD-73菌株(cry1Ac)对二点委夜蛾2龄幼虫的毒力(LC_(50)值为188.51μg/g)明显高于含多基因的Bt SC-40菌株(cry1Ac,cry2Ac,cry1I,vip3A)的毒力(LC_(50)值为418.13μg/g)。含有vip3A基因的Bt SC-40和BtHD-13营养期上清液对二点委夜蛾2龄幼虫表现出一定的杀虫活性(72 h死亡率分别达到42.5%和57.4%),而无vip3A基因的Bt HD-73营养期上清液未表现出明显的杀虫活性。【结论】由cry1Ac基因编码的Cry1Ac蛋白对二点委夜蛾幼虫具有特异杀虫活性,Vip3A蛋白对二点委夜蛾幼虫可能也有一定的杀虫活性。     

Rapid Identification of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 with Proteomic Analysis

This study was carried out to identify rapidly δ-endotoxin from Bacillus thuringiensis (Bt) subsp. kurstaki HD-1 with proteomic analysis. Protoxin was isolated from sporulated cells and purified by ultracen-trifugation using 40-70% sucrose density gradient. Protoxin was treated with trypsin to analyze digested peptides by liquid chromatographytandem mass spectrometry. The proteomic analysis for detected peptides revealed that this methodology is available for discriminating similar Bt strains by identifying Bt subsp. kurstaki HD-1-specific peptides, suggesting that proteomic analysis can be used for rapid identification of Bt strains.     

棉花抗虫工程菌对棉铃虫的离体及活体生物测定

棉花抗虫工程菌是以能在棉株体内定殖的优势内生细菌Bacillus cereus (Bc9102)为宿主菌,将Bt kurstaki的δ-内毒素基因cryIA©整合到其染色体上形成的内生工程菌。以棉铃虫Helicoverpa armigera为供试昆虫的离体生测结果表明：在同一剂量水平上,HE-1、HE-2、ME14-2、ME14-3、MK14-1等工程菌株对棉铃虫幼虫的毒力高于或等于Bt野生菌株HD-73,浓度最高时这几个工程菌处理的棉铃虫死亡率分别为96.7%、83.3%、93.3%、83.3%、80.0%,而HD-73为80.0%,死亡率与浓度呈正相关。在用上述工程菌接种棉花的活体生测中,注射处理和喷雾处理法杀虫效果均优于浸种处理法。以HE-1为例,注射、喷雾、浸种处理4周后棉铃虫校正死亡率分别达到90.0%、76.0%和23.3%。工程菌对棉铃虫的生长发育也有抑制作用。     

Effects of stilbene optical brighteners on the insecticidal activity of Bacillus thuringiensis and a single nucleopolyhedrovirus on Helicoverpa armigera

The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC₅₀ values) but their effect on Bacillus thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and occlusion bodies (OBs) of a Spanish isolate of H. armigera single nucleocapsid nucleopolyhedrovirus (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD-1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its insecticidal activity. Mixtures of HearNPV-SP1 OBs with Tinopal UNPA-GX or Leucophor UO resulted in 31.4- and 11.4-fold increases in pathogenicity, respectively, at 1%, and 11.4- and 6.3-fold increases in pathogenicity, respectively, at 0.1%, compared to the OBs alone. However, none of these brighteners increased Bt HD-1 activity. These results appear consistent with the hypothesis that the enhancement of HearNPV-SP1 pathogenicity and the null or antagonistic effects observed in Bt HD-1 against H. armigera were due to optical brightener-mediated degradation of the peritrophic membrane, but additional systematic studies involving a broad range of brighteners and electron microscope observations are required to confirm this premise.     

玉米内生杀虫工程菌对玉米螟的离体及活体生物测定

12#Bt/CXC是以能定植在玉米维管束系统的内生菌I>Clavibacter xyli subsp.Cynodon—tis(CXC)为宿主菌,将Bt urstaki的δ—内毒素基因cryIA?整合到其染色体上形成的内生工程菌。以玉米螟Ostrinia furnacalis Guenee为供试昆虫的生测结果表明：在同一浓度下,12^#菌株对玉米螟的毒力均高于野生型Bt菌株HD73和空白对照(最低浓度除外)。浓度最高时12^#处理的死亡率为63%,而m工73为53%,死亡率与浓度呈正相关,相关系数r₁₂^#大于r_HD—73。在用12^#Bt/CXC接种玉米的活体生测中,人工接虫4周后检测,注射接种法效果明显优于种子处理法。不同浓度的注射接种处理,玉米螟幼虫减少率最低70%,最高可达96%,与未处理对照相比差异显著。12#菌剂处理后对玉米螟的生长发育也有抑制作用,虫体重减轻26.4%～44.5%,处理株虫龄平均为2龄,而对照株为4龄。     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Cellular Fatty Acid Analysis of Isolates of Bacillus thuringiensis Serovar kurstaki,Strain HD-1

Whole-cell cellular fatty acid (CFA) composition was utilized to determine if Bacillus thuringiensis serovar kurstaki (HD-1) larvicides produced by different manufacturers could be distinguished from each other and to determine whether these larvicides were distinguishable from isolates deposited in public collections. This study analyzed Biobit, Dipel, Foray, Thuricide, and the non HD-1 larvicides Delfin and Javelin, as well as the 1971 and 1980 Standards of HD-1. Isolates of HD-1 deposited in the collections of the American Type Culture Collection, Bacillus Genetic Stock Center, Pasteur Institute, and United States Department of Agriculture (USDA) were also analyzed. The data were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages (UPGMA). Samples that linked at a Euclidean distance ≤2.0 units were considered to belong to the same fatty acid strain. Isolates of HD-1 from commercial products and deposits of HD-1 in the public collections and Standards were polytypic; 22 separate fatty acid strains were identified in the 1971 and 1980 Standards and 35 fatty acid strains were identified in the public collections. The type strain for Btk contained multiple fatty acid strains; three fatty acid strains were present in both the Bacillus Genetic Stock Center and the Pasteur Institute collections. In contrast, the type strain for serovar kurstaki in the USDA collection (HD-73) was monotypic and its fatty acid strain did not occur in other collections. We could distinguish between HD-1 and non-HD-1 larvicides using CFA composition. We conclude that CFA analysis may be used to identify commercial products.     

Intestine and Environment of the Chicken as Reservoirs for Extraintestinal Pathogenic Escherichia coli Strains with Zoonotic Potential

Although research has increasingly focused on the pathogenesis of avian pathogenic Escherichia coli (APEC) infections and the “APEC pathotype” itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (n = 121) with nonoutbreak strains, including fecal E. coli strains from clinically healthy chickens (n = 211) and strains from their environment (n = 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic E. coli (ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal E. coli strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.     

Transmission identification of Escherichia coli aerosol in chicken houses to their environments using ERIC-PCR

In order to study E. coli aerosol spreading from chicken houses to their surrounding air, air samples, including indoor and outdoor air (upwind 10 and 50 m as well as downwind 10, 50, 100, 200 and 400 m away) of 5 chicken houses were collected using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli concentrations (CFU/m3 air) collected from different sampling sites were calculated. E. coli strains from chicken feces samples were also isolated. Furthermore, the enterobacterial repetitive intergenic consensus (ERIC)-PCR method was applied to amplify the isolated E. coli strain DNA samples. Through the genetic similarity analyses of the E. coli obtained from different sampling sites, the spreading of bioaerosol from animal houses to the ambient air was characterized. The results showed that the isolated E. coli concentrations in indoor air (9―63 CFU/m3) in 5 chicken houses were higher than those in upwind and downwind air, but there were no significant differences between the indoor and downwind sites 10 m away from all the 5 houses (P>0.05). The phylogenetic tree indicated that a part of the E. coli (34.1%) isolated from indoor air had 100% similarity with those isolated from feces, and that most of E. coli isolated (54.5%) from downwind at 10, 50, 100 or even 200 m had 100% similarity with those isolated from indoor air or feces too. But those isolated from upwind air had a lower similarity (73%―92%) with corresponding strains isolated from indoor air or feces. Our results suggested that some strains isolated from downwind air and indoor air originated in the chicken feces, but most of isolates obtained from upwind air samples did not come from the chicken feces or indoor air. Effective hygienic measures should be taken in animal farms to prevent or minimize downwind spreading of microorganism aerosol.     

Methicillin (Oxacillin)-Resistant Staphylococcus aureus Strains Isolated from Major Food Animals and Their Potential Transmission to Humans

From May 2001 to April 2003, various types of specimens from cattle, pigs, and chickens were collected and examined for the presence of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). S. aureus was isolated and positively identified by using Gram staining, colony morphology, tests for coagulase and urease activities, and an API Staph Ident system. Among 1,913 specimens collected from the animals, 421 contained S. aureus; of these, 28 contained S. aureus resistant to concentrations of oxacillin higher than 2 μg/ml. Isolates from 15 of the 28 specimens were positive by PCR for the mecA gene. Of the 15 mecA-positive MRSA isolates, 12 were from dairy cows and 3 were from chickens. Antimicrobial susceptibility tests of mecA-positive MRSA strains were performed by the disk diffusion method. All isolates were resistant to members of the penicillin family, such as ampicillin, oxacillin, and penicillin. All isolates were also susceptible to amikacin, vancomycin, and trimethoprim-sulfamethoxazole. To determine molecular epidemiological relatedness of these 15 animal MRSA isolates to isolates from humans, random amplified polymorphic DNA (RAPD) patterns were generated by arbitrarily primed PCR. The RAPD patterns of six of the isolates from animals were identical to the patterns of certain isolates from humans. The antibiotypes of the six animal isolates revealed types similar to those of the human isolates. These data suggested that the genomes of the six animal MRSA isolates were very closely related to those of some human MRSA isolates and were a possible source of human infections caused by consuming contaminated food products made from these animals.     

Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5′ mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (±0.214) and 1.356 (±0.247) μm in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (±0.222) and 1.139 (±0.202) μm in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 × 10⁹, 1.3 × 10⁹), compared to HD-73-pEHchiA74 (4.9 × 10⁹, 5.3 × 10⁹) and HD-73 (6.8 × 10⁹, 8.8 × 10⁹) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.     

Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry⁻) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized (González and Carlton, 1980). The plasmid patterns of both Cry⁻ and Cry⁺ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry⁻ mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry⁻ mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (˜130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.     

Dual Insecticidal Activity of Spodoptera-Toxic Bacillus thuringiensis Strain Transformed with Lepidopteran-Specific Cry Toxin

The E. coli-B. thuringiensis shuttle vector for expression of cry1Ac, pHT1K-1Ac plasmid was introduced into acrystalliferous B. thuringiensis Cry⁻B and Spodoptera toxic STB-3 strain. The presence of a recombinant plasmid in transformants after electroporation was confirmed by PCR. The 1K-1Ac/Cry⁻B(Cry⁻B transformant) and 1K-1Ac/STB-3 (STB-3 transformant) produced bipyramidal-shaped parasporal inclusion that was 130 kDa in size as like B. thuringiensis subsp. kurstaki HD-73. In P. xylostella bioassay, these transformants showed significantly high toxicity than the wild-type recipients and further, in case of B. thuringiensis STB-3 transformant still had original Spodoptera toxicity. These results suggested that the pHT1K could be successfully applied for generating individual B. thuringiensis strains that produce various combinations of insecticidal proteins to expand their host spectrum and enhance insecticidal activity.     

Identification of the Most Abundant Lactobacillus Species in the Crop of 1- and 5-Week-Old Broiler Chickens

Bacteria from crops of 1- and 5-week-old broiler chickens fed with two brands (diets A and B) of wheat-based diets were isolated on Lactobacillus-selective medium and identified (n = 300) based on partial 16S rRNA gene sequence. The most abundant Lactobacillus species were L. reuteri (33%), L. crispatus (18.7%), and L. salivarius (13.3%). Regardless of farm and feed, L. reuteri was the most abundant species (P < 0.005) in the crops of the younger chickens. However, the amount of L. reuteri was significantly reduced in the crops of the 5-week-old chickens regardless of the feed (P = 0.016). The diversity of L. reuteri isolates was studied by fatty acid analysis, and the 94 L. reuteri isolates could be arranged into several clusters. The nisin sensitivities of the L. reuteri isolates were determined because nisin is a candidate coccidiostat. Sensitive isolates were found more frequently in younger chickens (77%) than in 5-week-old chickens (23%), whereas chickens fed with commercial feed B had a higher proportion of nisin-resistant isolates (73%) than did chickens fed with feed A (45%). Nisin-resistant strains are potential candidates for adjunct cultures for maintaining L. reuteri in its natural niche in the crop and are potential targets for genetic engineering with nisin-selectable food-grade vectors. The diversity of the L. reuteri population suggested that one should consider including several strains representing different clusters and nisin resistance phenotypes in candidate probiotic feed supplements for chickens.     

Scanning Electron Microscopy of Selected Members of the Streptomyces hygroscopicus Group

Distinctive variations in whole spore morphology and spore surface morphology of Streptomyces hygroscopicus strains, which had been observed previously by the authors by the preshadowed carbon replica technique, were confirmed by observations with the scanning electron microscope.     

Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis

Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective although, at the doses used, not all cells were killed. After 2 days of exposure to 0.8 pg/μl AA 1-9 or 0.06 pg/μl HD-73, columnar and goblet cell numbers declined to ca 20% of controls. In contrast, stem and differentiating cells increased to 140-200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves. Controls and cells treated with toxin from Bt strain AA 1-9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1). With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation. The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.     

A mid-gut microbiota is not required for the pathogenicity of Bacillus thuringiensis to diamondback moth larvae

The mode of action of the entomopathogenic bacterium Bacillus thuringiensis ( Bt ) remains a matter of debate. Recent reports have claimed that aseptic lepidopteran hosts were not susceptible to Bt and that inoculation with mid-gut bacteria restores pathogenicity. These claims are controversial because larvae were rendered aseptic by consuming antibiotics, although the effect of these antibiotics on Bt was not examined. We tested the generality of the mid-gut bacteria hypothesis in the diamondback moth, Plutella xylostella using properly controlled experiments that investigated the effect of antibiotic consumption and absence of gut microbiota separately. We found that purified Bt toxin and spore/toxin mixtures were fully pathogenic to larvae reared aseptically. Persistence of antibiotics in larval tissues was implicated in reducing host mortality because larval consumption of the antibiotic rifampicin reduced the pathogenicity of rifampicin-sensitive Bt strains but not rifampicin-resistant strains. Inoculating larvae with Enterobacter sp. Mn2 reduced the mortality of larvae feeding on Bt HD-1 and the presence of a culturable gut microbiota also reduced the pathogenicity of the Bt toxin Cry1Ac, in agreement with other studies indicating that an intestinal microbiota can protect taxonomically diverse hosts from pathogen attack. As ingestion of antibiotics suppresses host mortality the vegetative growth of Bt in the host must be important for its pathogenicity. Furthermore, claims that aseptic larvae are not susceptible to Bt must be supported by experiments that control for the effect of administering antibiotics.     

Diversity of Bacteroides fragilis Strains in Their Capacity To Recover Phages from Human and Animal Wastes and from Fecally Polluted Wastewater

Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains of Bacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 10¹ to approximately 10³ phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.     

Molecular Characterization and Genetic Diversity of Insecticidal Crystal Protein Genes in Native Bacillus thuringiensis Isolates

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.