Molecular characterization and insecticidal activities of Malian native crystalliferous Bacillus thuringiensis (original) (raw)
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The objective of this work was to select the most active Bacillus thuringiensis (Bt) isolated from agricultural soils of Mali through the molecular characterization and the determination of insecticidal activities of the protein crystals, produced by these native isolates. Crystal proteins were extracted from B. thuringiensis culture, and characterized using the SDS-PAGE techniques. Their insecticidal activities were tested using third-instar larvae of Helicoverpa armigera in bioassay tests. The results showed that, of 62 B. thuringiensis treated, 52 isolates showed fragments varying between 10 and 140 kDa on 12% polyacrylamide gel. Cry1 and Cry2 protein crystals were recognized to be effective against Lepidoptera's larvae, which were found in 21% of the tested isolates. In addition to these two expected crystal protein weights, other molecular weights were observed at different proportions, suggesting the presence of other cry genes in the local B. thuringiensis isolates. Four native B. thuringiensis isolates were able to kill 95 to 100% of H. armigera 3rd-instar larvae. Only one native of B. thuringiensis isolate was able to kill 100% of the H. armigera larvae. This is the first study for molecular characterization of Malian native B. thuringiensis isolates, showing the efficacy of the native B. thuringiensis against an important agricultural insect pest.
Diversity of Insecticidal Crystal proteins (ICPs) of indigenous Bacillus thuringiensis strains
Tribhuvan University Journal of Microbiology, 2018
Objectives: The purpose of this study was to characterize the indigenous Bacillus thuringiensis (Bt) isolated from the soil samples of Terai. Methods: A total of 50 soil samples were collected from cultivated and barren fi elds of Terai region. Isolation was carried out using the acetate selection protocol Nutrient broth (NB) was acetated by using 0.25M sodium acetate which is a selective enrichment method for isolation of Bt. Characterization of the isolate was done by phenotyping methods (microscopy and biochemical). Results: No distinct variation was observed between the isolates of cultivable and uncultivable lands. Bt were categorized into 7 different types based on colony morphology. The dominant colony was fried egg type identical with the reference strain, followed by fl at white type of colony. The result showed that even though the colony morphology was same but the ICPs (Insecticidal crystal proteins) shapes produced by them varied, rod shapes (53.57%), spherical (10.71%), ovoid (8.3%), amorphous (17.85%), capheaded (9.5%). ICPs morphology revealed the cry1, cry2, cry3, cry4, cry8, cry 9, cry10 and cry11 types of gene may be present in the native isolates. Conclusion: This study represents the fi rst report of several indigenous Bacillus thuringiensis strains with signifi cantly different ICPs producing strains from hot tropical climate.
The novel δ-endotoxin from Bacillus thuringiensis (Bt) is one of the alternative measures for lepidopteran pest control. A survey of 31 Bt isolates, obtained from Thailand and previously screened for their toxicity to lepidopterans, was conducted to determine the presence of cry1-type genes, using polymerase chain reaction -restriction fragment length polymorphism analysis. Seven distinct types of cry1 genes: cry1Aa, cry1B, cry1C, cry1Cb, cry1D, cry1E, and cry1F, were identified. The most common of the cry1-type genes was cry1Cb, followed by cry1C and cry1D, which covered 64.6, 48.4, and 25.8%, respectively. Besides cry1C and cry1Cb, two candidate cry genes, cry1E and cry1D, of isolate JC 190 (harbouring cry1C/1E), with cotton bollworm toxin specificity, showed 99% amino acid sequence identity to Cry1Ea of B. thuringiensis subsp. kenyae, while isolate JC 291 (containing cry1C/1Cb/1D), with Asian corn borer toxin specificity, harboured Cry1Dc, which exhibited only 84% amino acid sequ...
Egyptian Journal of Biological Pest Control
Background The indiscriminate use of synthetic insecticides is not affordable and poses collateral damage to humans, non-target organisms, and environmental health. Bacillus thuringiensis (Bt) is a cosmopolitan, antagonistic soil bacterium employed as an alternative to chemical insecticides in pest management. In this study, genetic diversity and insecticidal activity of 50 indigenous Bt isolates were investigated in order to find the effective Bt isolates with a broad spectrum of insecticidal activity against lepidopteran insects. Results Most of the Bt isolates investigated appeared as creamy white in colour and fried egg or mucoid-type colonies having a flat or raised elevation with entire or undulated margins. Out of 50 isolates investigated, crystal shapes varied from cuboidal (40.00%), spherical (36.00%), bipyramidal (30.00%), rectangular (10.00%), and minute crystal attached to the spores (28.00%). SDS-PAGE analysis revealed that the molecular weight of the crystal proteins o...
Applied and environmental microbiology, 1988
We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pi...
A novel Bacillus thuringiensis strain and its pathogenicity against three important pest insects
Journal of Stored Products Research, 2012
A highly pathogenic Bacillus thuringiensis (Bt) strain was isolated from a soil sample in Turkey and characterized in terms of both its 16S-ITS rDNA region and cry gene content. This strain (SY49.1) harboured several cry genes producing crystalline inclusions known to have toxicity on lepidopteran, dipteran and coleopteran pests. The 16S-ITS sequence analysis of Bt SY49.1 showed 98% similarity to Bt serovar andalousiensis BGSC 4AW1. The larvae of Ephestia kuehniella and Plodia interpunctella were treated with a spore-crystal mixture of this strain in the dose range of 50e1000 mg g À1. Mortality rates were higher than 90% at the highest concentration for these pests. For Tribolium castaneum much higher concentrations were needed than with lepidopteran pests larvae. At the highest concentration tested of 10 mg g À1 , about 62% mortality was obtained. It was concluded that this native strain of Bt could be used as an effective biocontrol agent against various lepidopteran pests.
Total of 553 soil samples were taken for the isolation of Bacillus thuringiensis (Bt) by two different methods using acetate selection process in order to understand their biodiversity and distribution in different soils. Out of these samples, 86 Bt like isolates were obtained and characterized based on PCR analysis using primers for genes encoding proteins active against Lepidoptera, crystal morphology, and protein band patterns as well as their insecticidal activity against Helicoverpa armigera. Out of these Bt isolates, 12 and 8 isolates were PCR positive for Lep-I and Lep-II gene specific primers, respectively. The insecticidal crystal protein bands size ranged from 24 to 130 kDa. These isolates may contain a new gene or genes that seem to be promising for biological control of insects and management of insecticide resistance. Based on morphological and molecular studies, 15 potentially lepidopteranspecific active isolates were selected for bioassay. Two of the isolates were found to be more toxic to H. armigera than the reference strain HD-1. Given the variability of insecticidal crystal proteins described to date, the isolation and characterization of various new subspecies of B. thuringiensis could contribute to the discovery of very effective biopesticide with higher insecticidal activities and broader host range against insect pests especially in the area of agriculture and forestry, and vectors of medically important diseases of human beings and other animals.
Environmental distribution and diversity of insecticidal proteins of Bacillus thuringiensis Berliner
Malaysian Journal of Microbiology
Bacillus thuringiensis Berliner based biopesticides have been successfully used world over for the control of agricultural pests and vectors of human diseases. Currently there are more than 200 B. thuringiensis strains with differing insecticidal activities are available as biocontrol agents and for developing transgenic plants. However, two major disadvantages are the development of insect resistance and high target specificity (narrow host range). Globally there is a continuous search for new B. thuringiensis strains with novel insecticidal activities. The present study aims to study the environmental distribution of B. thuringiensis and their toxic potential against insect pests. Soil and grain samples were collected from different environments and were processed by a modified acetate selection method. Initially B. thuringiensis isolates were screened on the basis of colony morphology and phase contrast microscopy for the presence of parasporal crystal inclusions. The population dynamics showed that B. thuringiensis is abundant in sericulture environment compared to other niches. Relative abundance of B. thuringiensis strains in sericulture environment shows the persistent association of B. thuringiensis with Bombyx mori (silk worm) as insect pathogen. The protein profiles of the selected strains were studied by SDS-PAGE. The protein profiles of majority of B. thuringiensis isolates from grain storage facilities predominantly showing the 130 kDa and 68 kDa proteins, which is characteristics of lepidopteran active B. thuringiensis. However, one isolate BTRX-4 has 80-85 kDa protein, which is novel in that, this strain also exhibits antilepidopteran activity, which is normally presented by B. thuringiensis strains having 130 kDa and 68 kDa proteins. The protein profile of B. thuringiensis isolates from sericulture environment shows two different protein profiles. B. thuringiensis isolates BTRX-16 to BTRX-22 predominantly show 130 kDa protein, however, the B. thuringiensis isolates BTRX-23 to BTRX-30 have two distinct protein profiles corresponding to 130 kDa and 68 kDa. These results show that there may be more than one B. thuringiensis strain that can infect Bombyx mori. The preliminary bioassay against second instar larvae of Heliothis armigera showed varying mortality rate. In conclusion, despite the ubiquitous presence of B. thuringiensis strains in different environments, specifically the sericulture environment supports B. thuringiensis in a significant manner compared to other environments. Further the ICPs produced by different strains of B. thuringiensis are unique in terms of the protein profile and hence may also differ in their insecticidal activities.
Isolation and characterization of Lepidopteran specific Bacillus thuringiensis
Seven Bacillus thuringiensis strains were isolated and selected on the basis of primary toxicity test from 22 representative soil samples of Middle Gujarat region of India. Characterization of the strains provided useful information on its distribution with a native cry gene. SDS-PAGE analysis of these isolates shows presence of major bands of Cry1 type of pattern, which was comparable with standard check of B. thuringiensis var. kurstaki HD-73. Only one isolate gave different banding pattern. Characterization of strains was based on PCR with general and specific primers for cry genes. Use of cry primers elucidated that majority of the isolates carried cry1 gene indicating their effectiveness against Lepidopteran pests while use of gene specific primers indicated preponderance of cry1A and cry1Ac genes and some isolates confirmed the presence of cry1Ab and cry1B, cry1C, cry1D and cry9 genes. Result of SDS-PAGE of whole-cell proteins indicated that all the isolates differed from each other including the standard, showing the usefulness of diversity studies. Bioassays with Helicoverpa armigera exhibited LD 50 of 1.73 x 10 7 spores-crystal mixture/cm 3 by isolate HP-4 in comparison LD 50 of 3.14 x 10 7 spores-crystal mixture/cm 3 of B. thuringiensis var. kurstaki HD-73.
Bacillus thuringiensis and its pesticidal crystal proteins
Microbiology and molecular biology reviews : MMBR, 1998
During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.