Diversity of Insecticidal Crystal proteins (ICPs) of indigenous Bacillus thuringiensis strains (original) (raw)
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Bacillus thuringiensis (Bt) is a gram positive, rod shaped spore forming bacterium. During sporulation, it produces intracellular crystal proteins (Cry proteins), which are toxic to specific insects among the orders Lepidoptera, coleopteran and Diptera. However it is still necessary to search for more toxins to control other insect orders and to provide alternatives for coping up with the problem of insect resistance. Each habitat may contain novel B. thuringiensis strains, which may have some toxic effects on target spectra of insects. In the present study, about 115 soil samples were collected from different areas under various environmental conditions. A total of 64 indigenous isolates that produce parasporal crystal inclusions were further characterized based on the morphological and biochemical analysis. Further characterization of the 64 Bt isolates for crystal protein profile through SDSPAGE revealed six different types of crystal protein profile via, 135, 97, 90, 85, 75and 50 kDa in size. Variation in the mass of crystal proteins showed the presence of diverse group of cry genes in various environmental conditions
Annals of Microbiology, 2009
Sichuan Basin situated in the west of China, the fourth-largest basin of China, is a special area with complicated geomorphology (mountain, pasture, gorge, virgin forest, highland, hurst, glacier, and plain), and contains a rich and unique biodiversity. In order to describe a systematic study of cry gene resources from Bacillus thuringiensis (Bt) strains of different ecological regions in Sichuan Basin, a total of 791 Bt strains have been screened from 2650 soil samples. The analysis of the cry genes was based on the method of PCR-restriction fragment length polymorphism (PCR-RFLP). cry1, cry2, cry3, cry4/10, cry9, cry30, and cry40-type genes were found in this basin. Strains containing cry1 genes were the most abundant in our collection (66%), and twenty-one different cry1-type gene combinations were found. Bt strains harboring cry2 genes were the second most abundant (39.5%), and the strains containing cry3, cry9, cry4/10, cry30, and cry40 genes were found in 2.5, 3.5, 4.2, 4.2, and < 1%, respectively. Furthermore, several novel haplotypes cry genes were found, and the full-length sequences of three novel cry genes were obtained, which were designated as cry52Ba1, cry54Aa1, cry30Fa1 by the B. thuringiensis Pesticide Crystal Protein Nomenclature Committee, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay of 80 strains which did not produce any PCR products indicated that these strains may harbour potentially novel Cry proteins. All these researches mentioned above revealed that the diversity and particularity of cry gene resources from B. thuringiensis strains in Sichuan Basin.
Molecular characterization of indigenous Bacillus thuringiensis strains isolated from Kashmir valley
3 Biotech
Bacillus thuringiensis (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, new indigenous B. thuringiensis strains were isolated and investigated so that these could be used as an alternative and/or support the current commercial strains/cry proteins in use. For this, 159 samples including soil, leaf and spider webs were collected from ten districts of Kashmir valley (India). Of 1447 bacterial strains screened, 68 Bt strains were identified with 4 types of crystalline inclusions. Crystal morphology ranking among the Bt strains was spherical (69.11%) > spore attached (8.82%) > rod (5.88%) = bipyramidal (5.88%) > spherical plus rod (4.41%) > spherical plus bipyramidal (2.94%) = cuboidal (2.94%). SDS-PAGE investigation of the spore–crystal mixture demonstrated Bt strains contained proteins of ...
African Journal of Biotechnology
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.
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.
Bacteria, especially Bacillus thuringiensis (Bt) and B. sphaericus (Bs), are the most potent and successful group of organisms for effective control of insect pests and vectors of diseases. Bt is widely known and exploited for δ-endotoxin which is produced in a distinct proteinacious structure known as crystal or insecticidal crystal protein. Diversities of Bt strains envisage selection and assay of different isolates and especially the native areas to develop indigenous system. Soil sample was collected from the fellow area of Ooty botanical garden Ooty. Staining characters of the organism were studied for vegetative, reproductive and crystal structure determination. The colony characters of the Bt cultures viz. TB 251 was circular, gummy white, raised and entire. But TB 252 colony was irregular, gummy white, raised and undulate. The host spectrum of the isolates needs to be assessed at the native areas for their effective exploitation. Bacillus thuringiensis (Bt) could be isolated from the soil and they are diverse in nature.
Tropical Agricultural Research and Extension, 2010
The protein toxins produced by Bacillus thuringiensis are the most widely used natural insecticides in vector and pest control in agriculture. B. thuringiensis strains present in surface and sub-surface soil samples collected from Nochchiyagama were isolated by 0.25M sodium acetate selection method. Isolated B. thuringiensis was grown on Luria Bertani agar medium and stained by Gram staining procedures. Sixty isolates of B. thuringiensis were identified by Coomassie Blue staining procedure and characterized based on colony morphology, crystal shape, plasmid profile and bioassay. Results revealed that sub-surface samples had more B. thuringiensis counts than surface soils. This study also indicated that B. thuringiensis was abundant in soils contaminated with animal wastes. All the isolates formed 'pan cake' shape circular colonies with smooth or serrate margins with varying diameter. Fifty five isolates were found to have rod shape crystals, 4 were spherical shape and only one isolate had rhomboidal shape crystal. Thirty six isolates were toxic to the third instar larvae of Aedes aegypti including the isolate which contained rhomboidal shape crystal. All the other isolates found toxic to the mosquito larvae consisted with rod shape crystal inclusion bodies. There were eight different B. thuringiensis strains among the isolates and 55% of these were B. thuringiensis israelensis.
Diversity of Bacillus thuringiensis cry genes in soils of Andhra Pradesh, India
2020
A total of 61 bacterial strains was isolated in soils from Chittoor and Kadapa districts of Andhra Pradesh, India using sodium acetate selection method and preliminarily identified as Bacillus thuringiensis based on Gram's staining, endospore staining and crystal protein stud. These strains were tested against the insect, Spodoptera litura under laboratory conditions and found 18 strains with ≥ 50% insect mortality. Further, these eighteen strains were analyzed for the insecticidal crystal protein composition with fourteen cry specific primers. The results indicated that, among all 14 cry genes, cry1I was the observed in 11 strains, followed by cry1Aa in 10 strains. At the same time a strain, F493 collected from Talakona area identified as effective against S. litura with 100 per cent larval death and which was also identified with 8 different crystal protein encoding genes (cry1Aa, cry1Ac, cry1Fa1, cry1I, cry2, cry2A(a)1, cry8, cry9Ca1).
Current Microbiology, 2012
Molecular characterization and genetic diversity among 82 soybean accessions was carried out by using 44 simple sequence repeat (SSR) markers. Of the 44 SSR markers used, 40 markers were found polymorphic among 82 soybean accessions. These 40 polymorphic markers produced a total of 119 alleles, of which five were unique alleles and four alleles were rare. The allele number for each SSR locus varied between two to four with an average of 2.97 alleles per marker. Polymorphic information content values of SSRs ranged from 0.101 to 0.742 with an average of 0.477. Jaccard's similarity coefficient was employed to study the molecular diversity of 82 soybean accessions. The pairwise genetic similarity among 82 soybean accessions varied from 0.28 to 0.90. The dendrogram constructed based on genetic similarities among 82 soybean accessions identified three major clusters. The majority of genotypes including four improved cultivars were grouped in a single subcluster IIIa of cluster III, indicating high genetic resemblance among soybean germplasm collection in India.