• Korean journal of applied entomology
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• v.32 no.3
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• pp.300-306
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• 1993

The CryIIA gene encoding the insecticidal crystal protein of Bacillus thuringiens!s subsp. kurstalri HD-l has been cloned in Escherichia col!, and its nucleotide sequences were determined completely. 5kb Hindlli fragment harboring CryIIA gene was screened in the large ca. 225kb plasmid DNA by southern blot. HindlIT digested 5kb fragment was ligated into pUC19 and transformed in E. coli. The 4kb BamHI-HindlIT fragment containing the CryIIA gene was subcloned and named pSKIIA. DNA sequence analysis demonstrates that pSKIIA is the gene of an operon which is comprised of Lhree open reading frames (designated orn, orf2 and or£3). The CrylIA gene is composed of 3,952bp-long BamHI-Hindill DNA restriction fragment. The orf3 code for a polypeptide of 633 amino acid residues. The protoxin protein has a predicted molecular weight of 70,780. The E. coli derived protoxin gene product is biologICally active against three species of Lepidopteran (Plu.lelia maculipennis, He/iolhis assulta, Spodoptera litura) and a species of Dip Leran( Culex pipines) larvae in bioassay.

• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.167-176
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• 2013

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

• The Korean Journal of Pesticide Science
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• v.11 no.3
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• pp.201-209
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• 2007

Characteristics of the 5 biopesticides that included Bacillus thuringiensis and on the domestic markets were investigated. These products were contained different strains of B. thuringiensis, for examples; product A and E was B. thuringiensis subsp aizawai; product B was B. thuringiensis; product C was B. thuringiensis Berline var. kurstaki; product D was B. thuringiensis var. kurstaki. Number of active spores were counted because they could influence the bio-activity against target pests. Only product C are contained the fixed quantity as its label, however, product D and E were a tenth part, and product A and B were a hundredth part of their descriptions. The pHs of product A and B were measured 3.67 and 3.73, and C, D and E were 5, respectively. Typical bypyramidal crystals produced from B. thuringiensis was found in only product C under a phase contrast microscope. For the uniform formulation of products that conformed whether B. thuringiensis were equally spreaded on the crops, B. thuringiensis in the C, D and E were equally grown on the nutrient agar medium As a results, product A were more different from product C than any other products. When product A and C were bioassayed against different larval stages of diamondback moth, their mortalities with spraying application were showed 100% after 48 hours.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2004.05a
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• pp.174-174
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• 2004

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.1
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• pp.18-27
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• 2009

B. thuringiensis strain LY-99 belonging to subsp. alesti (H3a3c), was isolated from Chinese tobacco warehouse and showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes from B. thuringiensis LY-99, an extended multiplex PCRrestriction fragment length polymorphism (PCRRFLP) method was established by using two pairs of universal primers based on the conserved regions of the cry1-type genes to amplify around 2.4 kb cry1-type gene fragments. Then the DNA fragment was cloned into pGEM-T Easy vector and digested with EcoRI and EcoRV enzymes. Through this method, a known cry1-type gene was successfully identified from the reference strain, B. thuringiensis subsp. alesti. In addition, the RFLP patterns revealed that B. thuringiensis LY-99 included a novel cry1A-type gene in addition to cry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novel cry1A-type gene was designated cry1Ah2 (Genbank accession No DQ269474). An inverse PCR method was used to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensis LY-99 plasmid DNA including 5' region and partial ORF was amplified, and sequence analysis revealed that cry1Ah2 gene from LY-99 showed 89.31% of maximum sequence similarity with cry1Ac1 crystal protein gene. In addition, the deduced amino acid sequence of Cry1Ah2 protein shared 87.80% of maximum identity with that of Cry1Ac2. This protein therefore belongs to a new class of B. thuringiensis crystal proteins.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.358-364
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• 1998

New microbial-control agents were prepared with B. thuringiensis strain NT0423 having unique properties which are different with other B. thuringiensis strains belonging to serotype 7[Kor. J. Appl. Entomol. 32: 426-432.]. Three B. thuringiensis formulations designated as BioBact 10%, 20% and 40%, were made with various combinations of adjuvants. These formulations showed good physical properties in wettability, suspensibility, particle size and adherence. In addition the result of SDS-PAGE analysis indicated that $\delta$-endotoxins remain stably in all formulations. Among the tested formulations, two wettable powder formulations, BioBact 20% and 40%, comprising 20% and 40% of B. thuringiensis technical powder showed the effective control against diamondback moth larvae (Plutella xylostella) in laboratory and field tests. Especially, when compared with commercial B. thuringiensis formulations (A and B commercial formulations) in field evaluation, BioBact 20% and 40% formulations showed equal activity up to 80% lethality and a good persistence effect which remain on leaves at least 7 days.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.322-326
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• 1994

Bacillus thuringiensis strain BT-14 was isolated from alfalfa dust in Korea. The strain BT-14 produced one bipyramidal crystal and one spore in the cell. The biochemical characteristics of the strain BT-14 were similar to those of Bacillus thuringiensis subsp. kurstaki HD-l. Examination of its antibiotic resistance revealed that while the strain BT-14 was less resistant than BTK HD-l to ampicillin, gentamycin, neomycin and tobramycin, it was more resistant to amikacin than BTK HD-l. The $\delta$-endotoxin crystal of strain BT-14 consisted of a single protein with a high molecular weight of ca 135 KD on a 10% SDS-PACE. The strain BT-14 contained at least nine different plasmids with sizes of 2.9, 5.3, 5.8, 6.2, 9.4, 15.1, 18.1, 23.1 and 79 Kb. In insect bioassay, the isolated strain BT-14 showed lethality of 67% against Plutella xylostella larvae at dilution of 5$\times$$l0^{-4}$ (5$\times$l0 to 3$\times$$l0^2$ spores/ml), which is, almost equivalent to that of BTK HD-l.

• Journal of Microbiology and Biotechnology
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• v.5 no.6
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• pp.359-363
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• 1995

The antigenic variation of B. thuringiensis subsp. satto have been investigated for 120 hours during sporulation and crystallization by using SDS-PAGE and Western blot. Most antigens of a vegetative cell were found to disappear as it was in sporulation and crystallization, but protein antigens of 46, 29, 27, and 21 kDa continued to be expressed. The new protein bands of 293, 138, 119, 75, and 68 kDa appeared on days 2 through 5 in modified GYS medium. They were thought to be involved in sporulation and crystallization. The protein of 138 kDa was found to be a major protein of both crystal and spore. The expression patterns were immunologically analyzed by Western blot. The polyclonal antisera against the intact crystal showed strong immunoreactivity to proteins with molecular masses of 293, 138, 68, and 46 kDa. The polyclonal antisera against the spore recognized proteins of 293, 138, 68, and 46 kDa. Both crystals and spores appeared to express the common protein antigens.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.685-691
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• 1998

Baculovirus Hyphantrin. cunea nuclear polyhedrosis virus (HcNPV) insecticide containing the insecticidal protein (ICP) gene from Bacillus thuringiensis subsp. kurstaki HD1 was constructed using a lacZ-HcNPV system. The ICP ($\delta$-endotoxin) gene was placed under the control of the polyhedrin gene promoter of the HcNPV. A polyhedrin-negative virus was derived and named ICP-HcNPV insecticide. Then, the insertion of the ICP gene in the ICP-HcNPV genome was confirmed by Southern hybridization analysis. Polyacrylamide gel electrophoresis (PAGE) analysis of the Spodoptera frugiperda cell extracts infected with the ICP-HcNPV showed that the ICP was expressed in the insect cells as 130 kDa at 5 days post-infection. The ICP produced in the cells was present in aggregates. When extracts from the cells infected with the ICP-HcNPV were fed to 20 Bombyx mori larvae, the following mortality rate was seen; 8 larvae at 1 h, 10 larvae at 3 h, and 20 larvae at 12 h. These data indicate that the B. thuringiensis ICP gene was expressed by the baculovirus insecticide in insect cells and there was a high insecticidal activity. The biological activities of the recombinant virus ICP-HcNPV were assessed in conventional bioassay tests by feeding virus particles and ICP to the insect larvae. The initial baculovirus insecticide ICP-HcNPV was developed in our laboratory and the significance of the genetically engineered virus insecticides is discussed.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.534-540
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• 1999

Thirty-seven strains of Bacillus thuringiensis were isolated from Korean soil and examined for H-antigen serotyping, toxicity, and different spectra of biological activities. The isolate HL-175 bore a specific H-antigen, different from the 51 known serotypes, a spherical $\delta$-endotoxin crystal, and minor different biochemical characteristics. It was resistant to ampicillin, colistin, and penicillin G. Therefore, it was classified as a new serotype, H52, with the name kim. The other 36 isolates also produced endotoxin crystals and endospores. The crystal shape of eight strains was cuboidal while the others were bipyramidal. Biochemical characteristics of the isolates were only slightly different from the known serotypes of B. thuringiensis. The flagellar (H) antigens of the 36 isolates were identified as: one colmeri (H21), three galleriae (H5a,5b); two pakistani (H13); one toumanoffi (H11a, 11b); and twenty-nine kurstaki (H3a,3b). All 36 isolates were resistant to ampicillin, colistin, penicillin, cephalothin, and chloramphenicol.