• Korean journal of applied entomology
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• v.62 no.4
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• pp.277-285
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• 2023

The onion moth, Acrolepiopsis sapporensis, was monitored in the farms cultivating the welsh onion, Allium fistulosum, using sex pheromone from transplantation to harvest. Two occurrence peaks were observed at early June and late July after the overwintering population. However, the population sizes were varied among different years and the cultivating environments. To effectively control A. sapporensis with microbial pesticides, different Bacillus thuringiensis strains were screened to select B. thuringiensis kurstaki (BtK). To enhance the insecticidal virulence of BtK, the culture broth of Photorhabdus temperata temperata (Ptt) was added to the BtK. This mixture of two entomopathogenic bacteria was called 'BtPlus', which was superior to BtK alone in the insecticidal virulence. The enhanced virulence was explained by the immunosuppressive activity of the secondary metabolites contained in the Ptt extract. The metabolites inhibited both cellular and humoral immune responses of A. sapporensis, resulting in the enhanced virulence of BtK. These results suggest that A. sapporensis occurs in the welsh onion fields and the resulting economic damage would be effectively prevented by BtPlus application.

• 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.

• 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.

• Korean journal of applied entomology
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• v.36 no.4
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• pp.341-350
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• 1997

We have now constructed a novel recombinant baculovirus producing fusion protein with Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin and Bacillus thuringiensis(Bt) cryIA(c) crystal protein. The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The N-terminal of cryIA(c) gene of Bt subsp. kurstaki HD-73 was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front of intact polyhedrin gene or by insertion into the HindIII site in polyhedrin gene. The recombinant baculoviruses were named as BtrusI or BtrusII, respectively. Although single transcript from the fusion protein gene was apparently observed. BtrusI was produced the two proteins, 92 kDa fusion protein and only polyhedrin. In addition, fusion protein produced by BtrusI did not form polyhedra. Interestingly, however, the cells infected with BtrusII did not show a 33 kDa polyhedrin band as a cells infected with BtrusI. Cells infected with BtrusII were only produced fusion protein, but the polyhedra formed by fusion protein was not observed. To determine the insecticidal toxicity of fusion protein, therefore, Sf9 cells infected with BtrusI were inoculated to Bombyx mori larvae. Sf9 cells infected with BtrusI that expressed the fusion protein caused larval mortality although the insecticidal toxicity was low. In conclusion, our results clearly demonstrated that the fusion protein with polyhedrin and Bt cryIA(c) crystal protein have a insecticida toxicity.

• Korean journal of applied entomology
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• v.34 no.3
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• pp.243-248
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• 1995

In order to isolate naturally occurring novel Bacillus thuringiensis strains, we investigated the distribution of B. thuringiensis from granary which exist in Kyong-gi province, Korea. A total of 146 strains of B. thuringiensis producing spore and crystal wre isolated. The toxicity of B. thuringiensis isolates was examined against lepidopteran larvae (Bombyx mori), dipteran larvae (Culex pipiens) and coleopteran larvae (Sitophilus oryzae.), respectively. The results showed that a large number of B. thuringiensis isolates from granary dusts were isolated and most isolates wer toxic to lepidopterous larvae. Also, non-toxic B. thuringiensis isolate was common. In order to isolate many B. thuringiensis, therefore, it suggested that granary is favorable locality.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1708-1716
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• 2016

Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned from Bacillus thuringiensis subsp. kurstaki, and wild-type (GDH-BT^WT) and His-tagged (GDH-BT^N-His, GDH-BT^C-His) enzymes were produced in Escherichia coli BL21 (DE3). All enzymes were produced in the soluble forms from E. coli. GDH-BT^WT and GDH-BT^N-His showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BT^C-His showed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BT^WT was stable up to 65℃, whereas GDH-BT^N-His and GDH-BT^C-His were stable up to 45℃. Gel permeation chromatography showed that GDH-BT^WT is a dimer, whereas GDH-BT^N-His and GDH-BT^C-His are monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BT^WT single subunit was constructed based on the crystal structure of Bacillus megaterium GDH (PDB ID 3AY6), showing that GDH-BT^WT has a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BT^WT and GDH-BT^N-His regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.

• Korean journal of applied entomology
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• v.50 no.3
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• pp.171-178
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• 2011

Bacillus thuringiensis (Bt) is a bacterial biopesticide against insect pests, mainly lepidopterans. Spodoptera exigua and Plutella xylostella exhibit significant decreases in Bt susceptibility in late larval instars. To enhance Bt pathogenicity, we used a mixture treatment of Bt and other bacterial metabolites which possessed significant immunosuppressive activities. Mixtures of Bt with culture broths of Xenorhabdus nematophila (Xn) or Photorhabdus temperata ssp. temperata (Ptt) significantly enhanced the Bt pathogenicity against late larval instars. Different ratios of Bt to bacterial culture broth had significant pathogenicities against last instar P. xylostella and S. exigua. Five compounds identified from the bacterial culture broth also enhanced Bt pathogenicity. After determining the optimal ratios, the mixture was applied to cabbage infested by late instar P. xylostella or S. exigua in greenhouse conditions. A mixture of Bt and Xn culture broth killed 100% of both insect pests when it was sprayed twice, while Bt alone killed less than 80% or 60% of P. xylostella and S. exigua, respectively. Other Bt mixtures, including Ptt culture broth or bacterial metabolites, also significantly increased pathogenicity in the semi-field assays. These results demonstrated that the Bt mixtures collectively names "Bt-Plus" can be developed into potent biopesticides to increase the efficacy of Bt.

• International Journal of Industrial Entomology and Biomaterials
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• v.11 no.2
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• pp.125-127
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• 2005

In order to improve the transformation efficiency of the Bacillus thuringiensis (Bt)-Escherichia coli (E. coli) shuttle vector, pHT3101, we intended to minimize replication origin of Bt in pHT3101. For this, two modified shuttle vectors, pHT1K and pHT261, in which 2.9 kb of replication origin of Bt were shortened to 1 kb and 261 bp, respectively as previously reported. Whereas the pHT1K could efficiently transform Bt into the antibiotic resistant, no transformants were obtained with pHT261. Furthermore, pHT1K showed higher transformation efficiency compared to that of parent vector, pHT3101. Therefore, pHT1K might be a very useful Bt-E. coli shuttle vector carrying minimal replication origin of Bt.

• International Journal of Industrial Entomology and Biomaterials
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• v.14 no.1
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• pp.33-36
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• 2007

To improve insecticidal activity of B. thuringiensis 2385-1 (Bt 2385-1), a recombinant plasmid, pHT1K-1Ac, was introduced into lepidopteran toxic Bt 2385-1 by electroporation. The presence of the recombinant plasmid in Bt 2385-1 after electroporation was confirmed by PCR. Bt 2385-1 transformant was named as Bt pHT1K-1Ac/2385-1 (1K-1Ac/2385-1). The 1K-1Ac/2385-1 transformant produced bipyramidal-shaped parasporal inclusion as like the wild-type strain, Bt 2385-1, and showed an 130 kDa band of Cry1Ac protein. The insecticidal activity of 1K-lAc/2385-1 against S. exigua was similar to that of Bt 2385-1 but the $LC_{50}$ value of transformant against P. xylostella was 1.8 times lower. Through these bioassay results, it was confirmed that toxicity of Bt 2385-1 transformant showed synergistic effect by introducing Cry1Ac. These results suggested that the multiple expressions of Cry proteins in a promising Bt strain may interact synergistically in insect midgut, resulting in increase of toxicity and expansion of host spectrum.

• Korean journal of applied entomology
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• v.48 no.1
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• pp.101-108
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• 2009

For biological control of larva of Cnaphalocrocis medinalis and Naranga aenescens do considerable damage to crops by folding and scraping the leaf tissue of rice, a large number of Bacillus thuringiensis isolates have been obtained from soil samples in Korea and the pesticidal activity was assayed against two insect pest species described above. Among 53 Bt isolates tested in bioassay, 18 and 13 isolates showed over 90% mortality against C. medinalis and N. aenescens, respectively. Some isolates (11 isolates including CAB141) presented dual activity against C. medinalis and N. aenescens. These isolates showed over 96% control effect in pest control in laboratory against larvae of C. medinalis. Also, it was investigated that pupation, pupal length, and adult emergence of larvae exposed to Bt suspension decreased. Especially, the pupal length of C. medinalis after being fed corn seedling leaves treated Bt suspension for 10 days, were much smaller than that of control.