• Journal of Sericultural and Entomological Science
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• v.33 no.1
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• pp.32-36
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• 1991

The study has been carried out to acquire some basic informations about Bacillus thuringiensis for developing the microbial pesticide. Three strains of Bacillus thuringiensis var. Kurstaki, dendrolimus and aizawai, were used in the experiments as follows. Growth characteristics of each strain were examined and parasporal protein crystals were isolated from the mixtures of spores and protein crystals by the new method of centrifugation in two-layer cushion of Renograffin using a fixed angle rotor. The results are as follows. 1. No difference was shown in growth characterestics among three strains of B. thuringiensis. In growth curves, all strains reached to exponential phase by 2 hr and stationary phase by 7-8 hr after inoculation. 2. The pH of the culture media during exponential growth stage decreased about 1.4 of a pH unit at the beginning of sporulation, but recovered during the early stage of sporulation and then remained nearly constant during the later stage. 3. As 10$m\ell$ sample was applied to two-layer cushion of Renograffin and then centrifuged for 1hr at 27,000g a fixed angle rotor, the purity and recovery ratio was 99.9% and 5.8%, respectively. It has been shown that the new method for the isolation of parasporal protein crystals was more efficient than any from the estabilished methods.

• The Korean Journal of Pesticide Science
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• v.6 no.4
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• pp.257-263
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• 2002

This study was carried out not only to investigate the toxicities of 12 registered insecticides on different developmental stages, but also to determine the sublethal effects on longevity and reproduction of newly emerged adult female and development of the next generation in the rice skipper, Parnara guttata. Fenitrothion, fenthion, cartap hydrochloride, ethofenprox highly suppressed egg-hatch. All insecticides treated showed high larvicidal activity on the 1st to 2nd instar larva. The insecticides showed higher larvicidal activities on the 5th instar larva were fenitrothion, fenthion, ethofenprox, fipronil, methoxyfenozide, tebufenozide and Bt. var. kurstaki. The sublethal doses of fenthion, tebufenozide, cartap hydrochloride, methoxyfenozide, ethofenprox, imidacloprid and fipronil shortened the longevities of newly emerged adult female from the treated larva ($3{\sim}4$ instar). BPMC, imidacloprid, ethofenprox, fipronil and methoxyfenozide delayed the preoviposition periods of adult females and decreased the number of eggs laid when they were treated at the larval stages of the previous generation. Ethofenprox caused severe sublethal effects on P. guttata offspring, completely blocking the production. All insecticides except fenitrothion affected the egg viability, and all eggs from the adult females emerged from the survivors treated larvae with imidacloprid or fipronil fail to hatch. IGRs, methoxyfenozide and tebufenozide showed an adverse effect on the development of next generation larva.

• Korean journal of applied entomology
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• v.59 no.1
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• pp.41-54
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• 2020

Two mosquito species were collected in still-water near farming area in Andong, Korea. Based on morphological characters, these two mosquitoes were identified as Aedes koreicus and Culex vagans, respectively. DNA barcode analyses supported the identification. An entomopathogenic bacterium, Bacillus thuringiensis subsp. israelensis (BtI), exhibited insecticidal activities against the two mosquito species and its virulence was more potent than that of B. thuringiensis subsp. kurstaki. It has been known that the bacterial metabolites of Xenorhabdus spp. suppress insect immunity and enhance pathogenicity of B. thuringiensis. This study tested the effect of the bacterial culture broth of Xenorhabdus spp. on enhancing BtI pathogenicity. Among three Xenorhabdus spp., culture broth of X. ehlersii (Xe) was relatively effective to enhance BtI pathogenicity against both mosquito species. Indeed, organic extracts of Xe culture broth suppressed the hemocyte-spreading behavior, suggesting the presence of immunosuppressant in the culture broth. These results suggest a formulation of BtPlus by mixing BtI spore and Xe culture broth to be applied to control the two mosquito species.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.348-351
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• 1988

The production of extracellular $\alpha$-amylase by Bacillus thuringiensis 19 serotypes was examined by the hydrolysis test of starch. Thirteen serotypes produced the amylase. B. thuringiensis serovar thuringiensis alesti, kurstaki, sotto, kenya, israelensis, morrisoni, entomocidus, tolworthi, thompsoni, toumanoffi, pakistani, and indiana produced tee enzyme. The amylase produced by B. thuringiensis serovar israelensis showed highest activity around pH 6.7 to 7.2 and 55$^{\circ}C$ to $65^{\circ}C$. The high production medium of the amylase was composed of 1% bactopeptone, 0.3% beef-extract, 0.3% yeast ex-tract, 0.5% NaCl, 0.3% $K_2$HPO$_4$, 0.1% KH$_2$PO$_4$, 0.2% Soluble Starch, 0.012% CaCl$_2$.2$H_2O$$_2$, 0.005% MnCl$_2$, and 0.03% MgCl$_2$.7$H_2O$. The highest production of the enzyme was observed at 4 hours culture in the soluble starch (0.6 units/$m\ell$) or glucose (0.43 units/$m\ell$) substrate.

• 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.

• KSBB Journal
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• v.6 no.3
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• pp.289-297
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• 1991

The insecticidal protein gene in the pKL-20-1 clone derived from Bacillus thuringiensis serovar. kurstaki plasmid was subcloned in the plant shuttle vector, pGA643. The 7.3 kb fragment was cloned in the BglII and Hpal sites of pGA643 vector and expressed in E. coli S17-1, which produced insecticidal proteins killing Bombyx mori larvae. The clone was named pHL-20. The protoplast formation, calli induction and plantlet regeneration of Nicotiana tabacum was carried out. A tremendous number of mesophyll protoplasts of N. tabacum were formed, up to 7$\times$105 protoplast per ml, for 20 hours in darkness in the enzyme solution of 0.5% cellulase and 0.1% macerosin, pH 5.8. The viabilities of the protoplasts were maintained above 80% for 6 days in the media containing 2mg/1 of NAA and 1mg/1 of kinetin. Calli were induced from the protoplasts and leaves of the N. tabacum on MS medium containing 0.5mg/1 BAP. Under the culture conditions the protoplasts underwent repeated cell division into calli. Plantlets were regenerated from callus cultures derived from protoplast and leaves. Shoots were induced in a medium containing 1mg/1 of BAP.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.295-300
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• 1989

Screening of Pseudomonas strains that can be used as hosts for expression of crystal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 was carried out. From rhizosphere soil of 7 kinds or crops as fluorescent Pseudomonas strains were isolated. A hybrie plasmid, pKTC1, composed of the broad host range vector pKT230 and the crystal protein gene was constructed and used for transformation of the 35 Pseudomonas strains. As the result, the crystal protein gene could be introduced into 4 isolates. Several methods including bioassay and immunochemical detection indicated that the crystall protein gene was expressed in the Pseudomonus isolates.

• 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.

• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.159-165
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• 1997

We collected 3,237 plant samples, mainly leaves of various trees, from many provinces in Korea and a total of 1,925 Bacillus thuringiensis isolates were obtained and characterized. The isolates were characterized in terms of crystal morphology, PAGE pattern of the toxin proteins, plasmids pattern, biochemical characteristics, and bioassay. The microscopic observation showed that 49.1% of the isolates have bipyramidal shape crystals, 7.1% of spherical shape crystals, 1.4% of rhomboidal shape crystals, and others have small or amorphous inclusions. The insecticidal activities of the spore-crystal mixtures of isolates were tested against Plutella xylostella, Bombyx mori, Culex pipiens, and Agelastica coerulea. Bioassay showed that 51.3% of the isolates were shown to be active; lepidopteran-specific (44.8%), dipteran-specific(4.9%) and coleopteran-specific (1.6%). The remainder(48.8%) did not show any activity against the insects we tested. Interestingly though, some of these non-active isolates were shown to have bipyramidal crystals. By serotyping 22 isolates of our collection, we found that there are various kind of subspecies such as aizawai, amagiens, canadensis, darmstadiensis, galleriae, finitimus, kurstaki, morrisoni and neoleonensis, and three isolates have been classified into a new serotype, H49, and one of them, the type strain, named subsp. muju. From this study it was found that phylloplane is a good source for the isolation of Bacillius thuringiensis, and Bacillus thuringiensis is distributed widely in Korea.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.240-245
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• 1991

We have constructed a promoter-probe vector pKU20 using pKT230, a derivative of broad-host-range plsmid RSF1010, as a base. The pKU20 contains structural gene for aminoglycoside phos-photransferase (aph), without promoter, and a multiple cloning site upstream the aph. Using this vector, a 412base pairs (bp) PstI fragment showing strong promoter activity both in Escherichia coli LE392 and Pseudomonas putida KCTC1644 has been cloned from Pseudomonas fluorescens chromosomal DNA on the basis of streptomycin resistance. The nucleotide sequence of the 412 bp fragment has been determined and the putative - 35 and -10 region was observed. Insecticidal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 inserted on downstream of the promoterlike DNA fragment was efficiently expressed in E. coli and P. putida. The toxin protein was efficiently synthesized in an insoluble form in both strains.