• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2000.05a
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• pp.114-114
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• 2000

• Korean journal of applied entomology
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• v.48 no.3
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• pp.385-392
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• 2009

Two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata, are known to be potent against the diamondback moth, Plutella xylostella, when the bacteria are injected into the hemocoel. This study investigated any pathogenic effect of their culture broth on P. xylostella by oral administration. Only culture broth of both bacterial species did not give enough pathogenic effects by the oral administration. However, when the culture broth was orally treated together with Bacillus thuringiensis (Bt), both cell-free culture broth significantly enhanced Bt pathogenicity against the 3rd instar larvae of P. xylostella. The culture broth was then fractionated into hexane, ethyl acetate, and aqueous extracts. Most synergistic effect on Bt pathogenicity was found in ethyl acetate extracts of both bacterial species. Thin layer chromatography of these extracts clearly showed that ethyl acetate extracts of both bacterial culture broths possessed metabolites that were different to those of hexane and aqueous extracts. These results suggest that the both entomopathogenic bacteria produce and secrete different factors to give significant synergistic effect on Bt pathogenicity.

• BMB Reports
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• v.37 no.3
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• pp.304-313
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• 2004

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B $\delta$-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel $\beta$-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting ${\alpha}4$ and ${\alpha}5$ of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.

• 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.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.110-116
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• 2004

The over-expression in E. coli of the pHLN1-SO(＋) and pHLN2-80(－) plasmids cloned an insecticidal crystal protein (ICP) gene (crylAal type) from Bacillus thuringiensis var. kurstaki HD 1 was investigated through in part, the deletion of -80 bp promoter and an alternative change of cloning vector system. Two recombinant plasmids were constructed in an attempt to analyze the over-expression of the ICP in relations to its gene structure possessing only -14 bp ［Shine-Dalgarno (SD) sequence of -80 bp promoter］. Also, anther two recombinant plasmids similarly cloned the icp gene in a different vector system. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone, pHLRBS1-14 clone in which only the SD sequence in the inverted orientation icp gene appeared, was more evident than the pHLRBS2-14 clone in which only the -14 bp SD sequence of the right orientated icp gene was shown to exist. The pHLN2-80(－) clone produced more ICP proteins than the pHLRBS1-14 clone. In the two clones, pHLNUC1-80 right-oriented icp gene and the pHLNUC2-80 clone inverted-orientation icp gene in a new different vector, the pHLNUC2-80 produced more ICP proteins in E. coli system. These results indicate that the P/ac promoter, the inverted icp gene insertion and -80 bp promoter (-66 bp part of the icp gene promoters), were concerned with the expression of the icp gene in the recombinant plasmids. In addition, the expression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.451-455
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• 1996

Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

• Korean Journal of Agricultural Science
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• v.38 no.1
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• pp.39-44
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• 2011

The changes in major moth populations were monitored by sex pheromone traps in pear orchards at Yuseong-Gu, Daejeon from 2008 to 2010. Among four major moths, Grapholita molesta and Caposina sasakii occurred most frequently. Their occurrences peaked 2 to 3 times during the growing season from May to September. G. molesta was exceptional, occurring until September. For the environmentally-friendly control of these moths, 9 control materials including insect pathogenic bacteria and environmentally-friendly agricultural materials, were examined on the larva of 4 kinds of moth and sprayed on pear leaves in the field. As the generalized results of bioassay, Bacillus thuringiensis subsp. kurstaki and Sophora flavescens extract were shown to have better control effects than any other control material.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1093-1098
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• 2001

The gene coding for a Lepidoptera-specific insecticidal crystalline (or control) protein (ICP), recognized as cryIAc, from Bacillus thuringiensis subsp. kurstaki HD-73, was cloned into the vector pBluscript ll SK-, and then transformed in Escherichia coli $DH5{\alpha}$. The clone was named EBtIAc and the chimeric phagemid, as pEBtIAc. Hyperexpression of CryIAc protoxin was observed in the extract of the culture of E. coli harboring pEBtIAc. Crystalline protoxin was purified by differential solubility. It was dissolved in alkaline pH, and exposed to trypsin to be activated. The molecular weights of the pro- and activated toxins on SDS-PAGE were estimated to be ca. 130 kDa and 60 kDa, respectively. The toxicity was tested by force-feeding larvae of gypsi moth (Lymantria diapar) with trypsinized protoxin. Using the batch of biologically active form of the toxin as an immunogen, anti-CryIAc antiserum was raised in a New Zealand white rabbit. Immunoglobulin G was fractionated from the seam by Protein-A sepharose affinity chromatography. Immunoreactivity of the antibody was examined by dot and Westerns blottings. It has been found that the anti- CryIAc antibody recognized the purified toxin at a level below a nanogram in terms of quantity. Using the antibody some of Bt-corns were able to be differentiated from tons of corn kernels which were imported from America as forage crops.

• The Korean Journal of Pesticide Science
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• v.2 no.1
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• pp.91-96
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• 1998

For the expression of the cry11Aa gene highly toxic to dipteran insects, we constructed two cyanobacteria-Escherichia coli(E. coli) shuttle vectors, pCYASK5-l and pCYASK5-2. These vectors were transformed into E. coli and selected with kanamycin. The expression of the cry11Aa gene in E. coli was characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Two E. coli transformants harboring pCYASK5-1 and pCYASK5-2 expressed the cry11Aa gene in size of 72 kDa and 64 kDa, respectively and showed over 89% mortality against Culex pipiens larvae.

• Korean journal of applied entomology
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• v.51 no.1
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• pp.47-58
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• 2012

Effect of a new crop protectant 'Bt-Plus' on natural enemies was analyzed in this study. Tested natural enemies included two parasitic species of $Aphidius$ $colemani$ and $Eretmocerus$ $eremicus$, and four predatory species of $Harmonia$ $axyridis$, $Orius$ $laevigatus$, $Amblyseius$ $swirskii$, and $Phytoseiulus$ $persimilis$. 'Bt-Plus' was formulated by combination of three entomopathogenic bacteria ($Xenorhabdus$ $nematophila$ (Xn), $Photorhabdus$ $temperata$ subsp. $temperata$ (Ptt), $Bacillus$ $thuringiensis$ (Bt)) and bacterial metabolite (BM). All three types of 'Bt-Plus' showed significantly higher toxicities against fourth instar $Plutella$ $xylostella$ larvae than Bt single treatment. Two types of bacterial mixtures ('Xn+Bt' and 'Ptt+Bt') showed little toxicity to all natural enemies in both contact and oral feeding assays. However, 'BM+Bt' showed significant toxicities especially to two predatory mites of $A.$ $swirskii$ and $P.$ $persimilis$. The acaricidal effects of different bacterial metabolites were evaluated against two spotted spider mite, $Tetranychus$ $urticae$. All six BM chemicals showed significant acaricidal effects. The BM mixture used to prepare 'Bt-Plus' showed a high acaricidal activity with a median lethal concentration at 218.7 ppm (95% confidence interval: 163.2 - 262.3). These toxic effects of bacterial metabolites were also proved by cytotoxicity test against Sf9 cells. Especially, benzylideneacetone, which was used as a main ingredient of 'BM+Bt', showed high cytotoxicity at its low micromolar concentration.