• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.2
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• pp.123-129
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• 2000

Expression of the crylAcl gene of an acrystalliferous Bacillus thuringiensis strain under the control of the native or $\alpha$-amylase gene promoter was investigated. The crylAcl gene was cloned in a B. thuringiensis - E. coli shutle vector, pHT3101, undder the control of either the native promoter (pProAc) or the $\alpha$-amylase promoter from Bacillus subtilis (pAmyAc). These two recombinant plasmids were successfully expressed in B. thuringiensis subsp. kurstaki Cry B. The first transformant (ProAc/CB), harboring pProAc, expressed an about 130 kDa protein begining 24 hr after inoculations just as in the case of the wild type of B. thuringiensis subsp. kurstaki HD-73. The second pAmyAc-transformant (AmyAc/CB) began to express the gene just 6 hr after inoculation, but Western analysis showed that the activity of the $\alpha$-amylase promoter was relatively weaker than that of the native promoter. As expected, their toxicity against Plutella xylostella larvae was dependent on the amount of Cry1Acl protein expressed.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.2
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• pp.145-149
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• 2004

The expression of a fusion protein comprised of the B. thuringiensis crystal protein, Cry1Ac, and enhanced green fluorescent protein (EGFP) in Escherichia coli XLl-blue was examined. Three recombinant plasmids were transformed into E. coli XL1-blue and named as ProAc/Ec, MuEGFP/Ec and ProMu-EGFP/Ec, respectively. All transformants were observed by light and fluorescence microscopy at mid-log phase. The expression in E. coli transformants, ProMu-EGFP/Ec and MuEGFP/Ec, exhibited bright enough fluorescence to be observed. Furthermore, ProMu-EGFP/Ec produced fluorescent inclusions, which may have been recombinant crystals between EGFP and Cry1Ac while MuEGFP/Ec expressed soluble EGFP in cell. In SDS-PAGE, ProAc/Ec had 130 kDa crystal protein band and MuEGFP/Ec had thick 27 kDa EGFP band. However, ProMu-EGFP/Ec had about 150 kDa fusion protein band. Accordingly, these results indicated that a fusion protein between the B. thuringiensis crystal protein and a foreign protein under the lacZ promoter was successfully expressed as granular structure in E. coli. It is suggested that the E. coli expression system by N-terminal fusion of B. thuringiensis crystal protein may be useful as excellent means for fusion expression and characterization of B. thuringiensis fusion crystal protein.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.168-173
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• 1988

Immunological analysis between endotoxin proteins produced by Bacillus thuringiensis serovar. kurstaki HD1 and HD73 have been investigated by using polyclonal antibodies. The antisera against the endotoxin proteins were prepared from rabbits injected with the endotoxin protein antigens. When about 2mg/$m\ell$ of the antigens were injected for 7 times, the titers were highest. The stability of the antigens was reduced to about 50% after 9 days incubation at 4$^{\circ}C$. The sensitibity of endotoxin protein from B. thuringiensis HD1 and HD73 by indirect ELISA was 50ng/$m\ell$ and 400ng/$m\ell$, respectively. The cross reaction of antiserum appeared that anti-HD1 partialy reacted with crystal protein but anti-HD73 reacted with HD1 endotoxin about 100%.

• Korean journal of applied entomology
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• v.52 no.1
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• pp.35-43
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• 2013

All tested Korean populations of the diamondback moth, Plutella xylostella, are known to be resistant especially against pyrethroid insecticides by mutation in its molecular target, para-sodium channel. Moreover, P. xylostella is able to develop resistance against most commercial insecticides. This study was performed to develop an efficient control technique against P. xylostella by a combined treatment of an endoparasitoid wasp, Cotesia plutellae, and a microbial insecticide, Bacillus thuringiensis. To investigate any parasitism preference of C. plutellae against susceptible and resistant P. xylostella, five different populations of P. xylostella were compared in insecticide susceptibilities and parasitism by C. plutellae. These five P. xylostella populations showed a significant variation against three commercial insecticides including pyrethroid, organophosphate, neonicotinoid, and insect growth regulator. However, there were no significant differences among five P. xylostella populations in their parasitic rates by C. plutellae. Moreover, parasitized larvae of P. xylostella showed significantly higher susceptibility to B. thuringiensis. As an immunosuppressive agent, viral ankyrin genes (vankyrins) encoded in C. plutellae were transiently expressed in nonparasitized larvae. Expression of vankyrins significantly enhanced the efficacy of B. thuringiensis against the third instar larvae of P. xylostella. Thus an immunosuppression induced by C. plutellae enhanced the insecticidal efficacy of B. thuringiensis. These results suggest that a combined treatment of C. plutellae and B. thuringiensis may effectively control the insecticide-resistant populations of P. xylostella.

• Applied Biological Chemistry
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• v.39 no.3
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• pp.177-182
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• 1996

Effects of 14 carbohydrates supplied as carbon sources on cell growth and sporulation of, and the production of insecticidal crystal proteins by Bacillus thuringiensis strains were investigated in liquid cultures. Strains grew well in media containing any one of the 14 carbohydrates supplied, reaching maximum cell densities of $10^7{\sim}10^8\;cells/ml$ in 16.7 to 22 hours after inoculation depending on the strain. Spores first appeared in 16.7 to 24.7 hours after inoculation, and 80% sporulation was reached in 28 to 51.3 hours after inoculation depending on the strain. No change in pH of media was observed after cell multiplication. The production of total protein was highest when supplied with sucrose and was lowest with starch. More insecticidal crystal proteins were produced when supplied with glucose, lactose, maltose, or sucrose. The amount of insecticidal crystal proteins produced by the strains was proportional to that of the total protein. The relative amount of individual insecticidal crystal protein species produced by B.t. kurstaki and B.t. israelensis was not influenced by the carbohydrates supplied.

• Korean journal of applied entomology
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• v.37 no.2
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• pp.187-191
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• 1998

For efficient and economical production of Btrc,illus tlz~rr.ingi~r1~sstirsa in NT0423 as amicrobial-control agents, a new culture medium and culture condition were developed. Five mediadesignatzd as SWI I , SW14, SW23. SW32 and SW4I were prepared ~ : i t hv arious mixture ratio ofsoybean cake and wheat bran. It was founcl that in terms of the cell growth rate and development ofsporulation of B, thrri.il~girrl.sis strain NT0423 in all SW culture media were more efficient than those inGYS and in LB media. Total cell number in all SW media showed similar values, hut SW32 lnediilm wasthe most efficient in the development of spore, which amo~~ntetod 3.7 x 10XC FUImI. Also. at the pHranging frorn 6.2 to 7.3 in the mediiun~ no ad\:erse effect was not made in the culture of B. thur-ingicnsisstrain NT0423. The optimal volume (%) of SW32 mecliuni in a 5 1 fernientor was determined as 4 8\rolume of total niediuni. resulting ill growth (4.2 x 1OTCFUlrnl) of H. t1~~irir1,yirrz.ssit.vr ain NT0423. As H.t l i ~ t r i t ~ g iw~ a~s~ csuil~tu rcd in the shakc-flash and 5 1 fcrnientor. bacterial cells were yielded to 1 X 10"CFUIml and 5 x I O1oCFLJlml.FUIml and 5 x I O1oCFLJlml.

• Journal of environmental and Sanitary engineering
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• v.13 no.2
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• pp.40-46
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• 1998

For a biological control on a plant pathogen, Pryicularia Oryzae, and a mosquito, Aedes aegypti, Bacillus thuringiensis strain AF6 which produces parasporal inclusion, delta-endotoxin, was isolated. The B. thuringiensis strain AF6 was produced not only an antifungal substance(AFS) against P. oryzae, but also a mosquitocidal delta-endotoxin. The AFS of the strain AF6 in more stable at pH 4.0 than pH 10.0. At the mode of action, the AFS of the strain AF6 was inhibited hypha growth on potato agar plate(pH 5.0), and degraded cell walls of P. oryzae.

• Journal of Microbiology
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• v.34 no.4
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• pp.370-373
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• 1996

Three strains (KW-1, KW-14, KW-15) of Bacillus thuringiensis were isolated from soil in Wonju area and characterized. The three strains produced parasporal inclusion bodies (crystals) and spores in their cells. The KW-1 strain produces spherical crystals. The crystals of strain KW-14 are bipyramidal crystal. The KW-15 strain harbors irregular crystals. Only minor biochemical characteristics of the three isolates were different and distinctive, however general characteristics were similar to the known serotypes of B. thuringiensis. Three strains were resistant to penicilin G, oxacillin and cephalothin. Three strains were highly toxic to Bombyx mori larvae, but not to the Culex pipiens larvae.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.99.2-99
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• 2003

An attempt was made to screen antifungal activities of Bacillus thuringiensis strains on various plant pathogens, Botryosphaeria dothidea, Diaporthe actinidiae, Botrytis cinerea, Glomerella cingulata, Colletorichum cocodes, Sclerotinia scierotiorum, Alternaria alternata, Helicobuidium mompa, Bipolaris coicis, Fusarium graminearum and Rhizoctosnia solani. Ten and forty-five strains of B. thuringiensis were isolated from animal feces in Korea and Japan, respectively. Inhibitory effects of the strains on the mycelial growth of the pathogens were examined on the mixed media of potato dextrose agar and nutrient agar. Approximately half of the strains inhibited the mycelial growth of one or more pathogens. Most of the pathogens were inhibited by any of the strains but Fusarium graminearum and Rhizoctonia solani were not inhibited at all. This is the first report that B. thuringiensis shows a potent antifungal activity on plant pathogens in Korea.