Bacillus thuringiensis와 Neem oil에 대한 배추좀나방의 살충활성

조민수(Min-Su Cho),최수연(Su-Yeon Choi),김택환(Tae-Whan Kim),박찬(Chan Park),김담아(Dam-A Kim),김영림(Young-Rim Kim),오세문(Se-Mun Oh),김성우(Sung-Woo Kim),윤영남(Young-Nam Youn),유용만(Yong-Man Yu) 한국농약과학회 2009 농약과학회지 Vol.13 No.4

For the environmental friendly management of diamondback moth, Plutella xylostella (L.), Bacillus thuringiensis subsp. kurstaki (30×108 cfu/mg) and neem oil (0.5% azadirachtin) were used as green control agencies with mixed and alternative treatments on the chinese cabbage. When Bacillus thuringiensis subsp. kurstaki was applied to 1st and 2nd larva of P. xylostella with recommended concentration, their mortalities were reached to 100% by 2 days after treatment. In case of azadirachtin, its effect of mortality was continued for 7 days, and reached to 100% mortality. LC?? values of Bacillus thuringiensis subsp. kurstaki and azadirachtin against 1st, 2nd, 3rd and 4th larva of P. xylostella were 2.8×10⁴, 3.1×10⁴, 3.4×10⁴ and 1.5×10? cfu/ml, and 2.7, 3.9, 4.7 and 7.1 ppm, respectively. The number of laid eggs of P. xylostella was reduced to 57.5 at 25 ppm of azadirachtin compared with control treatment. The hatch ratio was not significantly different with Bacillus thuringiensis subsp. kurstaki treatment in comparison with control treatment. However, when azadirachtin was applied, their hatch ratio were reduced to 25.8 and 45.4% at 25 and 50 ppm, respectively. On the other hand, emergence rate of eggs was not different with Bacillus thuringiensis subsp. kurstaki treatment, but 45.4% was shown in azadirachtin treatment with 50 ppm in comparison with control. When the mixture with Bacillus thuringiensis subsp. kurstaki and azadirachtin was applied to adults of P. xylostella, their mortality was higher than Bacillus thuringiensis subsp. kurstaki treatment only. These results are supposed that the mixture of Bacillus thuringiensis subsp. kurstaki and azadirachtin might be used as green control agents for reducing the demage of diamondback moth in the Chinse cabbage.

Evaluation of SERS Nanoparticles to Detect Bacillus cereus and Bacillus thuringiensis

( Jeehwa Hong ),( Jianwei Qin ),( Jo Ann S. Van Kessel ),( Mirae Oh ),( Sagar Dhakal ),( Hoonsoo Lee ),( Chansong Hwang ),( Diane E. Chan ),( Dongho Kim ),( Hyunjeong Cho ),( Moon S. Kim ) 한국농업기계학회 2018 바이오시스템공학 Vol.43 No.4

Purpose: This research evaluated five types of nanoparticles to develop a surface-enhanced Raman spectroscopy (SERS) method for the rapid detection of two Bacillus species (Bacillus cereus and Bacillus thuringiensis) that are commonly found on fresh produce, which can cause food poisoning. Methods: Bacterial concentrations were adjusted to a constant turbidity, and a total of 30 μL of each Bacillus cell suspension was prepared for each nanoparticle. A point-scan Raman system with laser light source of wavelength 785 nm was used to obtain SERS data. Results: There was no qualitative difference in the SERS data of B. cereus and B. thuringiensis for any of the five nanoparticles. Three gold nanoparticles, stabilized in either citrate buffer or ethanol, showed subtle differences in Raman intensities of two Bacillus species at 877.7 cm-1. Conclusions: Among the three types of nanoparticles, the gold nanoparticles stabilized in citrate buffer showed the lowest standard deviation, followed by gold nanoparticles stabilized in ethanol. This result supports the potential application of gold nanoparticles for SERS-based detection of B. cereus and B. thuringiensis.

Bacillus thuringiensis 항원들의 면역학적 분석

정재득,박정선,조영수,홍순복,이형환,조명환 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.1

B. thuringiensis 향원들의 면역학적 특성을 살펴 보기 위해 초음파를 이용하여 B.t 항원들을 분쇄하여 전기영동을 시행하였다. 단백질 양상은 균주들간에 아주 유사했으나, 밴드들의 양적인 차이와 몇 개의 분자량에 차이가 있음을 보여주었다. 단백질 양상들을 비교분석한 결과 B.t subsp. canadensis을 포함한 11균주는 45 kd을 공통항원으로 지니고 있었고, B. thuringiensis subsp. canadensis 및 galleriae는 혈청학적으로도 구별하기 어렵지만 전기영동을 통해 두 균주들간의 차이는 양적인 변화로 쉽게 구별할 수 있는 것은 균주들을 동정하는데 하나의 유용한 방법임을 제시해 준다. 조사된 모든 균주들은 전기영동상에서 유사한 폴리펩티드 양상들을 보여준 반면, western blot에서는 항원들의 면역 반응에는 차이가 나타났다. 항원들의 분포를 살펴보기 위해 간접면역형광법을 이용한 결과 B. thuringiensis subsp. thuringiensis와 israelensis는 편모항원과 세포표면항원에 반응한 것으로 확인되었다. 본 연구에서는 SDS-PAGE와 western blot 분석은 아종들을 동정하는데 유용한 도구가 된다는 것을 제시해 주고 있다. This study was carried out to immunologically characterize Bacillus thuringiensis (B.t) antigens. Protein patterns of ultrasonicated- antigens of B. thuringiensis subspecies using SDS-PAGE revealed marked similarities among all the strains analyzed except for the difference between quantative variations of bands and some protein antigens. The comparison of the protein patterns showed that the protein antigen of 45 kilodalton (kd) was common in 11 strains and that the difference between B. thuringiensis subsp. Canadensis and galleriae was noticed in quantative variations of bands despite of ambiguous serogrouping, suggesting a useful method for identification. All strains examined similar antigenic patterns in SDS-PAGE, while immunodominant bands differed in antigenic reactivity in western blot using polyclonal antibodies. Polyclonal antibody to B. thuringiensis subsp. thuringiensis and israelensis in indirect immunofluorescence assay reacted with flagella and cell surface antigens. The present study indicates that SDS-PAGE and western blot analysis may be used as tools for differentiation and identification of B. thuringiensis subspecies.

Bacillus thuringiensis isolates from Korean forest environments

이대원,제연호,Young Ho Koh 한국응용곤충학회 2012 Journal of Asia-Pacific Entomology Vol.15 No.2

We investigated the distribution, toxicity, morphology, and protein profiles of Bacillus thuringiensis isolates from forests in Korea to isolate naturally occurring novel B. thuringiensis. A total of 170 B. thuringiensis isolates were obtained from 832 samples producing spore and parasporal inclusion bodies. In toxicity tests for lepidopteran,dipteran, and coleopteran insects, 57.6% isolates were toxic only to Lepidoptera, 5.3% were toxic only to Diptera, and 24.1% were toxic to both Diptera and Lepidoptera. The remaining collections (13.0%)were not toxic to the tested insects. The shapes of the parasporal crystals produced in B. thuringiensis isolates were bipyramidal, spherical, ovoid, or irregular. As their toxicities varied with parasporal crystal shape, B. thuringiensis isolates possessing bipyramidal or irregular parasporal crystals were largely toxic to lepidopteran species whereas those producing spherical parasporal crystals were mainly toxic to dipteran species. B. thuringiensis toxic to both dipteran and lepidopteran insects contained 130- and 70-kDa parasporal crystals,whereas B. thuringiensis toxic to lepidopteran insects expressed 130-kDa parasporal crystals. The results suggest that forest areas in Korea are a rich source of B. thuringiensis and need to be further explored to discover novel B. thuringiensis isolates.

Expression of a Recombinant Cry1Ac Crystal Protein Fused with a Green Fluorescent Protein in Bacillus thuringiensis subsp. kurstaki $Cry^-B$

Roh Jong Yul,Lee In Hee,Li Ming Shun,Chang Jin Hee,Choi Jae Young,Boo Kyung Saeng,Je Yeon Ho The Microbiological Society of Korea 2004 The journal of microbiology Vol.42 No.4

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

Bacillus thuringiensis BT-1, BT-2가 생산하는 δ-endotoxin의 특성 규명

김영민,최홍서,정건섭,Kim, Young-Min,Choi, Hong-Seo,Chung, Kun-Sub 한국생명과학회 2007 생명과학회지 Vol.17 No.5

B. thuringiensis는 많은 해로운 곤충들을 박멸시키는데 널리 사용되는 생물학적 살충제로 잘 알려져 있다. 그것은 측포자 형태의 결정체($\delta$-내독소)를 생산하고 내생포자들을 형성한다. 본 논문에서는 B. thuringiensis BT-1과 BT-2에 의해 생산되는 내독소의 특성을 주사전자현미경(SEM), 투과전자현미경(TEM), SDS-PACE, 알칼리 용액에서의 용해 활성을 통하여 규명하였다. BT-1, BT-2 균주는 GBY 배지에서 배양되었고, 두 균주의 내독소는 당밀도구배법을 이용하여 정제되어, 주사전자현미경(SEM), 투과전자현미경(TEM)으로 관찰하였다. 형태학적으로, BT-1의 내독소는 사각형 및 납작형이고 크기는 $1.73{\mu}m{\times}0.7{\mu}m$, 그리고 BT-2 내독소는 구형이며 $1.1{\mu}m$, 폭이 $0.9{\mu}m$인 것으로 밝혀졌다. SDS-PACE 방법으로 분석한 결과, BT-1의 분자량은 28 kDa, 21 kDa, 반면에 BT-2의 분자량은 50 kDa, 35 kDa, 22 kDal으로 밴드가 형성되었다. 이들의 결정체는 알칼리 완충용액 내에서 시간에 지나감에 따라서 점차로 용해되었으며 3시간 후에는 거의 완전하게 용해되었다. 이 결과들을 통하여 BT1과 BT-2 결정체의 내독소가 곤충의 중장 내에서 시간이 흐름에 따라 비활성 상태에서 활성상태로 전환되는 것으로 보여진다. Bacillus thuringiensis is a well-known species of entomophathogenic bacteria that is widely used as a biopesticide against many insect pests. It produces parasporal crystals ($\delta$-endotoxin) and endospores during sporulation. In this report, the $\delta$-endotoxin produced by Bacillus thuringiensis BT-1 and BT-2 were characterized by Scanning Electron Microscope(SEM), Transmission Electron Microscope(TEM), SDS-PACE, and solubilization activity by alkaline solution. BT-1, BT-2 were cultured in the GBY medium, and the $\delta$-endotoxin of them was purified with discontinuous sucrose density gradient centrifugation. Their $\delta$-endotoxin was observed by SEM and TEM. Morphologically, the $\delta$-endotoxin of BT-1 was a square and flat type, whose size was $1.73{\mu}m{\times}0.7{\mu}m$, and the $\delta$-endotoxin of the BT-2 was spherical form whose size was $1.1{\mu}m{\times}0.9{\mu}m$ determined by SEM and TEM. The $\delta$-endotoxin of the BT-1 was composed of 28 kDa and 21 kDa, however, it of the BT-2 was composed of 50 kDa, 35 kDa, and 22 kDa bands determined by SDS-PACE. The purified crystals of BT-1 and BT-2 were dissolved gradually in alkaline solution as time goes by, and it was perfectly dissolved after 3 hours. It is supposed that the $\delta$-endotoxin of crystal was converted to a state of activation in the course of time in the intestines of insect.

Cyanobacterium Synechocystis PCC6803 내에서 Bacillus thuringiensis subsp. morrisoni PG-14 cryIVD 유전자의 발현

이대원,박현우,진병래,정영호,박영목,강석권 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.2

미생물 살충제로 사용되고 있는 B. thuringiensis를 모기유충방제에 적용하기 위한 시도는 B. thuringiensis가 수서생태계에서 부유하지 못하고 가라앉으며, 생활환을 이루지 못한다는 문제점으로 인해 적용에 어려움이 있다. 따라서 본 연구는 모기유충에 강한 독성을 갖는 B. thuringiensis subsp. morrisoni PG-14의 cryIVD 유전자를 모기유충의 먹이인 cyanobactrium에 도입하기 위하여 발현벡터 pCYASK 5-1을 제작하고, cyanobacterium Synechocystis PCC6803에 형질전환시켜, 세포내에서 cryIVD 유전자의 발현과 뇌염모기에 대한 독성을 조사하였다. 그 결과 형질전환체 내에서 cryIVD 발현은 immunoblot 분석을 통해 B. thuringiensis subsp. morrisoni PG-14에서 발현된 단백질과 같은 분자량으로 발현되는 것을 확인하였으며, 모기유충에 대한 형질전환체의 독성은 1.40×10^6 cells/ml 농도에서 약 80%의 치사율을 나타내었다. Bacillus thuringiensis subsp. morrisoni PG-14 is a gram-positive soil bacterium producing mosquitocidal parasporal inclusions composed of several crystal proteins. Among these crystal protein genes, cryIVD gene is one of major component which has 72 kDa in size. However, these parasporal inclusions sink quickly from the surface of water where mosquito larval feeding occurred. To develope mosquitocidal cyanobacterium, therefore, we constructed the expression vector, pCYASK 5-1 harboring cryIVD gene. The expression vector, pCYASk5-1 was transformed into the cyanobacterium Synechocystis PCC6803 reported as a natural mosquito larval food source and the transformants were selected with kanamycin. Expression of cryIVD gene in transformant was characterized by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis. The mosquitocidal activity of a transformant was determined electrophoresis (PAGE) and immunoblot analysis. The mosquitocidal activity of a transformant was determined with Culex tritaeniorhynchus. The results showed that the transformed cyanobacterium is toxic to mosquito larvae and will be expected as a potential agent that is used for mosquito control.

파밤나방에 활성이 있는 Bacillus thuringiensis 균주와 tannic acid와의 균주특이성조사

곽창순,진나영,오산나,이유경,전미진,서미자,윤영남,유용만 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.05

Bacillus thuringiensis(이하 B. thuringiensis) subsp. kurstaki KB100은 protease inhibitor중의 하나인 tannic acid를 혼합처리 함으로서 파밤나방의 살충활성에 상승효과를 가져왔다. 이를 바탕으로 tannic acid가 B. thuringiensis균주 특이성을 나타내는지 확인하기 위해, 국내 토양으로부터 분리하여 실험실에 보관중인 B. thuringiensis중 파밤나방에 높은 살충활성을 보이는 6개의 균주와 기준균주인 B. thuringiensis subsp. kurstaki HD-1을 선발하여 총 7종의 균주에 각각 tannic acid를 농도별로 처리하여 2령 파밤나방에 대해 생물검정을 실시하였다. 또한 선발된 7개의 균주들의 살충성단백질의 패턴과 파밤나방 중장액에 대한 tannic acid의 단백질분해 저해정도를 알아보기 위해 SDS-PAGE를 수행하였다. B. thuringiensis KB100은 tannic acid의 농도를 높일수록 사충이 증가하다가 80mM농도에서 낮아지는 반면에 B. thuringiensis KB098은 tannic acid의 농도를 높일수록 오히려 사충률의 감소를 보였고, 이외 5개 균주는 tannic acid의 농도와 상관없이 뚜렷한 사충률의 변화를 보이지 않았다. 각 균주의 Parasporal inclusion에 tannic acid를 처리하여 파밤나방 중장액에 반응시켜 parasporal inclusion의 분해정도를 SDS-PAGE로 확인한 결과, 133kDa크기의 단백질밴드를 나타내는 각각의 균주가 생성한 parasporal inclusion은 tannic acid처리 농도에 따라 살충활성을 나타내는 70~60kDa단백질크기로의 분해정도가 각각 다른 것으로 나타났다. 이는 Tannic acid가 B. thuringiensis균주에 따른 특이적인 효과를 나타낼 수 있는 가능성을 보여준다.

화학살균제가 미생물살충제(Bacillus thuringiensis)에 미치는 영향

한은정,박종호,홍성준,윤여준,지형진 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.05

Bacillus thuringiensis(B.t.)는 해충방제용으로 가장 많이 사용되고 있는 미생물제이며, 배추재배 농가현장에서 B. thuringiensis를 제형화한 미생물 살충제와 함께 배추 병 방제를 위하여 화학살균제를 사용하고 있다. B. thuringiensis의 미생물 살충제를 안정적으로 사용하기 위하여 이들 농약과 B. thuringiensis간의 상호작용을 규명하고 혼용 및 교호살포하기에 적절한 농약을 선발하기 위하여 본 연구를 수행하였다. 현재 유통되고 있는 B. thuringiensis 살충제 7종에 대하여 수행되었으며, 화학살균제는 배추에서 노균병과 무름병에 등록되어 있는 코퍼하이드록 사이드 수화제 등 12종을 대상으로 수행하였다. 추천농도의 화학농약을 첨가한 TSA배지에 대상미생물살충제를 도말하여 형성된 콜로니를 비교하였으며, 대상 미생물살충제 7종과 화학살균제를 각각 추천농도로 혼합하여 30분 shaking 한 후 배추 잎침지하여 배추좀나방에 대한 살충력을 petri-dish에서 조사하였다. 7종의 B. thuringiensis 살충제는 화학농약을 함유한 배지에서의 B. thuringiensi의 콜로니는 옥시테트라사이클린 수화제, 스트렙토마이신 수화제, 발리다마이신에이 수화제, 옥솔린산 수화제를 함유한 TSA배지에서 콜로니가 형성되지 않았다. 미생물 살충제와 화학살균제의 혼합액을 배추좀나방에 처리한 결과 5종의 B. thuringiensis 살충제(TB-WP, SC, SB, BG)는 12종의 화학살균제의 혼합에 따른 살충력에 차이가 없었으나, TB-SO와 플루오피콜라이드 ․ 프로파모카브하이드로 클로라이드 액상수화제, TH와 코퍼하이드록사이드 수화제의 혼합액처리에서 배추좀나방의 살충력이 유의하게 감소하는 결과를 보였다.

Evaluation of SERS Nanoparticles to Detect Bacillus cereus and Bacillus thuringiensis

홍지화,Jianwei Qin,Jo Ann S. Van Kessel,오미래,Sagar Dhakal,이훈수,황찬송,Diane E. Chan,김동호,조현정,Moon S. Kim 한국농업기계학회 2018 바이오시스템공학 Vol.43 No.4

Purpose: This research evaluated five types of nanoparticles to develop a surface-enhanced Raman spectroscopy (SERS) method for the rapid detection of two Bacillus species (Bacillus cereus and Bacillus thuringiensis) that are commonly found on fresh produce, which can cause food poisoning. Methods: Bacterial concentrations were adjusted to a constant turbidity, and a total of 30 μL of each Bacillus cell suspension was prepared for each nanoparticle. A point-scan Raman system with laser light source of wavelength 785 nm was used to obtain SERS data. Results: There was no qualitative difference in the SERS data of B. cereus and B. thuringiensis for any of the five nanoparticles. Three gold nanoparticles, stabilized in either citrate buffer or ethanol, showed subtle differences in Raman intensities of two Bacillus species at 877.7 cm-1. Conclusions: Among the three types of nanoparticles, the gold nanoparticles stabilized in citrate buffer showed the lowest standard deviation, followed by gold nanoparticles stabilized in ethanol. This result supports the potential application of gold nanoparticles for SERS-based detection of B. cereus and B. thuringiensis.