Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. B. thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

곤충병원성세균의 Bacillus thuringiensis(이하 B.t)는 친환경농업의 주요 농자재로 사용되는 생물농약 중의 하나이다. B.t는 그람양성세균이며 spore, crystal를 형성하고 parasporal inclusion를 형성을 한다. 포자 형성기에는 균체 내에 δ-내독소라는 독소단백질을 생성한다. δ-내독소는 곤충이 섭식 시 중장세포막에 binding 된 후 중장을 파괴하여 치사에 이르게 한다. 본 연구에서는 경상북도 김천 지역의 밭과 과수원 토양으로부터 총 8곳에서 sample을 채취하여 살충활성이 우수한 B.t를 분리 선발하고자 하였다. 토양희석액을 nutrient agar plate에 고르게 도말한 후 27℃에서 3~4일간 배양한 후 형성된 colony들 중에서 배양특성이 B.t와 유사한 80개의 colony를 선발하였다. 위상차현미경으로 spore형성과 crystal의 형태를 확인하는 과정에서 내독소 단백질 결정체를 형성하는 16개의 B.t를 확보할 수 있었고, 그중bipyramial형 15개와 irregular형 1개가 확인되었다. 분리선발된 16개 균주의 살충활성을 확인하기 위해, 실내 누대 사육한 나비목 담배거세미나방(Spodoptera litura)에 실험한 결과, 높은 사충율을 나타냈다. 추후에 나비목 파밤나방(Spodoptera exgua)에 대한 살충활성을 확인한 후 SDS-PAGE를 통하여 단백질패턴을 분석할 계획이다.

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.

Bacillus thuringiensis subsp. aizawai KB098 (이하 KB098 균주)은 파밤나방에 높은 살충 활성을 보인다. 해충에 살충 활성을 나타내는 내독소 단백질은 cry gene에 의해 발현이 되는데 cry gene은 주로 Bacillus thuringiensis의 plasmid DNA 상에 존재한다. KB098의 plasmid DNA를 추출하여 PCR을 수행한 결과, Cry1Aa, Cry1Ab, Cry1C, Cry1D 총 4개의 cry gene을 가지고 있는 것으로 확인되었다. KBM-1은 KB098균주를 curing한 mutant 균주로, KB098균주를 42℃조건에서 48시간 배양하고, UV를 조사하여 확보하였다. KBM-1의 plasmid DNA를 PCR한 결과 KB098에서처럼 Cry1Aa, Cry1Ab, Cry1C, Cry1D의 cry gene이 증폭됨을 알 수 있었다. KBM-1은 KB098처럼 crystal을 암호화하는 plasmid DNA를 가지며, cry gene을 보유하고 있다. 반면 KB098과는 달리 KBM-1 균주는 내독소 단백질을 생성하지 못하며 파밤나방의 생물 활성 검정에서 독성을 나타내지 않았다. 이러한 원인을 밝히고자 KBM-1 plasmid DNA의 특정 cry gene의 PCR 결과, 증폭된 cry 유전자 내에서 염기서열의 변화에 기인할 것이란 가정하에, KB098과 KBM-1 균주의 특정 plasmid DNA 상에서 증폭된 cry gene의 염기서열을 비교분석 할 것이다. 이를 통해 crystal 형성에 관여하는 유전자의 변이 유무를 확인할 계획이다.

나비목유충 소화액의 다양한 Protease는 Bacillus thuringiensis(이하 B. thuringiensis)가 생성한 protoxin의 활성을 결정하는데 가장 중요한 소화효소로 알려져 있다. 나비목유충의 소화효소 중 Serine protease인 trypsin은 단백질 가수분해과정에 주요한 역할을 하는 것으로 알려져 있다. 그러나 Protease의 지속적인 가수분해결과 독소 단백질의 불활성화를 초래하여 B. thuringiensis의 살충활성에 부정적인 영향을 초래할 수 있다. 이전 실험에서도 Bacillus thuringiensis subsp. kurstaki KB100과 protease inhibitor중의 하나인 tannic acid를 혼합하여 중장액에 처리하였을 때, 파밤나방에 대한 살충활성이 높아진 원인으로 protease activity의 감소를 예상할 수 있었다. 따라서 본 실험은 다양한 Protease가 있는 중장액에 protease 특이적기질로 tannic acid가 어떤 종류의 protease activity를 낮추는지 확인하고자 하였다. 파밤나방 중장액과 농도별(10, 20, 40, 80mM) Tannic acid와의 protease activity를 측정 한 결과 tannic acid의 농도가 높아 질 수 록 protease activity(%control)는 각각 83.1±2.1, 77.6±1.6, 68.0±0.4, 40.1±2.2로 감소됨을 확인 하였다. 파밤나방 중장액과 serine(azocasein), trypsin(BApNA, BPVApNA), chymotrypsin(BTpNA, SAAPPpNA, AAVApNA), elastase(SAAApNA, SAAPLpNA) 와의 기질 반응을 분석 한 결과 trypsin 기질에서 protease activity가 높음을 확인 하였다. 추후 연구에서 파밤나방 중장액과 Tannic acid를 반응 시킨 후 기질과의 protease activity를 측정 하고 zymogram을 통해 protease 활성 부분을 연구 할 것 이다.

An assessment was made of the toxicity of 12 insecticides, three essential oils and Bacillus thuringiensis var. israelensis (Bti) alone or in combination with the oil major constituents, (E)-anethole (AN), (E)-cinnamaldehyde (CA) and eugenol (EU), to third instars from bamboo forest collected Aedes albopictus and rice paddy field collected Anopheles sinensis resistant to various groups of insecticides. The toxicity of the test insecticides, essential oils and binary mixtures of Bti and the oil constituents (1:1 ratio) was evaluated using a direct-contact mortality bioassay. Binary mixtures of B.t.i. and CA, AN or EU were significantly more toxic against Ae. albopictus larvae (LC50, 0.0084, 0.0134 and 0.0237 mg/l) and An. sinensis larvae (0.0159, 0.0388 and 0.0541 mg/l) than either B.t.i. (1.7884 and 2.1681 mg/l) or CA (11.46 and 19.43 mg/l), AN (16.66 and 25.11 mg/l) or EU (24.60 and 32.14 mg/l) alone. Based on the co-toxicity coefficient (CC) and synergistic factor (SF), the three binary mixtures operated in a synergy pattern (CC, 140.7–368.3 and SF, 76–213 for Ae. albopictus CC, 75.1–245.3 and SF, 40–136 for An. sinensis).The binary mixtures of Bti and essential oil constituents described, particularly (E)-cinnamaldehyde, merit further study as potential mosquito larvicides for the control of malaria vector mosquito populations in light of global efforts to reduce the level of highly toxic synthetic insecticides in the aquatic environment.

 ,  , The entomopathogenic bacterium, Xenorhabdus sp., was isolated from an entomopathogenic nematode, Steinernema monticolum. When these bacteria were injected into the hemocoel of the diamondback moth, Plutella xylostella, they caused significant mortality. However, the bacterium was not pathogenic when it was administered orally. This study showed that Xenorhabdus sp. significantly enhanced oral pathogenicity of Bacillus thuringiensis (Bt) against the last instar larvae of P. xylostella. Different ratios of culture broth of Xenorhabdus sp. and Bt showed significantly different pathogenicities against P. xylostella. In field tests, the optimal bacterial mixture significantly enhanced control efficacy against P.xylostella compared to Bt treatment alone. These results demonstrated that Xenorhabdus sp. culture broth can be developed as a potent biopesticide by enhancing the insecticidal efficacy of Bt.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.

Bacillus thuringiensis (Bt) is characterized by its ability to synthesize crystal toxins and also able to produce bacteriocins such as thuricin, tochicin, entomocin and bacthuricin. The present work, for the first time, describes the biological activity of bacteriocins from B. thuringiensis subsp. cameroun (Btc). Supernatant which was produced from a liquid culture of Btc had antimicrobial activity against various Bt subspecies, ending up to making a inhibition zone on an agar medium. A significant reduction in antimicrobial activity was observed when the supernatant was exposed to heat at 47~50°C for 15 min. Proteins were separated from the supernatant by a fast protein liquid chromatography (FPLC) given the thermal instability. A group of FPLC fractions had antimicrobial activity against Bt subsp. palmanyolensis, israelensis, 1-3, morrisoni, toguchini and kurstaki and a Bacillus. cereus ATCC21768, ATCC14579 and NRRLB-569. Interestingly, when the supernatant was individually incorporated into the liquid cultures of Bt subsp. israelensis (Bti) and mogi (Btm) with mosquitocidal activity, a vegetative cell growth was observed only in the Btm culture 10 h post-incubation. A possible recovery of vegetative Btm cell growth was observed, compared to a control without the supernatant. These results suggest that Btc produced proteinous antimicrobial substances, one of which may be used as a selection marker to separate Btm after possibly conjugating the two mosquitocidal strains.

Bacillus thuringiensis(B.t.)에서 cry gene은 plasmid DNA상에 존재하고 대상 곤충에 살충활성을 나타내는 내독소단백질 형성에 관여하는 주요 유전자이다.파밤나방(spodoptera exigua)에 높은 살충활성을 보이는 B.t. subsp. aizawai KB098 균주의 cry gene이 위치하는 plasmid DNA를 찾기 위해 curing 방법을 사용하였다. KB098균주는 cry1Aa, cry1Ab, cry1C, cry1D 4개의 cry gene을 가지고 있으며, Plasmid DNA는 7개가 확인되었다. Cry gene을 암호화하는 plasmid DNA를 찾기 위한 curing 방법은 LB배지에 KB098균주를 희석 후, 27℃ 진탕배양기에서 24시간 배양한 뒤 NA배지에 spreading 한 후, 42℃조건으로 48시간 배양하여 단일 colony를 얻었다. 위상차현미경으로 관찰했을 때 내독소단백질을 형성하지 않는 colony를 NA배지에 streaking하여 27℃ 조건으로 4일간 배양하였다. 위상차현미경관찰을 통해 내독소단백질을 형성하지 않는 colony를 선발하였다. Curing과정이 제대로 수행되었는지 확인하기 위해 SDS-PAGE를 통하여 분자량 130kDa의 내독소단백질 band가 형성되지 않음을 확인하였으며, PCR증폭을 통해 acrystalliferous균주가 KB098 균주가 가지고 있는 4개의 cry gene을 가지고 있지 않음을 확인하였다. cry gene을 암호화 하는 plasmid DNA를 찾기 위해 KB098균주와 5개의 acrystalliferous균주와의 plasmid DNA pattern을 비교하였다. acrystalliferous균주에서 결실된 plasmid DNA만을 gel elution 한 후에 PCR을 통해 cry gene의 존재를 확인하였다.

Tannic acid가 파밤나방의 중장액에 존재하는 단백질분해효소의 활성을 저해함으로 처리된 Bacillus thuringiensis(이하 B. thuringiensis) subsp. kurstaki KB100균주의 insecticidal crystal proteins(ICPs)의 과분해를 억제하며 살충활성을 상승시키는 효과가 있다는 연구결과를 기초로, tannic acid의 protease inhibition여부 및 tannic acid가 B. thuringiensis KB100균주의 parasporal inclusion에 미치는 영향을 조사하였다. Tannic acid와 파밤나방 중장액을 먼저 혼합하여 반응시킨 후 B. thuringiensis KB100의 parasporal inclusion에 처리하여 SDS-PAGE를 통해 단백질밴드를 확인한 결과, 중장액내의 단백질분해효소에 의해 정상적으로 분해되어 60KDa의 살충활성을 나타내는 독소단백질 밴드를 나타냈다. 따라서 tannic acid에 의한 살충활성증대의 정확한 영향을 확인하기 위해, B. thuringiensis의 parasporal inclusion과 먼저 혼합하여 반응시킨 후 파밤나방 중장액을 처리하고 단백질 밴드패턴을 확인한 결과 전독소인 130kDa의 단백질이 거의 분해되지 않고 나타났다. B. thuringiensis KB100의 parasporal inclusion과 tannic acid을 먼저 혼합하여 반응시킨 것을 위상차현미경하에서 확인한 결과, tannic acid의 농도가 높아질수록 parasporal inclusion이 응집하여 있는 것이 확인되어, tannic acid가 파밤나방 중장 내 소화효소에 반응하는 영향보다 B. thuringiensis 독소단백질의 응집을 통해 소화효소의 분해저해 원인 가능성을 확인하였다.

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 ins tar 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.

Bacillus thuringiensis (Bt) is characterized by its ability to synthesize crystal toxins and also able to produce bacteriocins such as thuricin, tochicin, entomocin and bacthuricin. The present work, for the first time, describes the biological activity of bacteriocins from B. thuringiensis subsp. cameroun (Btc). Supernatant which was produced from a liquid culture of Btc had antimicrobial activity against Bacillus cereus, ending up to making a inhibition zone on an agar medium. A significant reduction in antimicrobial activity was observed when the supernatant was exposed to heat at 75~100°C for 15 min. Proteins were separated from the supernatant by a fast protein liquid chromatography (FPLC) given the thermal instability. A group of FPLC fractions had antimicrobial activity against Bt subsp. palmanyolensis, israelensis, 1-3, morrisoni, toguchini and kurstaki, and B. cereus ACTC21768, ATCC14579 and NRRLB-569. Interestingly, when the supernatant was individually incorporated into the liquid cultures of Bt subsp. israelensis (Bti) and mogi (Btm) with mosquitocidal activity, a vegetative cell growth was observed only in the Btm culture 10 h post-incubation. A possible recovery of vegetative Btm cell growth was observed, compared to a control without the supernatant. These results suggest that Btc produced proteinous antimicrobial substances, one of which may be used as a selection marker to separate Btm after possibly conjugating the two mosquitocidal strains.

cry gene은 Bacillus thuringienesis(B.t.)에서 대상 곤충에 살충활성을 나타내는 내독소단백질을 형성하는 주요 유전자로 특정 plasmid DNA상에 암호화되어 있 다. B.t. subsp. aizawai KB098 균주는 파밤나방(spodoptera exigua)에 높은 살충 활성을 보이는 균주로 본 연구에서는 이 균주의 cry gene이 위치하는 plasmid DNA를 찾고자 하였다. 본 실험에 이용된 KB098균주는 cry1Aa, cry1Ab, cry1C, cry1D 4개의 cry gene을 가지고 있으며, Plasmid DNA는 7개가 확인되었다. Cry gene을 암호화하는 plasmid DNA를 찾기 위해 acrystalliferous균주가 필요하였으 며, 42℃조건으로 48시간 열처리 후 새로운 배지에 27℃ 조건으로 3일간 재배양 하여 sporulation단계에서 위상차현미경관찰을 통해 내독소단백질을 형성하지 않 는 colony를 autolysis 단계까지 배양한 후에 위상차현미경으로 재확인하여 확보 할 수 있었다. 획득한 14개의 acrystalliferous를 균주 중, 서로 다른 pattern의 plasmid DNA를 갖는 5개의 균주를 선발하였고, acrystalliferous재확인을 하기 위 해 SDS-PAGE를 통하여 내독소단백질의 분자량인 130kDa의 band가 형성되지 않음을 확인하였으며, PCR증폭을 통해 acrystalliferous균주가 KB098 균주가 가 지고 있는 4개의 cry gene을 가지고 있지 않음을 확인하였다. cry gene을 암호화 하는 plasmid DNA를 찾기 위해 KB098균주와 선발한 5개의 균주와의 plasmid DNA pattern을 비교한 결과, 두 개의 plasmid DNA band에서의 차이가 확인되 었다.

Bacillus thuringiensis (Bt) strain K4 was isolated from fallen leaves which had been collected at a forest stand in Mungyeong city, Republic of Korea. The flagellated vegetative cells of Bt K4 were agglutinated with the H3 reference antiserum among 55 reference H-antisera. In a further test to identify subfactors, 3b and 3d monospecific antisera were reactive to the cells, followed up with introducing a novel serogroup of 3a3b3d, designated as serovar mogi. The strain K4 had mosquitocidal activity against Dipteran larvae, Anopheles sinensis and Culex pipiens pallens, with no Lepidopteran toxicity observed. The SDS-PAGE profile of K4 crystal protein, ovoidal-shaped, included several bands ranging from 30-75 kDa. Four putative peptides, Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected from the bands by a nano-LC-ESI-IT MS analysis. Through a thermal asymmetric interlaced PCR, cry19Ba, cry40ORF2 and cry27Aa genes were partially cloned from K4 strain. Three cry genes were further found in the strain by a 454 pyrosequencing, ending up to showing 58%, 39% and 84% homology in amino acids with Cry56Aa, Cry8Ba and Cry39ORF2 toxins, respectively. This novel 3a3b3d type strain, B. thuringiensis subsp. mogi, can be used as a good resource for studying unknown mosquitocidal cry genes.

The impact of transgenic Bt maize plant contained Cry1F was evaluated on the oat aphid Rhopalosiphum padi as a non-target insect species. Slightly reduced rates of survival and alata vivipar production were observed on Bt maize than on the non-Bt maize. In addition, slightly low preference to Bt maize plant was observed. Aphid fecundity, measured as the number of offspring produced for 7 days, was higher on Bt maize than on non-Bt maize but not different significantly. ELISA test using Cry1F-antibody revealed that 26% of Cry1F protein compared to the positive control was detected from the whole body of R. padi when the insects were fed Bt maize for 50 days, showing that R. padi can carry Cry1F protein to the higher trophic level when exposed to Bt maize. Taken together, the Bt maize plant is not likely to cause any negative side impacts on non-target insect R. padi but Bt toxin can be transferred to higher predators via R. padi as it carries the toxin.

해충에 대한 화학농약의 오남용으로 인하여 해충의 저항성이 발달되고 환경오염, 인축에 대한 위험 등의 이유로 화학농약의 사용을 줄이게 되었다. 그래서 친환경적인 방제인자인 미생물농약 Bacillus thuringiensis(이하 B.t)가 현재 전 세계적으로 널리 사용되고 있다. B.t는 그람 양성의 호기성 간균으로 토양, 곡물의 분진, 낙엽, 곤충의 사체 등 다양한 곳에서 분리되는 것으로 알려져 있는데, 본 연구에서는 풍뎅이류 곤충의 사체로부터 분리한 B.t subsp. kurstaki CAB530 균주가 나비목 해충에 대해서 살충활성을 나타내는 것을 확인하였으며, 특히 난방제 해충인 파밤나방에 살충활성이 뛰어난 KB099 균주와 비교하기 위하여 SDS-PAGE와 PCR을 수행하였다. 파밤나방에 대한 생물활성 검정 결과, CAB530 균주는 처리 48시간 후에 90%가 넘는 사충률을 보여주었며, 다른 B.t subsp. kurstaki SDS-PAGE 수행 결과 파밤나방에 살충활성이 있는 B.t subsp. kurstaki와 비슷한 130kDa의 밴드를 나타내었다. 또한 파밤나방 중장액으로 소화를 시킨 후에 약 65kDa의 활성독소를 확인할 수 있었다. 파밤나방에 6%의 사충율을 보이며 낮은 살충활성을 보인 CAB533 균주는 65kDa의 독소단백질 밴드를 나타냈지만 파밤나방에 대해서 살충활성이 높지는 않았다. PCR수행 결과 파밤나방에 대해서 가장 활성이 높은 CAB530 균주는 Cry1Aa, 1Ab, 1Ac, 1B, 1D, 1E, 1F, 1I, Cry2 유전자가 존재하는 것으로 밝혀졌다.

The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.

Large amounts of genetically modified (GM) grains, including maize, cotton and soybean, have been imported to Korea for food, feed and processing (FFP). To evaluatethe environmental impacts, particularly on non-target insects, of FFP GM grains of unknown source, it is a prerequisite to identify Cry protein types in the test GM grains and to establish proper risk assessment protocols. Imported GM maize grains were randomly obtained and their Cry toxins were analyzed by ELISA using Cry1A, Cry1F, and Cry3A antibodies. Since all tested GM maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as a non-target insect species. A domestic maize strain was used as a non-GM control, which did not show any differences in major nutritional composition from the GM maize grain. Slightly increased survival rate and head capsule width of T. molitor larvae were observed when reared on GM maize powder, demonstrating no sub-chronic adverse effects of GM maize on T. molitor larvae. Head capsule width of T. molitor neonate increased steadily from hatch to 70-day-old, regardless of being fed Bt or non-Bt maize. ELISA test using Cry1A-antibody revealed that concentration of Cry1A protein slowly increased in the whole body of T. molitor from 0 to 50 post-feeding days when the insects were fed GM maize but rapidly decreased within 5 days when Bt maize-fed larvae were transferred to non-Bt maize, showing that the Cry toxin is not accumulated inside the body of T. molitor once the exposure source is removed. In addition, no Cry protein was detected in the hemolymph of the larvae reared on Bt maize, suggesting little possibility of Cry toxin exposure to higher tropic level. Taken together, the imported GM-maize grains is not likely to cause any side impacts on non-target insect T. molitor.

Among 18 Bacillus thuringiensis isolates with spherical parasporal inclusion from soils, B. thuringiensis subsp. israelensis CAB199 was selected. It was showing over 90% mortality against Aedes aegypti and Culex pipiens moletus. It was confirmed that this B. thuringiensis subsp. israelensis CAB199 isolate also had a insecticidal activity against Culex inatomii that was occurred in the marsh. Because most of mosquito larva were primarily situated or shifted from under- to surface water, we need to select long floating formulations on surface water for controlling mosquito larva. It was tested the pesticidal and control effects in the laboratory and wetland with two formulation types of B. thuringiensis subsp. israelensis, for example, wettable power (WP) and suspension concentrate type (SC). Laboratory test showed that SC formulation type was relatively faster and more effective against 3 tested mosquito species, C. pipiens, Aedes aegypti, and C. inatomii. Otherwise, the control efficacy of SC formulation type was more rapidly appeared against C. inatomii in the wetland.