Eight Bacillus thuringiensis strains activated against mosquito larva were compared their characterization. Spherical-shaped parasporal inclusion of B. thuringiensis subsp. tohokuensis CAB167 was observed by phase-contrast microscopy and scanning electron microscopy. LC₅₀ values of B. thuringiensis subsp. tohokuensis CAB167 against Culex pipiens molestus, Culex pipiens pallens, and Aedes aegyti were 173, 190 and 580 ng/㎖, respectively. B. thuringiensis subsp. tohokuensis CAB167 had a parasporal inclusion containing 4 major protein components, for example, 135, 80, 49 and 28-kDa by SDS-PAGE. Otherwise, after trypsin digestion of parasporal inclusion, SDS-PAGE was showed new protease-resistant peptides at 72 and 63-kDa. Activated toxins of isolated CAB167 were different from other reference strains on a serological by immuno-diffusion test.

국내 토양에서 분리된 Bacillus thuringiensis 중에서 나비목 해충에 대해 살충활성 가능성을 가지며 이중피라미드형 내독소 단백질을 가진 균주들을 대상으로 실내에서 살충효과를 검정한 결과, 실험에 이용한 Bt 54균주 중, 7개 균주가 벼혹명나방, 벼애나방 두 종 해충 모두에 90% 이상의 살충율을 보였다. 높은 살충율을 보인 균주들을 대상으로 실내에서 방제실험을 실시하여 무처리구와의 피해정도를 비교한 결과, 균주에 따라 차이를 보이지만 피해엽률에 있어 50%이상의 차이를 보이며, 살충효과를 확인할 수 있었다. 반면, 살충활성은 거의 나타나지 않았지만, 처리한 식물체에서 섭식저해 현상을 보이는 유충을 계속적으로 사육하면서 용화율, 우화율 및 번데기 길이를 조사함으로, Bt가 대상 해충의 생물학적, 생태학적 특성에 미치는 영향을 조사한 결과, 살충율의 차이에 따라 균주별 차이가 존재했으나, 처리 후 조사기간 동안 생존한 개체라 할지라도 용화, 우화율이 정상개체에 비해 30~40%이상 낮게 나타났다. 또한 번데기 길이에서도 정상개체와 처리 후 생존개체가 차이를 보였다. 따라서 직접적인 살충효과가 낮게 나타난 균주라 하더라도, 용화율, 우화율에 영향을 미쳐 다음세대의 개체군 크기를 감소시킬 수 있는 부수적인 해충방제능력을 기대해 볼 수 있을 것으로 본다.

벼에 발생하여 엽육을 전체적으로 식해해서 피해를 주는 혹명나방(Cnaphalocrosis medinalis)과 벼애나방(Naranga aenescens)의 곤충병원성미생물을 이용한 친환경적 방제를 위해, 국내 논토양에서 분리된 Bacillus thuringiensis 중에서 위 두 종 해충 모두에 높은 살충효과를 보이는 균주를 선발하였다. 선발된 HARI004균주는 혹명나방, 벼애나방 두 종 모두에 1차생물검정에서 90%이상의 살충율을 나타내며 높은 살충활성을 보였으며, 실내에서 방제실험을 수행한 결과, 포자 및 크리스탈 현탁액을 처리한 후 3일째부터 무처리구와 유충에 의한 피해엽수에 차이를 보이며 살충효과가 나타나기 시작하여 피해엽비율을 약 58%이상 줄이며 방제효과를 나타낸 것을 확인할 수 있었다. 또한 SDS-PAGE를 통한 HARI004균주의 내독소단백질의 패턴을 분석한 결과, B. thuringiensis subsp. kurstaki와 유사한 주요 단백질 밴드를 확인할 수 있었으나, 혈청학적인 분류를 통해 정확하게 동정해야 할 것으로 보인다.

Bacillus thuringiensis 1-3 (Bt 1-3), isolated from Korean soil sample, showed high insecticidal activity against Plutella xylostella. Recently, we improved plasmid capture system donor-s (PCS-S) by inserting attB sites including lacZ between transposable elements (designated as pTroy), to reduce background and construct E. coli-Bt shuttle vector. Through in vitro transposition with total plasmid DNA of Bt 1-3, at least 6 different size plasmids of Bt 1-3 were cloned. Among them, 47 clones which have approximately 10 kb plasmid in size were sequenced and 5 contigs were assembled. These contigs showed partial similarity with two known plasmids, pGI3 or pBMB175, separately. These cloned plasmids will acquire erythromycin resistance by BP recombination reaction with pDonrattPEm vector. After transformation into Bt cells, final erythromycin resistant Bt cell might contain novel E. coli-Bt shuttle vector. This scheme proposes that pTroy and pDonr-attPEm system can easily construct new shuttle vector by in vitro transposition, BP reaction, and erythromycin selection with any Bt plasmids.

Bt crystal proteins, encoded by cry genes, are a group of insecticidal proteins unique in the Gram-positive and spore-forming bacterium, Bacillus thuringiensis. These cry genes are widely applied as one of the most successful candidates for constructing transgenic crops resistant to pest insects. In our previous report, we found Cry1-5 had high insecticidal activity against Spodoptera larvae although its amino acid sequences showed high similarity (95.6%) to those of Cry1Ac which had low activity. In comparison with Cry1Ac, Cry1-5 had 12 different residues on domain I and II. In order to convert these residues to Cry1-5 randomly, 10 mutagenic primers were designed. Through multi site-directed mutagenesis, we mutated the modified cry1Ac gene by plant codon usage in pOBI-Modcry1Ac based on cry1-5 and constructed 63 mutant cry genes. Among them, 10 mutant cry genes on domain II were selected and their recombinant proteins were expressed by baculovirus expression system. From bioassay results to P. xylostella and S. exigua, we found some mutants have high insecticidal activities to be applicable to transgenic crops.

To develop an advanced baculovirus insecticide with additional advantages, such as higher toxicity and recovering to wild-type baculovirus, a novel recombinant baculovirus, NeuroBactrus was constructed. Bacillus thuringiensis crystal protein gene (cry1-5) and an insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of poyhedrin gene promoter, and by fusion of orf603 partial genes and AaIT under the control of early promoter of ORF3006 from Cotesia plutellae bracovirus. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by NeuroBactrus was occluded into the polyhedra, and activated as about 65 kDa of crystal protein when treated with trypsin. RT-PCR analysis indicated that transcription of AaIT gene occurs by 2 h postinfection (p.i.) and increased at 16 h p.i.. NeuroBactrus showed high toxicity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcNPV. Re-recombinants derived from NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro. This result showed that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 genes.

The effects of pollens of Bt Chinese cabbage (Brassica campestris), expressing the insecticidal protein Cry1Ac toxin (Bt Chinese cabbage) on Bombix mori larvae, were determined in laboratory studies. When reared on an artificial diet containing pollens of Bt Chinese cabbage, decreased survival rate and body weight of B. mori larvae were observed. ELISA test using Cry1Ac-antibody implied that the Cry toxin was detected not only in the whole body and alimentary canal but also in the hemolymph and remaining body parts, suggesting that the ingested Cry1Ac protein is distributed inside the body through epithelial membrane of the midgut of B. mori. Taken together, it was suggested that the pollens of Bt Chinese cabbage adversely affect non-target insect B. mori larvae.

A new Bacillus subtilis isolate showed high anti-fungal activities (more than 80% control efficacy) against several plant diseases such as rice blast (Magnaporthe grisea), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans) and wheat leaf rust (Puccinia recondita). We tried to confer an insecticidal activity to this B. subtilis isolate for constructing a recombinant strain which has dual functions, anti-fungal and insecticidal activity. The insecticidal cry1Ac gene of B. thuringiensis was constructed under its own promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K-1Ac). The plasmid, pHT1K-1Ac was introduced into B. subtilis isolate by electroporation and the transformant was confirmed by PCR with cry1Ac specific primers. B. subtilis transformant produced a parasporal inclusion in the cells as in B. thuringiensis and the size of that protein was appox. 130 kDa. The insecticidal activity of the transformant was checked against lepidopteran pest, Plutella xylostella. This result suggests that this recombinant B. subtilis strain shows the possibility of controlling harmful insect pests as well as plant fungal diseases simultaneously at one crop, and both culture broth and harvested cells of this strain can be used as individual biological control agents separately for integrated crop protection.

To screen highly active Bacillus thuringiensis isolates against Spodoptera litura (Lepidoptera, Noctuidae), 46 B. thuringiensis was isolated from 115 samples obtained from several crop soils. Especially, B. thuringiensis subsp. kurstaki CAB133 and CAB162 isolates showed 100% mortality against S. litura. LD50 values of CAB133, CAB162 and HD-1 strains of B. thuringiensis subsp. kurstaki were 0.089, 3.144 and 0.513 ㎍/㎖ against 2nd larva of μ S. litura, respectively. The weight of 3rd larva of S. litura which were fed crystal inclusion protein (1.267 ㎍/㎖) with B. thuringiensis subsp. kurstaki CAB133 was about 30 times lass than control group. CAB133 and CAB 162 strains of B. thuringiensis subsp. kurstaki which were taken a highly toxity against S. litura were analyzed by SDS-PAGE, and estimated the molecular weight of the Cry proteins. Their serological identification by H serotypes were showed B. thuringiensis subsp. kurstaki (3abc) type.

A B. thuringiensis kurstaki was first discovered by H. Dulmage in 1970 and commercialized as a DipelTM due to powerful toxicity to various Lepidoptera. Previously we isolated B. thuringiensis kurstaki on the basis of plasmid DNA profiling and H-antigen serotyping. The aims of this study were to screen larvicidal activities and select the highly active B. thuringiensis isolates against the important polyphagous pests of mandarine oranges and vegetables. The colony forming unit (CFU/ml) of each of culture mixtures was determined to estimate the δ-endotoxin concentration. The bioassay against artificial diet-rearing insects was conducted by surface contamination methods using the Petri dishes specially designed and manufactured by SPL Lifesciences. The insecticidal activities to the natural diet-rearing insect were measured by the application of spore and crystal mixtures to the leaf discs of the chinese cabbage with Potter spray tower. The following insects were used for the larvicidal activities of B. thuringiensis isolates: beet armyworm Spodoptera exigua, diamondback moth Plutella xylostella, giant looper Ascotis selenaria, tobacco cutworm Spodoptera litura, and variegated cutworm Peridroma sucia.

Bt crystal proteins, encoded by cry genes, are a group of insecticidal proteins unique in the Gram-positive and spore-forming bacterium, Bacillus thuringiensis. These cry genes are widely applied as one of the most successful candidates for constructing transgenic plants resistant to pest insects. In our previous report, we found Cry1-5 had high insecticidal activity against Spodoptera larvae although its amino acid sequences showed high similarity (97.9%) to those of Cry1Ab which had low activity. In comparison with Cry1Ac, Cry1-5 had 12 different residues in domain Ⅰ and domain Ⅱ, and we focused on domain Ⅰand domain Ⅱ regions and designed 10 mutagenic primers to change 12 residues. Through multi site-directed mutagenesis, we mutated the modified cry1Ac gene by plant codon usage in pOBⅠ-Mod-cry1Ac based on cry1-5 and constructed 63 various mutant cry genes. In the further study, we will express those mutant proteins as a fusion form with polyhedrin using baculovirus expression system and subsequently do bioassay to Spodoptera larvae.

A strain of Bacillus thuringiensis, named Bt 1-3, was isolated from Korean soil sample and it showed high insecticidal activity against Plutella xylostella. Bt 1-3 was deterimined to belong to ssp. aizawai (H7) by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins. PCR analysis with specific cry gene primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2Ab genes. In addition, this isolate showed high uptake rate of foreign plasmid by electroporation. Based on these characteristics of Bt 1-3, we tried to construct a spore-free Bt 1-3 mutant by knock-out sigG gene, which is known as a key transcription factor during sporulation. First, we constructed a basal vector, named pDST, consisting of erythromycin resistant gene (EmR), partial polyhedrin gene and temperature sensitive origin of replication gene (Orits). Subsequently, according to the chromosomal DNA sequence of Bt subsp. konkukian 97-27, we amplifed upstream and downstream regions of Bt 1-3 sigG, and cloned into pDST (pDST-G). So far, several EmR colonies were obtained by electroporating into the wildtype Bt 1-3 and crossover by homologous recombination is going on.

To develop an improved baculovirus insecticide with additional advantages, a novel recombinant baculovirus, AcB5B-AaIT was constructed. B. thuringiensis crystal protein gene (cry1-5) and insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of polyhedrin (polh) gene promoter, and AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus, respectively. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by AcB5B-AaIT was occluded into the polyhedra produced by the recombinant virus, and activated as about 65 kDa of crystal protein when treated with gut-juice of Bombyx mori. The AcB5B-AaIT showed about 50% reduced LT50 value compared to that of the recombinant virus, Ap1Ac, expressing Cry1Ac against Plutella xylostella larvae. In addition, Spodoptera exigua larvae fed the recombinant polyhedra of AcB5B-AaIT showed about 4 fold higher refusing diet effect compared S. exigua larvae fed the recombinant polyhedra of the recombinant virus, Ap1C, expressing Cry1C. AcB5B-AaIT could be transferred to wild-type baculovirus along with serial passage by the homologous recombination between two polyhedrin genes contained in polh-cry1-5-polh fusion protein gene. These results suggested that the novel recombinant baculovirus, AcB5B-AaIT, could be applied as advanced viral insecticide.

벼에 발생하여 엽육을 전체적으로 식해해서 피해를 주는 혹명나방(Cnaphalocrosis medinalis)과 벼애나방(Naranga aenescens)의 곤충병원성미생물을 이용한 친환경적 방제를 위해, 국내토양에서 분리된 Bacillus thuringiensis 중에서 나비목 해충에 대해 살충활성 가능성을 가지며 이중피라미드형 내독소 단백질을 가진 균주들을 대상으로 생물검정을 수행하였다. 실험에 이용한 Bt 30균주 중, 약 14개 균주가 혹명나방에 50% 이상의 살충율을 나타내었고, 7개 균주가 벼애나방에 살충활성을 보였는데, 두 종 모두에 80% 이상의 살충율을 보이는 균주도 3균주나 확인되었다. 이들 균주는 두 종 해충 뿐만 아니라 배추좀나방, 담배거세미나방에도 높은 살충활성을 보인 것으로 나타나 생물농약으로서의 활용가능성을 보여 주었다. 반면, 이중피라미드형 내독소단백질을 가지고 있으나 두 종 모두에 살충활성이 전혀 나타나지 않은 균주도 확인되었다. 또한 살충활성은 거의 나타나지 않았지만, 처리한 식물체에 섭식저해 현상을 보이는 유충을 계속적으로 사육하면서 용화율, 우화율 및 번데기 길이를 조사함으로, Bt가 대상 해충의 생물학적, 생태학적 특성에 미치는 영향을 조사한 결과, 처리 후 조사기간 동안 생존한 개체라 할지라도 용화, 우화율이 정상개체에 비해 30~40%이상 낮게 나타났다. 특히나 혹명나방의 경우, 번데기 길이에서도 정상개체와 처리 후 생존개체가 차이를 보이며 섭식에 저해를 받은 것을 확인할 수 있었다.

Bacillus thuringiensis with selected high toxicities against tobacco cutworm, Spodoptera litura were isolated from domestic soils. When being observed under a phase-contrast microscope, the insecticidal crystal proteins were showed a bipyramidal crystal types. New CAB 109 isolate was identified to B. thuringiensis subsp. aizawai in the H serotype. As a results of insecticidal activities between CAB 109 isolate and 3 existing ready-made products against 3rd larva of S. litura, CAB 109 isolate showed 100% mortality with spore concentration (1.3×10⁷ cfu/㎖). It was a very high insecticidal activity compared with a existing ready-made B. t. products. LD₅₀ values of CAB 109 isolate was 9.78×10⁵, 6.87×10⁶ and 1.83×10⁷ cfu/㎖ spore concentration against 2nd, 3rd and 4th larva of S. litura, respectively. Unlike Plutella xyfostella, S. litura was slowly died after application up to 7 days. The weight of S. litura larva applied with CAB 109 isolate were 6-7 times less than controlled group. Even though it didn"t die, it did not grow into next larva. The result observed with scanning electron microscope was that CAB 109 isolate of B. t. aizawai formed a typical bipyramidal crystal protein type. Otherwise, when CAB 109 isolate was examined with SDS-PAGE and with trypsin, there was no difference between CAB 109 strain and ready-made products of B. thuringiensis.

Bacillus thuringiensis strains were isolated from the domestic soil and a strain was selected that had a new host range and high toxicity against agriculture insect pest. The 142 samples of soil were sampled from the mountains, paddy fields and patches, in Daejon, Chungnam, Chungbuk and Jeonbuk and used for the investigation. Sixteen B. t strains were isolated from 12 samples among collected samples. There were 11 strains that showed toxical activity on Plutella xylostella (Lepidoptera: Yponomeutidae), 7 strains on Spodoptera litura (Lepidoptera: Noctuidae), 5 strains on Arcte coerulea (Lepidoptera: Noctuidae), 5 strains on Culex pipiens pallens (Diptera: Culicidae) among the 16 isolated B. t strains. But there were not any strains that showed activity against Hyphanria cunea (Lepidoptera: Arctiidae) and Sitophilus oryzae (Coleoptera: Rhynchophoridae). And also some of B. thuringiensis strains showed insecticidal activity with 2, 3 or 4 kinds of insects. But there were also 3 strains that did not show any activities to the 6 insects which were used in the experiment. When examined with a phase-contrast microscope, the insecticidal crystal protein produced from 16 selected strains had 13 bipyramidal and 3 spherical shapes. The insecticidal bioactivity of the S. litura showed 100% mortality when there were 1.3×10⁷ (cfu/㎖) of CAB109 isolates.