Bacillus thuringiensis subsp. kurstaki KB100 isolated from the domestic soil have the most effective activity against the beet armyworm, Spodoptera exigua larva. The tannic acid as protease inhibitor might be increased the efficacy of sublethal concentrations of B. thuringiensis. The tannic acid was identified as a protease inhibitor that could increased the efficacy of sublethal concentrations of B. thuringiensis. Mixture of B. thuringiensis and tannic acid was investigated the mortality of S. exigua larva in the laboratory and field. When B. thuringiensis treated to 2nd larva of S. exigua, mortality was shown 54.4%. However, mixtures of B. thuringiensis with 4 and 40 mM tannic acid were increased mortalities to 2nd larva of S. exigua as 64.0 and 95.5%, respectively. Also, synergy effect of mixture of B. thuringiensis and 40 mM tannic acid was increased the mortality of S. exigua 3rd larva to 93.3%, even though 60.0% mortality with only B. thuringiensis treatment. On the other hand, the mortality of mixture with B. thuringiensis and 80 mM tannic acid was 53.3% lower than B. thuringiensis single treatment. In the welsh onion field, the accumulated mortalities of 3 times replicated with mixture of B. thuringiensis and 40 mM tannic acid were 83.9, 89.4 and 66.8% compare with 61.8, 80.4 and 47.3% as only B. thuringiensis treatment, respectively.

토양에서 분리한 B.thuringensis 균주 들 중 Spodoptera litura에 활성이 높은 CAB133을 선발하였다. H serotype을 동정한 결과 혈청형이 3abc인 kurstaki로 동정되었다. 전형적인 이중 피라미드형의 결정성 내독소 단백질과 형태적인 유사성을 보였고, S. litura 유충에 대한 생물활성 결과 kurstak HD-1균주에 비해 높은 활성을 보였다. 혈청학적으로 동일한 균주일지라도 각각의 균주가 생산하는 결정성 독소단백질의 유전자가 다르기 때문에 해충의 살충성에는 차이가 나타나므로 분자생물학적인 특성을 알아보았다. 내독소 단백질 특성을 비교하기 위하여 SDS-PAGE를 수행한 결과, kurstak HD-1은 130kDa과 약 60kDa의 주요 단백질 밴드를 나타내었으나 CAB133균주는 130kDa의 주요 단백질 밴드만을 가지고 있었다. 균주의 전체 plasmid profile을 분석한 결과 kurstak HD-1균주는 7개, CAB133균주는 10개의 plasmid를 포함하고 있었으며 crystal을 형성하지 못하는 Cry-균주를 배양하여 확인한 결과 Cry+와 하나의 밴드가 없는 9개의 plasmid를 가지고 있음을 확인하였다. 균주의 crystal toxin을 확인하기 위해 usual primer로 PCR을 수행한 결과 kurstak HD-1은 cry1Aa,cry1Ab, cry1Ac, cry1I, cry2 gene을 CAB133은 cry1Aa, cry1Ab, cry1Ac, cry1C, cry1D, cry2 gene이 있음을 확인하였다.

파밤나방은 파, 배추, 수박 등에 다 발생하여 큰 피해를 주는 대표적인 농업 해충이며 화학농약에 대한 저항성이 강한 대표적인 해충으로, 본 연구에서는 생물적 방제인자로 방제하고자 Bacillus thuringiensis(이하 Bt) 균주를 선발하여 살충효과를 검정 하였다. 파밤나방의 살충효과검정에서 내성이 강한 해충과 비슷한 양상으로, 나비목 해충과 다르게 Bt에 대한 살충활성이 늦게 나타나는 지효성을 나타냈다. 이러한 원인으로 유충 중장 프로테아제에 의해 Bt의 cry 독소가 과분해 되는 것으로 가정하고 protein inhibitor로 알려져 있는 tannic acid를 선발하여 실내 실험과 야외 포장 실험을 수행하였다. 그 결과 Bt 단독처리보다 Bt와 tannic acid를 혼합처리 했을 때 더 높은 사충율을 나타냈다. 이 같은 결과에 대해 원인을 규명하고자, 가설을 세워 실험을 수행 하였다. 중장 프로테아제가 130kDa의 불활성 독소를 60kDa의 활성 독소로 분해한 후, 더 과분해 하는 것을 tannic acid가 억제할 것이다 라고 가설을 세운 후 실험을 수행 하였다. SDS-PAGE를 하여 tannic acid의 억제정도를 확인했고, 이 결과를 토대로 40mM tannic acid를 전 처리 한 후 Bt처리를 한 결과 동시처리보다 약 5% 더 높은 사충율을 나타냈다. Bt와 중장 프로테아제, tannic acid의 반응에 있어서 형태적인 면을 확인하기 위해 전자현미경을 관찰 하였다. 중장 프로테아제의 증명을 위해 자이모그램 실험을 수행 하였다.

For the environment-friendly control of beet armyworm, Spodoptera exigua, in spring onion fields, we have examined an alternative application method. Twenty-five insecticides registered for spring onion were tested for control effect against the beet armyworm in the laboratory, then the best 9 chemical and a single biological insecticides were selected and compared with 2 new isolates of Bacillus thuringiensis in a polyvinyl house. After that, 4 chemical and 3 B. thuringiensis treatments were used in the field for the control of beet armyworm in the spring onion. Two application methods are used: one is triple treatments with the same chemical and the other is alternative application with different chemicals and B. thuringiensis for 7 days intervals. Indoxacarb WP – chlorfluazuron EC – B.t. var. kurstaki CAB141 and indoxacarb WP – methoxyfenozide + spinosad SC – Chlorfluazuron EC – B.t. var. aizawai CAB109, B.t. var. kurstaki CAB141 showed greater than 78% mortality of beet armyworm larvae and greater than 43% damage decrease in spring onions infested by beet armyworm. These results showed that alternative applications had higher control effect than any other applications. It was suggested that alternative applications with microbial biological agents such as B. thuringiensis might minimize the development of insecticide resistance and might be used as the environment-friendly control of the beet armyworm.

파밤나방은 파, 배추, 수박 등에 다발생하여 큰 피해를 주는 대표적인 농업 해충이며 화학농약에 대한 저항성이 강한 대표적인 해충으로, 본 연구에서는 생물적 방제인자로 방제하고자 Bacillus thuringiensis(이하 Bt) 균주를 선발하여 살충효과를 검정 하였다. 파밤나방은 살충효과검정과정에서 내성이 강한 해충과 비슷한 양상으로, 다른 나비목 해충과 다르게 Bt에 대한 살충활성이 늦게 나타나는 지효성을 나타냈다. 이러한 원인으로 유충 중장의 프로테아제에 의해 cry 독소가 과분 해 되는 것으로 가정하고 protein inhibitor로 알려져 있는 tannic acid를 선발하 여 실내 실험과 야외 포장 실험을 수행하였다. 그 결과 Bt 단독처리보다 Bt와 tannic acid를 혼합처리 했을 때 더 높은 사충율을 나타냈다. 이 같은 결과에 대해 원인을 규명하고자, 몇 가지 가설을 세워 실험을 수행 하였다. 첫 번째 tannic acid가 Bt균 자체에 영향을 미치는 영향을 확인 하기 위해, Nutrient Agar(NA)배지에 tannic acid를 처리했을 때, Bt균의 생장여부와 생장정도를 확 인하고, 중장액과 Bt, tannaic acid를 각각 또는 혼합처리 했을 때의 중장내 Bt crystal의 형태적 변화를 관찰하기 위해 주사전자현미경 관찰을 수행 하였다. 두 번째로 중장 프로테아제가 130kDa의 불활성 독소를 60kDa의 활성독소로 분해한 후, 더 과분해 하는 것을 tannic acid가 억제할 것이라는 가설을 증명하 기 위해 SDS-PAGE를 수행하여 tannic acid의 억제정도를 확인했고, 이 결과를 토대로 40mM tannic acid를 전처리 한 후 Bt처리를 했을 때, 동시처리보다 약 5% 더 높은 사충율을 나타냈다.

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.

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.

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.

For the development of friendly environmental control of the beet armyworm, Spodoptera exigua that is too hard to control in the field, 25 insecticides were chosen from 58 registered to the beet armyworm, and bioassayed. There are 12 insecticides with neurotoxical activities, 10 with insect growth regulators and 3 Bacillus thuringiensis products. Among 12 insecticides with neurotoxical activities, mortality of S. exigua was 100% with emamectin benzoate (EC) and indoxacarb (WP) within 3 and 5 days after application, respectively. Otherwise, WG and SC of indoxacarb, Indoxacarb + etofenprox (WP) and pyridalyl (EW) were showed up to 91%. Methoxyfenozide + spinosad (SC) was better than any other insect growth regulator as 100% mortality within 3 days after application. And methoxyfenozide (WP), tebufenozide (WP) and methoxyfenozide (SC) were 92% by 5 days. However, 3 kinds of B. thuringiensis products were showed under 35% mortality within 5 days from first spray.

Bioassay of mosquito, Culex pipiens molestus, larva was investigated by several environment-friendly insecticides. These insecticides were Novaluron as chitin synthesis inhibitor, Metho-xyfenozide as ecdysone agonist, Pyriproxyfen as juvenile hormone mimic, and Spiromesifen as lipid biosynthesis inhibitor. The 50% lethal concentration (LC₅₀) of these insecticides were 0.00039, 0.07193, 0.65006 and 0.04839 ppm, respectively. Novaluron has lower concentration than any other insecticide. To determine the treatment time against larval stages, insecticides were applied to different larval stages of C. pipiens molestus. Mortality ratios of mosquito larva treated with Novaluron were 100.0, 84.5, 71.0 and 48.5% on 2, 4, 7 and 10 days after hatching from eggs, respectively. Otherwise, with the other insecticides, mortality ratios were under 80% with 2 days old larva. When exposure periods were tested to 3 or 4 days old larva against 4 insecticides, at least 3 hours were needed to 100% control effect against Novaluron, and over 12 hours with other insecticides.

The aim of this work is to assess the safety of fungicide tolclofos-methyl, difenoconazole and azoxystrobin in ginseng sprayed by safe use guideline. When tolclofos-methyl was sprayed on ginseng by safe use guideline, the residue amounts (MRL) of it in ginseng was 0.13 mg/kg which is below than 0.3 mg/kg, maximum residue limit established by Korea Food & Drug Administration (KFDA). The residue amounts of ginseng parts, head part was 0.37 mg/kg and main body part was 0.13 mg/kg. In case of difenoconazole, the residue amounts in ginseng was 0.81 mg/kg. which was exceed the MRL, 0.2 mg/kg. By the analyze results of ginseng part, the residues of head and main body part were 3.01 and 0.40 mg/kg, respectively. In experiment of vinyl mulching, the residue amount of difenoconazole in ginseng was 0.05 mg/kg. The residue amounts of azoxystrobin in ginseng sprayed by safe use guideline was 0.14 mg/kg. This residue was not exceed the MRL 0.5 mg/kg. The residue amounts by ginseng parts was 0.51 mg/kg for head part and 0.28 mg/kg for main body part. In case of vinyl mulching, the residue amount of azoxystrobin was 0.02 mg/kg.