• Proceedings of the National Institute of Ecology of the Republic of Korea
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• 제2권3호
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• pp.210-218
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• 2021

Recently, pest-resistant living modified (LM) crops developed using RNA interference (RNAi) technology have been imported into South Korea. However, the potential adverse effects of unintentionally released RNAi-based LM crops on non-target species have not yet been reported. Coccinella septempunctata, which feeds on aphids, is an important natural enemy insect which can be exposed to the double-stranded RNA (dsRNA) produced by RNAi-based LM plants. To assess the risk of ingestion of Snf7 dsRNA by C. septempunctata, we first identified the species through morphological analysis of collected insects. A method for species identification at the gene level was developed using a specific C. septempunctata 12S rRNA. Furthermore, an experimental model was devised to assess the risk of Snf7 dsRNA ingestion in C. septempunctata. Snf7 dsRNA was mass-purified using an effective dsRNA synthesis method and its presence in C. septempunctata was confirmed after treatment with purified Snf7 dsRNA. Finally, the survival rate, development time, and dry weight of Snf7 dsRNA-treated C. septempunctata were compared with those of GFP and vATPase A dsRNA control treatments, and no risk was found. This study illustrates an effective Snf7 dsRNA synthesis method, as well as a high-concentration domestic insect risk assessment method which uses dsRNA to assess the risk of unintentional released of LM organisms against non-target species.

• 환경생물
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• 제17권3호
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• pp.321-330
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• 1999

우리나라의 다양한 지역에서 100개의 토양과 곤충사체에서 누에 trap을 이용하여 곤충병원성 선충을 분리하였으며, 위생해충인 털검정파리유충(Calliphora vomitoria)과 농업해충인 멸강나방유충(Pseufaletia separata), 북방풀노린재 (Palomena angulosa) 그리고 왕풍뎅이유충(Melolontha incana) 에 대하여 20∼80%의 살충력을 나타내며, 누에(Bombyx mori mori)유충과 번데기에 대한 살충력은 100%인 선충 30계통을 선발하였다. 선발한 선충 30계통은 형태적 특징에 따라 Rhabditidae, Heteror-habditidae, Diplogasteridae, Steinernematidae, Tylenchidae의 5개 분류군으로 나뉘어졌다. 선충의 동정법 개선 및 분자생물학적 분류기준의 검토를 위하여 random primer 130개 중에서 20개의 primer를 선발하고, genomic DNA의 RAPD-RCR을 수행한 결과 유사도 0.853에서 형태적 분류기준과 일치하였다. 유사도계수를 이용한 5개 분류군에 속하는 선충 계통간의 유전적 근연관계는 Rhabditida에서 Steiner-nematidae는 경우 동일 목의 Heterorhabditidae및 Rhabditidae와는 유사도 0.819로서 거리가 멀고, 다른 목인 Tylenchidae와는 0.827로서 보다 더 근연이었다. 또한 Rhabditidae분류군은 유사도 0.863에서 다시 2개의 분류군으로 나누어졌으나 형태적 차이는 명확하지 않았다. 형태적 분류를 참조한 genomic DNA의 RAPD결과 유사도의 적절한 응용은 생물적 방제를 위한 곤충병원성 선충의 선발 및 동정에 있어서 형태적 단순성에 기인하는 자연분류의 한계를 보완 또는 극복할 수 있는 분자생물학적 동정법으로서 유용할 것이다. 또한 RAPD에 의한 동정은 생태환경에서 다수의 개체를 확보하기 쉬운 dauer larvae 또는 infective juvenile 등 유충기의 선충을 이용할 수 있다. 그러므로 노력이 소요되는 성충의 채집과 표본제작 등이 생략되므로 선충의 동정에 매우 안정적이며 효율적인 방법으로 판단되었다.

• 한국잠사곤충학회지
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• 제50권2호
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• pp.53-62
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• 2012

누에 BmNPV는 잠사업에 있어서 가장 위해한 바이러스로써 익히 보고되었으며, 종종 잠사업의 심각한 경제적 손실을 야기하기도 한다. 곤충의 박테리아, 곰팡이 그리고 원생동물과 같은 다양한 병원체에 대응하는 곤충의 생체 방어기작에 대한 연구가 많이 알려져 있지만, 항바이러스 기작에 대한 연구는 매우 부족한 실정이다. 따라서 본 연구에서는 누에서 처음으로 누에의 BmNPV에 대한 생체방어 관련 유전자를 선발하기 위하여, 누에에 인위적으로 BmNPV를 주사하여 면역을 유도한 다음, 이로부터 cDNA 유전자은행을 제작하였다. 제작된 cDNA 유전자은행으로부터 무작위로 3,332개의 cDNA 클론을 선발하여 정상 누에에 비하여 BmNPV에 의해 면역이 유도된 누에에서 차별화 발현되는 109종의 잠정 항바이러스 유전자 클론을 차별화선별법에 의해서 분리하였다. 본 연구를 통해 확보한 109개의 유전자 정보는 누에의 바이러스에 대한 면역반응뿐만 아니라 최근에 개발된 누에 형질전환 기술을 이용하여 BmNPV 저항성 누에 품종을 개발하는데 중요한 기초 자료를 제공할 것으로 기대되며 또한, 인간의 중요한 항바이러스제 개발을 위한 모델 곤충으로써 누에를 이용하는데 기초 자료로 활용될 것으로도 기대된다.

• 한국응용곤충학회지
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• 제59권4호
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• pp.281-293
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• 2020

고려엉겅퀴는 강원도에서 곤드레라는 음식 재료로 재배하는 작물이다. 고려엉겅퀴에 대한 정기적인 병해충 모니터링 중 우리대벌레의 국부적인 발생으로 인한 큰 피해를 발견하였고, 우리대벌레의 동정과 생태학적 특성을 보고한다. 본 조사는 2019년 5월 28일부터 동년 10월 1일까지 강원도 정선군에 있는 3곳의 고려엉겅퀴 재배지에서 실시하였다. 해당 포장에서 채집한 우리대벌레의 형태적, 분자 생물적 분석으로 대벌레과(Phasmida) 계열의 Ramulus koreanus Kwon Ha et Lee로 동정하였다. 우리대벌레는 6월 11일부터 8월 22일까지 고려엉겅퀴 포장에서 발생하였으며, 7월 23일에 최대 발생 밀도를 보였다. Taylor's power law와 Greens index를 이용하여 공간분포를 분석한 결과 무작위로 분포하는 것을 확인하였다. 우리대벌레의 고려엉겅퀴 일일 섭식량은 성충 기준 60.98 ± 4.35 ㎠ 였으며, 주요 기주식물은 가래나무와 아로니아였다. 우리대벌레 발생 메커니즘과 우리대벌레가 농업과 산림 생태계에 미칠 수 있는 영향 및 농작물 피해를 줄이기 위해 그 방법을 추구할 것이다.

• International Journal of Industrial Entomology and Biomaterials
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• 제25권2호
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• pp.195-203
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• 2012

In reared populations of Allomyrina dichotoma, commercial insects, the skin of last instar larvae was changed softer with opaque white, and infested grubs eventually died. To clarify the cause of the symptom, we collected the larvae of A. dichotoma from five farms and examined their intestinal bacterial florae using pyrosequencing technique. From those results, a member of Paenibacillus was found only in the larvae showing the symptom of disease. Through PCR analysis using a Paenibacillus specific primer set, we obtained the partial 16S rRNA gene sequence and confirmed the microbe as Paenibacillus sp. For clear identification, a whole guts was extracted from each larva showing the sign of the disease and incubated at $70^{\circ}C$ for 15 min to isolate spore forming bacteria. After then, each content of guts was cultured on $MYPGP_{NAL}$ agar medium($12.5{\mu}g/ml$ of nalidixic acid) at $30^{\circ}C$. The 16S rRNA gene sequence analysis for the isolated bacteria showed that they were closely related to P. rigui(97.9% similarity), to P. chinjuensis(96.1% similarity), and to P. soli(95.3% similarity). Additional tests including API test and cellular fatty acid composition analysis were performed, but the strain couldn't be identified at species level, suggesting it may represent novel species of the genus Paenibacillus.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.314-322
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• 2009

Four different bacterial colonies were isolated from an old stock of an entomopathogenic nematode, Steinernema monticolum. They all showed entomopathogenicity to final instar larvae of beet armyworm, Spodoptera exigua, by hemocoelic injection. However, they varied in colony form, susceptibility to antibiotics, and postmortem change of the infected host insects. Biolog microbial identification and 16S rDNA sequence analyses indicate that these are four different species classified into different bacterial genera. Owing to high entomopathogenicity and a cadaver color of infected insect host, Serratia sp. was selected as a main symbiotic bacterial species and analyzed for its pathogenicity. Although no virulence of Serratia sp. was detected at oral administration, the bacteria gave significant synergistic pathogenicity to fifth instar S. exigua when it was treated along with a spore-forming entomopathogenic bacterium, Bacillus thuringiensis. The synergistic effect was explained by an immunosuppressive effect of Serratia sp. by its high cytotoxic effect on hemocytes of S. exigua, because Serratia sp. caused septicemia of S. exigua when the bacterial cells were injected into S. exigua hemocoel. The cytotoxic factor(s) was present in the culture medium because the sterilized culture broth possessed high potency in the cytotoxicity, which was specific to granular cells and plasmatocytes, two main immune-associated hemocytes in insects.

• Mycobiology
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• 제41권2호
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• pp.86-93
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• 2013

Sequence characterized amplified region (SCAR) markers are one of the most effective and accurate tools for microbial identification. In this study, we applied SCAR markers for the rapid and accurate detection of Phytophthora katsurae, the casual agent of chestnut ink disease in Korea. In this study, we developed seven SCAR markers specific to P. katsurae using random amplified polymorphic DNA (RAPD), and assessed the potential of the SCAR markers to serve as tools for identifying P. katsurae. Seven primer pairs (SOPC 1F/SOPC 1R, SOPC 1-1F/SOPC 1-1R, SOPC 3F/SOPC 3R, SOPC 4F/SOPC 4R, SOPC 4F/SOPC 4-1R, SOPD 9F/SOPD 9R, and SOPD 10F/SOPD 10R) from a sequence derived from RAPD fragments were designed for the analysis of the SCAR markers. To evaluate the specificity and sensitivity of the SCAR markers, the genomic DNA of P. katsurae was serially diluted 10-fold to final concentrations from 1 mg/mL to 1 pg/mL. The limit of detection using the SCAR markers ranged from $100{\mu}g/mL$ to 100 ng/mL. To identify the limit for detecting P. katsurae zoospores, each suspension of zoospores was serially diluted 10-fold to final concentrations from $10{\times}10^5$ to $10{\times}10^1$ zoospores/mL, and then extracted. The limit of detection by SCAR markers was approximately $10{\times}10^1$ zoospores/mL. PCR detection with SCAR markers was specific for P. katsurae, and did not produce any P. katsurae-specific PCR amplicons from 16 other Phytophthora species used as controls. This study shows that SCAR markers are a useful tool for the rapid and effective detection of P. katsurae.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1281-1290
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• 2015

Thermolysin and its homologs are a group of metalloproteases that have been widely used in both therapeutic and biotechnological applications. We here report the identification and characterization of a novel thermolysin-like protease, BtsTLP1, from insect pathogen Bacillus thuringiensis serovar Sichuansis strain MC28. BtsTLP1 is extracellularly produced in Bacillus subtilis, and the active protein was purified via successive chromatographic steps. The mature form of BtsTLP1 has a molecule mass of 35.6 kDa as determined by mass spectrometry analyses. The biochemical characterization indicates that BtsTLP1 has an apparent K_m value of 1.57 mg/ml for azocasein and is active between 20℃ and 80℃. Unlike other reported neutral gram-positive thermolysin homologs with optimal pH around 7, BtsTLP1 exhibits an alkaline pH optimum around 10. The activity of BtsTLP1 is strongly inhibited by EDTA and a group of specific divalent ions, with Zn²⁺ and Cu²⁺ showing particular effects in promoting the enzyme autolysis. Furthermore, our data also indicate that BtsTLP1 has potential in cleaning applications.

• International Journal of Industrial Entomology and Biomaterials
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• 제48권1호
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• pp.42-47
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• 2024

The value of silkworms as functional health food materials has increased, as has the interest in its disease control for stable production, and in the economic value of entomopathogenic microorganisms. In this study, we isolated and identified disease-causing fungi from white muscardine silkworms, and confirmed whether this strain could produce white muscardine silkworms. For the analysis of the cause of white muscardine disease in the infected silkworms, the fungi and prokaryotes causing the disease were identified, isolated, and identified using metagenome analysis. Metagenomic analysis detected a large amount of the fungus Metarhizium rileyi in silkworms, and a large amount of the bacterium Enterococcus mundtii, which was presumed to be the causative agent of the disease. For accurate identification of the fungi, these were purified by culture medium, and sequencing and phylogenetic tree analyses were performed using an internal transcribed spacer. As a result, M. rileyi, Cladosporium cladosporioides, and C. tenuissimum were identified. In general, M. rileyi is known to form green conidia, but in this study, white-yellow conidia were formed, indicating that the exact causative agent of the fungal disease cannot be estimated by diagnosing the symptoms. Thus, a diagnostic method is necessary for the continuously collection of required pathogens, and identifying their morphological and genetic characteristics.

• 대한본초학회지
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• 제39권2호
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• pp.1-9
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• 2024

Objectives : Paeng-hwal is described as an insect herbal medicine used for digestive diseases in the Dong-ui-bo-gam. The origin of this herbal medicine is limited to several small crabs, such as Helice tridens. These crab species cohabitat in the same environment and share similar morphological characteristics, making it very difficult to distinguish and collect the individual species for use in dietary supplements or herbal medicines. This study was conducted to develop a genetic identification tool for discriminating among these closely related small crab species. Methods : CO1 DNA barcode regions of 15 samples from 6 species of small crabs were analyzed to obtain the individual sequences. To identify the correct species, comparative analyses were carried out using the database of the NCBI GenBank and the NIBR. SCAR primers were designed to develop simple and rapid assay methods using inter-species specific sequences. Optimal SCAR assay conditions were established through gradient PCR, and the limit of detection (LOD) was determined. Results : Six species of small crabs (Helicana tridens, Macrophthalmus abbreviatus, Helicana tientsinensis, Helicana wuana, Chiromantes dehaani, and Hemigrapsus penicillatus), which are distributed as Paeng-hwal, were identified through CO1 sequences analysis. We also developed SCAR markers to distinguish between six small crabs at the species level. Furthermore, we established the optimal PCR assay methods and the LOD of each individual species. Conclusions : The rapid and simple SCAR-PCR assay methods were developed to identify the species and control the quality of herbal medicines for Paeng-hwal based on the genetic analyses of CO1 DNA barcodes.