• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.131.1-131
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• 2003

This study reports the identification of species of Colletotrichum strains originating from red-pepper and Chinese matrimony vine in Cheongyang. Ninteen isolates of red-pepper and 26 Coiletotrichum isolates of Chinese matrimony vine were compared with 5 isolates of strawberry representing C. gloeosporioides, by use of morphological and cultural criteria. Twenty three isolates among 26 isolates from Chinese matrimony vine were identified as C. acutatum, characterized by the low growth rates and the low sensitivity to carbendazim and diethofencarb. Also, all the isolates of red-pepper were identified as C. acutatum, showing the same characteristics as those of Chinese matrimony vine. Three and five isolates from Chinese matrimony vine and strawberry, respectively, were identified as C. gloeosporioides, characterized by the high growth rates and the high seneitivity to carbendazim and diethofencarb. There were differences in colony color and pathogenicity between Chinese matrimony vine isolates and red-pepper isolates of C. autatum. The isolates of C. acutatum from Chinese matrimony vine producing orange colored colonies with abundant spores showed the strong pathogenicity to Chinese matrimony vine, although they could not infect fruits of red-pepper by the wound inoculation. However, red-pepper isolates of C. acutatum producing gray colonies showed the strong pathogenicity to Chinese matrimony vine as well as red-pepper. Furthermore, comparative study on PCR amplification of ITS regions of rDNA was carried out using a number of Colletotrichum isolates. A species-specific primer could be used for the identification of C. acutatum from red-pepper and Chinese matrimony vine.

• Applied Biological Chemistry
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• 제47권2호
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• pp.182-186
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• 2004

아바스큘라 내생균근(arbuscular mycorrhizae, AM) 균은 대부분의 육상식물 뿌리와 공생하며, 식물의 성장에 도움을 주는 균이다. 인삼은 다년생 식물로서 뿌리를 약재로 사용하는 대표적인 약재 중 하나이다. 본 연구에서는 우리나라 8개 지역에서 인삼을 채집하여 AM 균을 염색을 통하여 형태적으로 관찰하고, 분자생물학적인 방법을 사용하여 뿌리내에 공생하는 다양한 AM 균을 확인하였다. 인삼의 뿌리에 감염되어 있는 AM 균을 염색하여 형태적으로 관찰한 결과 감염률이 낮고 흐리게 염색되었다. 또한 균사와 vesicle 이 발견되었고 이들은 Arum type 으로 판단되지만 arbuscule을 관찰하지 못했기 때문에 Arum-type으로 단정하기는 어려웠다. 식물 내에 공생하는 AM균들의 종류를 확인하기 위하여 채집된 인삼의 뿌리에서 내생균근 균의 DNA를 AM균에 특이적인 18s rDNA primer를 이용한 nested PCR을 수행하여 AM 균의 18S rDNA중 일부를 확보하였다. 증폭된 DNA는 클로닝을 통해서 개별 내생균근 균 종의 염기서열들로 분리하였고, 이들은 AluI, HinfI과 같은 제한 효소를 사용하여 RELP하였다. RELP 패턴에 따라 그룹을 나누어, 각 그룹에서 1개씩 염기서열 분석을 수행하였다. 염기서열은 기존의 서열과의 유사성과 계통 관계의 분석을 통하여 모두 AM균인 것으로 확인되었고, 다음 2개의 종과 가장 유사한 것으로 판단되었다; Paraglomus brasilianum, Glomus spurcum이 중 Paraglomus에 속하는 종인 P. brasil-ianum. 이 인삼의 뿌리에서 공통적으로 관찰되어 이들 균과 인삼과의 특이적 관계에 관하여 추측할 수 있었고, G. spurcum은 유사한 것으로 분석된 염기서열들이 계통도 상에서 특이한 분지를 형성하는 것으로 나타났는데, 이들 분지에 대해서는 확충된 염기서열들과 비교 분석과 같은 연구가 필요한 것으로 생각된다.

• Parasites, Hosts and Diseases
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• 제51권3호
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• pp.269-277
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• 2013

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

• Mycobiology
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• 제48권4호
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• pp.313-320
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• 2020

In Pleurotus sp., green mold, which is considered a major epidemic, is caused by several Trichoderma species. To develop a rapid molecular marker specific for Trichoderma spp. that potentially cause green mold, eleven Trichoderma species were collected from mushroom farms and the Korean Agricultural Culture Collection (KACC). A dominant fungal isolate from a green mold-infected substrate was identified as Trichoderma pleuroticola based on the sequences of its internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1) genes. In artificial inoculation tests, all Trichoderma spp., including T. atroviride, T. cf. virens, T. citrinoviride, T. harzianum, T. koningii, T. longibrachiatum, T. pleurotum, and T. pleuroticola, showed pathogenicity to some extent, and the observed symptoms were soaked mycelia with a red-brown pigment and retarded mycelium regeneration. A molecular marker was developed for the rapid detection of wide range of Trichoderma spp. based on the DNA sequence alignment of the ITS1 and ITS2 regions of Trichoderma spp. The developed primer set detected only Trichoderma spp., and no cross reactivity with edible mushrooms was observed. The detection limits for the PCR assay of T. harzianum (KACC40558), T. pleurotum (KACC44537), and T. pleuroticola (CAF-TP3) were found to be 500, 50, and 5 fg, respectively, and the detection limit for the pathogen-to-host ratio was approximately 1:10,000 (wt/wt).

• 한국자원식물학회지
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• 제34권1호
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• pp.1-16
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• 2021

남부 지방의 밀양 근교에 자생하고 있는 야생 동충하초 균주(Cordyceps sp., Paecilomyces sp., Beauveria sp., Aranthomyces sp., Isaria sp., Himenostilbe sp.)를 채집 분리하여 rDNA 및 internal transcribed spacer (ITS) 부위의 염기서열을 비교하였다. ITS 영역에 특정적인 프라이머인 ITS1과 ITS4를 이용하여 PCR을 수행하여 증폭하였다. 다양한 균주에서 같은 크기의 PCR 생산물을 얻을 수 있었고, 이들의 서열분석을 위하여 pGEM-T easy 벡터에 클로닝하였으며, ITS1, 5.8S, ITS2 영역 부위의 염기서열들을 BLAST 수행하여 유연관계를 분석하였다. 밀양 근교에서 분리한 32개의 균주 중에 Cordyceps militaris는 서열이 등록된 유전정보 AY49191, EU825999, AY491992와 100% 일치하였으며, 몇 개의 종은 보고된 서열과 모두 일치하지는 않았다. 예를 들어 strain P17은 울주군 가지산에서 분리한 P. tenuipes로서 밀양시 조천읍 가지산에서 분리한 P. tenuipes와는 서열상에서 다른 부분들이 있었다. 결론적으로 밀양 근교의 균주들을 분리하는데 ITS영역분석이 분류와 검정에 효율적이고, 본 연구를 통하여 동충하초의 생태학적인 유전자원들을 확보할 수 있었다.

• 환경생물
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• 제32권4호
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• pp.382-388
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• 2014

최근 뱀장어 양식장에서 사육 중이던 동남아산 뱀장어에 몸통 근육의 요철현상을 나타내면서 폐사를 일으키는 질병이 발생하였다. 병어의 몸통 근육 내 환부는 흰색 또는 황색으로 변해있었다. 병리조직학적인 변화는 근조직내에 수많은 포자와 크고 작은 시스트들이 변형된 근육근섬유 내에 관찰되었다. 병어의 근육 환부를 취하여 PCR을 실시하여 H. anguillarum이 가지는 특정 유전자인 Small subunit ribosomal RNA (SSU-rRNA)를 증폭하였다. 좀 더 정확한 동정을 위해 PCR product를 Cloning 후 Sequencing하여 서열들을 분석한 결과 H. anguillarum의 Small subunit ribosomal RNA (Gene bank accession number: AB623036) 서열과 일치하게 나타남을 확인할 수 있었다. primer 18F과 1537R의 PCR product는 약 1366 bp 길이가 일치하였으며, V1과 1392R를 이용한 PCR product는 1200 bp가 일치하게 나타났다. 또한 H1과 H2의 PCR product의 경우는 800 bp 정도 일치하였으며 이의 서열은 나머지 2개의 product가 공통적으로 가지고 있는 서열이었다. 이를 토대로 본 연구에서는 동남아산 뱀장어에 감염된 포자충은 H. anguillarum임을 동정할 수 있었다.

• 한국식품위생안전성학회지
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• 제35권3호
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• pp.234-242
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• 2020

본 연구는 최근 소비가 크게 증가하고 있는 가정간편식의 원료에 대한 모니터링을 수행하였다. 다양한 유형의 가정간편식 제품을 구입하여 112개 원료의 DNA 바코드를 분석하였다. 원재료의 종을 동정하기 위하여 DNA 바코드 증폭에 주로 이용되는 미토콘드리아의 16S ribosomal RNA 유전자 부위를 증폭하는 프라이머 세트를 이용하였다. PCR 산물은 정제하여 염기서열을 분석한 후, 이를 이용하여 미국국립보건원에서 제공하는 BLAST search를 수행하였다. GenBank에 등록되어 있는 종의 염기서열과 유사도(Identity)와 매치 점수(Match score)를 비교하여 원료의 종을 판별하였다. 112개의 원료에서 24개의 종(Species)과 3개의 속(Genus)를 동정하였다. 3개의 속은 Identity의 기준이 되는 98% 이내에 해당하는 종이 다수 존재하여 속 수준에서 판별하였다. 판별 결과를 「식품의 기준 및 규격(제2019-57호)」 중 '(별표 1) 사용할 수 있는 원료 목록'에서 제시하는 사용 가능한 원료와 비교하여 국명 및 섭취 가능 여부를 판단하였으며, 등재되어 있지 않은 6개 종은 국제적으로 공인된 기구에서 어획량에 대한 정보를 확인하고, 식용 근거, 학명·이명 등을 확인하여 식용 가능 여부를 판단하였다.

• The Plant Pathology Journal
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• 제33권1호
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• pp.43-52
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• 2017

A survey was conducted to determine the status of Lucerne transient streak virus (LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh, Qassim, and Hail) during 2014. Three hundred and eight symptomatic alfalfa, and seven Sonchus oleraceus samples were collected. DAS-ELISA indicated that 59 of these samples were positive to LTSV. Two isolates of LTSV from each region were selected for molecular studies. RT-PCR confirmed the presence of LTSV in the selected samples using a specific primer pair. Percentage identity and homology tree comparisons revealed that all Saudi isolates were more closely related to each other but also closely related to the Canadian isolate-JQ782213 (97.1-97.6%) and the New Zealand isolate-U31286 (95.8-97.1%). Comparing Saudi isolates of LTSV with ten other sobemoviruses based on the coat protein gene sequences confirmed the distant relationship between them. Eleven out of fourteen plant species used in host range study were positive to LTSV. This is the first time to document that Trifolium alexandrinum, Nicotiana occidentalis, Chenopodium glaucum, and Lathyrus sativus are new host plant species for LTSV and that N. occidentalis being a good propagative host for it.

• 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.

• ALGAE
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• 제36권1호
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• pp.37-50
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• 2021

Heterotrophic dinoflagellates Gyrodinium spp. are one of the major grazers of phytoplankton in many coastal waters. Gyrodinium dominans, G. jinhaense, and G. moestrupii have similar morphologies but different edible prey species. To explore the variations in the ecological niches of these three species, we investigated their spatial-temporal distributions in Korean waters. Because of the high similarity in morphology among these three Gyrodinium species, we used real-time polymerase chain reactions to quantify their abundance in water samples that were seasonally collected from 28 stations along the Korean Peninsula from April 2015 to October 2018. Cells of G. dominans were found at all sampling stations, G. jinhaense at 26 stations, and G. moestrupii at 22 stations, indicating that all three species were widely distributed in Korea. Furthermore, all three species displayed strong seasonal distributions. The largest numbers of the stations where G. dominans and G. jinhaense cells were present were found during the summer (26 and 23 stations, respectively), but that for G. moestrupii was found in the autumn (15 stations). The abundance of G. dominans was positively correlated with that of G. jinhaense, but not with that of G. moestrupii. The highest abundances of G. dominans (202.5 cells mL-1) and G. jinhaense (20.2 cells mL-1) were much greater than that of G. moestrupii (1.2 cells mL-1). The highest abundances of G. dominans and G. jinhaense were found in July, whereas that of G. moestrupii was found in March. The abundances of G. dominans and G. jinhaense, but not G. moestrupii, were positively correlated with water temperature. Therefore, the spatial-temporal distributions of G. dominans and G. jinhaense were closer than those of G. moestrupii and G. dominans or G. jinhaense. This differs from results based on the relative differences in ribosomal DNA sequences and the types of edible prey reported in the literature. Thus, the variations in spatial-temporal distributions and prey species of these three Gyrodinium species suggest that they may have different ecological niches in Korean coastal waters.