Survey of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus incidence in Korea by Duplex RT-PCR

이승열,예미지,백창기,최광식,강인규,이수헌,정희영 한국식물병리학회 2014 Plant Pathology Journal Vol.30 No.4

The incidence of Cherry necrotic rusty mottle virus(CNRMV) and Cherry green ring mottle virus (CGRMV)have recently been occurred in Korea, posing a problemfor sweet cherry cultivation. Since infected trees havesymptomless leaves or ring-like spots on the pericarp,it is difficult to identify a viral infection. In this study,the incidence of CNRMV and CGRMV in sweet cherryin Gyeongbuk province was surveyed using a newlydeveloped duplex reverse transcriptase polymerasechain reaction (RT-PCR) method that can detect bothviruses in a single reaction. CNRMV and CGRMVco-infection rates were 29.6%, 53.6%, and 17.6%,respectively, in samples collected from three differentsites (Daegu, Gyeongju and Gyeongsan) in Gyeongbukprovince during 2012 and 2013. This duplex RT-PCRmethod offers a simple, rapid, and effective way ofidentifying CNRMV and CGRMV simultaneously insweet cherry trees, which can aid in the management ofviral infections that could undermine yield.

Immunocapture RT-PCR을 이용한 박과작물 종자전염 바이러스의 검출

이혁인,김정희,예미지 한국식물병리학회 2010 식물병연구 Vol.16 No.2

Immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) was applied to the detection of Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), and Zucchini green mottle mosaic virus (ZGMMV) on Cucurbitaceae crops. These seed-borne tobamoviruses were accurately detected from the infected leaves and seeds by IC-RT-PCR. This method was estimated to be about 100 times more sensitive than ELISA, and also it allowed the direct confirmation of ELISA results by using the captured antigens from a completed ELISA microwell. This convenient and reliable method could be used routinely for large-scale field surveys or seed tests of Cucurbitaceae crops. 박과작물에 발생하는 종자전염 바이러스 3종(CGMMV, KGMMV, ZGMMV)에 대한 검출을 위해 IC-RT-PCR의 적용 가능성을 시험하였다. IC-RT-PCR을 이용하여 감염 종자와 잎으로 부터 바이러스의 특이적인 검출이 가능하였으며, 검출 민감도는 ELISA 보다 100배 이상 높았다. 또한 반응을 마친 ELISA 마이크로플레이트에 남아있는 항원을 IC-RT-PCR의 template로 사용할 경우 ELISA 결과를 곧바로 확인할 수 있었다. 따라서 IC-RT-PCR에 의한 본 검정방법은 박과작물의 대규모 포장검사 및 종자검사에 편리하게 사용될 수 있을 것으로 기대된다.

Polymerase Chain Reaction(PCR)을 이용한 오동나무, 라일락, 미역취의 Phytoplasma 검출 및 유연 관계

이준탁,김은화,예미지,권오유 한국식물병리학회 1996 Plant Pathology Journal Vol.12 No.2

칠레산 수입포도 ‘레드글로브’ 품종에 발생한 부패병 병원균의 동정

송민지,이혁인,예미지,김대호,홍승범,차재순 한국식물병리학회 2012 식물병연구 Vol.18 No.3

Post-harvest rot of grape causes a severe economic loss and lower of the grape quality. It is also one of the important limiting factors for grape export. Grape rots and their casual agents on ‘Red Globe’ variety imported from Chile were identified. Grapes shown rotting symptom were collected from the storages near the import harbor. The 3 different rots were identified on the imported ‘Red Globe’; melting decay, gray mold, and blue mold. A bacterium that isolated from a typical melting decay symptom was identified as Gluconobacter cerinus on basis of its nucleotide sequence of 16S rDNA and fatty acid profile. By inoculation on grape, it caused cracking and dissolution of epidermis of grape which were the characteristics of melting decay. Botrytis cinerea and Penicillium expansum were isolated from grapes showing gray mold and blue mold. The 2 fungal isolates were identified on basis of their morphological characteristics and nucleotide sequence of their beta-tubulin genes. They showed strong pathogenicity on ‘Campbell Early’ variety that is a major table grape in Korea. 0

토마토 종자로부터 PCR을 이용한 Pseudomonas syringae pv. tomato의 검출

조정희,임규옥,이혁인,예미지,차재순 한국식물병리학회 2011 식물병연구 Vol.17 No.3

The bacterial speck of tomato caused by Pseudomonas syringae pv. tomato leads to serious economic losses especially on fruits of susceptible genotype. Thus, Pseudomonas syringae pv. tomato is a plant quarantine bacterium in many countries including Korea. In this study, we developed specific PCR assays for detection of the bacterium from tomato seeds. A specific primer set is designed from the hrpZ gene for specific detection of Pseudomonas syringae pv. tomato. A 501 bp PCR product corresponding to hrpZ gene was amplified only form Pseudomonas syringae pv. tomato strains, but no PCR product was amplified from other tomato bacterial pathogens, such as Pseudomonas syringae pv. glycinea, P. syringae pv. maculicola, P. syringae pv. atropurpurea, P. syringae pv. morsprunorum, and from other P. syringae pathovar strains. The nested-PCR primer set corresponding to an internal fragment of the 501 bp sequence (hrpZ) gine was used to specific detection of Pseudomonas syringae pv. tomato in tomato seed. A 119 bp PCR product using nested PCR primer was highly specific and sensitive to detect low level of Pseudomonas syrigae pv. tomato in tomato seeds. We believe that the PCR assays developed in this study is very useful to detect Pseudomonas syringae pv. tomato from the tomato seeds. P. syringae pv. tomato는 토마토에서 bacterial speck병을 일으키는 종자전염 세균으로, 감수성 품종에서 주로 발병하여 경제적으로 큰 손실을 입힌다. 따라서 P. syringae pv. tomato는 한국을 비롯한 많은 나라에서 식물 검역대상 세균으로 지정하여 관리되고 있다. 본 연구에서 우리는 토마토 종자로부터 PCR을 이용하여 Pst를 검출할 수 있는 방법을 개발하였다. P. syringae pv. tomato의 hrpZ 유전자에서 특이적인 프라이머를 개발하였다. 개발된 프라이머는 P. syringae pv. tomato에서만 501 bp 크기의 특이적 DNA를 증폭하였으며, P. syringae pv. glycinea, P. syringae pv. maculicola, P. syringae pv. atropurpurea, P. syringae pv. morsprunorum와 같은 다른 토마토 세균병원균과 P. syringae pathovar 균주들에서는 증폭되지 않았다. Nested PCR 프라이머를 이용한 PCR에서도 오직 P. syringae pv. tomato에서만 119 bp 크기의 특이적 DNA가 증폭되었고, 토마토 종자에서 P. syringae pv. tomato을 정확하고 민감하게 검출하였다. 본 연구는 현재까지 사용되고 있는 Pst의 검출방법의 민감도를 비교한 최초의 보고로 본 연구에서 개발된 PCR방법들은 토마토 종자에서 Pst을 검출하는 매우 유용한 방법으로 생각된다.

Development of a Species-specific PCR Assay for Three Xanthomonas Species, Causing Bulb and Flower Diseases, Based on Their Genome Sequences

백창기,이승열,이부자,예미지,김상목,강인규,차재순,정희영 한국식물병리학회 2015 Plant Pathology Journal Vol.31 No.3

In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP), X. hyacinthi (XH) and X. campestris pv. zantedeschiae (XCZ), based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of 1 pg/μl per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

사과나무에서 가지검은마름병 억제를 위한 효율적 가지치기

한규석,유지강,이한별,오창식,예미지,이종호,박덕환 한국식물병리학회 2016 식물병연구 Vol.22 No.4

Black shoot blight disease caused by Erwinia pyrifoliae have damaged economic loss to apple and pear growers until now since it was firstly reported in 1995 in Korea. This study was performed to reduce economic loss by mandatory eradication of all infected trees in case of more 10% disease incidence per orchard as official control. It also aims to set up effective management protocol for this disease by examining how far bacterial pathogen is present from the border of symptomatic and asymptomatic regions in infected apple twigs. Colony-PCR using isolated bacterial cells instead of genomic DNA was used to identify bacterial pathogen, EpSPF/EpSPR primer designed in enterobacterial repetitive intergenic consensus (ERIC) region was selected as specific for E. pyrifoliae. As results of monitoring of this disease during April to October in 2014–2015 by colony-PCR, occurrence of this disease was frequent from mid-May to early-July, when daily average temperature was around 25oC. Moreover, bacterial cells were continuously detected only in symptomatic regions and also asymptomatic regions of less than 20 cm from symptomatic regions. Therefore, we concluded that pruning of infected twigs at the region of more than 20 cm from symptomatic regions might be effective to manage black shoot blight disease in apple trees.

Validation of A Multiplex PCR Detection Kit for Screening of Herbicide-Tolerant Genes in Genetically Modified Crops

김재환,김해영,Eun-Hee Kim,예미지 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.2

A multiplex polymerase chain reaction (PCR) detection kit for screening of four herbicide-tolerant genes (cp4 epsps,mepsps, pat, and bar) in genetically modified (GM) crops was developed. The kit was validated by three different laboratories,and the expected targets were specifically observed in 14 different herbicide-tolerant GM events. This method can be effectively and conveniently used to monitor approved and unapproved GM crops containing four herbicide-tolerant genes.

Duplex PCR을 이용한 국내 미승인 유전자변형 감자(EH92-527-1)의 검사법 개발

유명렬(Myung-Ryul Yoo),김재환(Jae-Hwan Kim),예미지(Mi-Chi Yea),김해영(Hae-Yeong Kim) 한국식품과학회 2013 한국식품과학회지 Vol.45 No.2

우리나라에서 미승인 품목인 유전자변형 감자 EH92-527-1를 검출하기 위한 duplex PCR 검사법이 개발되었다. 감자의 내재유전자로 UDP-glucose pyrophosphorylase (UGP)가 선별되었고, 14개 다른 작물을 이용하여 특이성이 확인되었다. 유전자변형 감자에 삽입된 T-DNA 영역과 감자 게놈 사이의 연결 부위를 증폭하도록 프라이머 EH92-F/R 쌍이 제작되었고, 몇 개의 다른 유전자변형 작물을 이용하여 특이성이 확인되었다. 서론에서 언급한 바와 같이 BASF사에서 각 개발된 유전자변형 감자 EH92-527-1과 BPS-A1020-5가 GBSS 유전자를 동일하게 포함하고 있으나 본 연구에서 개발한 검사법은 event-specific primers를 이용하였기 때문에 유전자변형 감자 EH92-527-1에만 특이성을 나타낸다. 이와 같이 개발된 duplex PCR 검사법의 검정한계치는 약 0.05%이다. 이러한 duplex PCR 검사법이 우리나라에 미승인 유전자변형 감자의 모니터링에 유용하게 사용될 것으로 판단한다. A duplex polymerase chain reaction (PCR) method was developed to detect unapproved genetically modified (GM) potato (EH92-527-1) in Korea. The UDP-glucose pyrophosphorylase (UGP) gene was selected as an endogenous reference gene for potato and used to validate the specificity for 14 different crops. The primer pair EH92-F/R was designed to amplify the junction sequence between the genome and transgenic region introduced in GM potato. Its specificity was also validated using several different GM events. The detection limit of the duplex PCR method is approximately 0.05%. This duplex PCR method could be useful for monitoring cultivation of unauthorized GM potato in Korea.

Multiplex PCR Assay for Simultaneous Detection of Korean Quarantine Phytoplasmas

Young-Hwan Kim,정희영,Nang Kyu Win,Chang-Gi Back,예미지,임규옥 한국식물병리학회 2011 Plant Pathology Journal Vol.27 No.4

Multiplex PCR assays were developed for the simultaneous detection of ten important Korean quarantine phytoplasmas. The species-specific primers were designed based on ribosomal protein, putative preprotein translocase Y, immunodominant protein, elongation factor TU, chaperonin protein and the 16S rRNA genes of ‘Candidatus (Ca.) Phytoplasma’ species. Three main primer sets were prepared from ten designed primer pairs to limit nonspecific amplification as much as possible. The multiplex PCR assay using the three primer sets successfully amplified the correct conserved genes for each ‘Ca. Phytoplasma’ species. In addition,ten important ‘Ca. Phytoplasma’ species could be easily determined by recognizing band patterns specific for each phytoplasma species from three primer sets. Moreover, a high sensitivity of multiplex PCR for each primer set was observed for samples containing a low DNA concentration (10 ng/μl). This study provides the useful multiplex PCR assay as a convenient method to detect the presence of ten important quarantine phytoplasmas in Korea.