• Mycobiology
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• 제49권5호
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• pp.461-468
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• 2021

The genus Laccaria (Hydnangiaceae, Agaricales) plays an important role in forest ecosystems as an ectomycorrhizal fungus, contributing to nutrient cycles through symbiosis with many types of trees. Though understanding Laccaria diversity and distribution patterns, as well as its association with host plants, is fundamental to constructing a balanced plant diversity and conducting effective forest management, previous studies have not been effective in accurately investigating, as they relied heavily on specimen collection alone. To investigate the true diversity and distribution pattern of Laccaria species and determine their host types, we used four different approaches: specimen-based analysis, open database search (ODS), NGS analysis, and species-specific PCR (SSP). As a result, 14 Laccaria species have been confirmed in Korea. Results regarding the species distribution pattern were different between specimen-based analysis and SSP. However, when both were integrated, the exact distribution pattern of each Laccaria species was determined. In addition, the SSP revealed that many Laccaria species have a wide range of host types. This study shows that using these four different approaches is useful in determining the diversity, distribution, and host of ECM fungi. Furthermore, results obtained for Laccaria will serve as a baseline to help understand the role of ECM fungi in forest management in response to climate change.

• The Plant Pathology Journal
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• 제15권3호
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• pp.137-143
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• 1999

A novel chromosomal gene tagging technique using a specific fragment of the fatty acid desaturase-like open reading frame (des-like ORF) from the tox-argK gene cluster of Pseudomonas syringae pv. phaseolicola was developed to identify Xanthomonas spp.released into the environment as biocontrol agents. X. campestris pv. convolvuli FB-635, a pathogen of Convolvulus arvensis L., (bindweed), was chosen as the organism in which to develop and test the system. A 0.52 kb DES fragment amplified from P. syringae pv. phaseolicola C-199 was inserted into pGX15, a cosmid clone containing a 10.3 kb Eco RI-HindIII fragment derived from the xanthomonadin biosynthetic gene cluster contained in plasmid pIG102, to create a pigG::DES insertion. The 10.8 kb EcoRI-BamHI fragment carrying the pigG:: DES insertion was cloned into pLAFR3 to generate pLXP22. pLXP22 was then conjugated into X. campestris pv. convolvuli FB-635 and the pigG::DES insertion integrated into the bacterial chromosome by marker exchange. Rifampicin resistant, tetracycline sensitive, starch hydrolyzing, white colonies were used to differentiate the marked strain from yellow pigmented wild-type ones. PCR primers specific for the unique DES fragment were used for direct detection of the marked strain. Result showed the marked strain could be detected at very low levels even in the presence of high levels of other closely related or competitive bacteria. This PCR-based DES-tagging system provides a rapid and specific tool for directly monitoring the dispersal and persistence of Xanthomonas spp.released into the environment.

• Fisheries and Aquatic Sciences
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• 제6권1호
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• pp.13-19
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• 2003

Genomic DNA from the muscle of marsh clam (Corbicula leana) from Gochang, Muan and a Chinese site was extracted to identify genetic differences and similarity by randomly amplified polymorphic DNAs-polymerase chain reaction (RAPD- PCR). Out of 20 primers, seven primers produced amplified fragments which were consistently polymorphic. A total of 1,246 amplified products were produced of which 530 were polymorphic $(42.5\%)$. The number of polymorphic bands produced per primer varied from 40 to 122 with an average of 75.7 in marsh clam from Gochang. 3.28 of the 23.0 polymorphic bands per lane were found to be polymorphic. Also, about $4.34\%$ of total polymorphic bands were specific to marsh clam from Gochang. The major common bands of 0.28 kb generated by primer OPB-15 (GGAGGGTGTT) were present in every individuals, which were polymorphic. This common bands in every individuals should be diagnostic of specific strains, species and/or their relatedness. Primer OPB-19 (ACCCCCGAAG) produced the highest number of 12 specific bands. The intra-population variation was revealed in the band patterns identified by this primer. The random primer OPB-12 (CCTTGACGCA) yielded the amplified fragments which were consistently polymorphic between the marsh clams from Gochang and from Muan. This primer produced a total of 77 polymorphic bands: 31 bands from Gochang, 14 from Muan and 32 from the Chinese populations. An average of polymorphic bands were 1.8 from Gochang and 2.5 from the Chinese populations. This value obtained from the Chinese population was higher than those from the two domestic populations. Generally, the RAPD polymorphism generated by these primers may be useful as a genetic marker for strain or population identification of marsh clam.

• The Plant Pathology Journal
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• 제29권1호
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• pp.99-104
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• 2013

To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was $51.9^{\circ}C$. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.

• 한국균학회지
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• 제26권4호통권87호
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• pp.574-582
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• 1998

1996년부터 속리산 법주사 주변과 그 외의 지역에서 Pholiota species 5종을 채집하여 Pholiota adiposa와 Pholiota species로 동정하였다. 이 버섯들을 다른 버섯들과 random primer에 의한 RAPD를 실시하여 형태적 분류와 연관시켜 본 결과, primer #28(1.5kb와 1.0kb 사이)에서 Pholiota adiposa와 Pholiota sp.의 공통밴드가 형성되었고, primer #36(750과 500bp 사이)와 OPD-18(760 bp)에서는 검은비늘버섯만의 특이적 밴드가 나타났다. 모균주와 이를 접종원으로 하여 수확한 자실체의 DNA에서는 동일한 모양의 다형적 밴드가 형성되었다. 또한 채집된 5종의 검은비늘버섯은 약간씩 다른 밴드적 차이를 나타내었다. 검은비늘버섯의 포자지문법을 통한 단포자분리에 의한 mating 결과 tetrapolar형을 관찰하였는데, 이는 Arita와 Mimura(1969)가 이미 P. adiposa와 P. nameko가 bipolar 형이라고 한 것과 다른 결과이다. 본 실험은 PCR-RAPD를 이용하여 하나의 버섯에서 얻은 담자포자의 단핵(monokaryon)과 이핵(dikaryon) 균사간의 차이를 보기 위한 기초실험이며, 또한 육종 계획에 대한 응용을 위한 실험이다.

• Journal of Plant Biotechnology
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• 제49권3호
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

• Applied Biological Chemistry
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• 제48권4호
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• pp.335-338
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• 2005

국내에서 개발된 GM 쌀인 밀양 204호, 익산 483호의 정성 PCR 분석법의 개발을 위해 쌀의 내재 유전자로써 SAMDC1(S-adenosylmethionine decarboxylase)에 특이적인 primer (OsSAMDC1-5'/3')쌍을 제작하여 쌀을 포함한 20개 작물에 대해 PCR을 수행하여 쌀에 특이적으로 증폭되는 것을 확인하였다. 또한, 밀양 204호에 삽입된 GUS 유전자와 NOS terminator 연결 부위를 증폭시켜 172 bp의 PCR 산물을 얻을 수 있는 primer(Os204-5'/OsNOS-3')와 익산 483호에 삽입된 bar 유전자와 NOS terminator 연결 부위를 증폭시켜 161 bp의 PCR 산물을 얻을 수 있는 primer(Os483-5'/OsNOS-3')을 이용하여 국내 개발된 GM 쌀인 밀양 204호, 익산 483호의 PCR 정성 분석법을 확립하였다.

• 한국자원식물학회지
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• 제27권6호
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• pp.680-686
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• 2014

Acanthopanacis Cortex has been used for oriental medicinal purposes in Asian countries especially in Korea and China. In the Korean Pharmacopeia, the cortexes of the dried roots, stems and branches of all species in Eleutherococcus and Eleutherococcus sessiliflorus are known as 'Ogapi'. Mostly the cortexes of E. gracilistylus roots and E.senticosus roots were used as 'Ogapi' in China and Japan, respectively. Therefore, the purpose of this study was to determine and compare the molecular authentication of Korean 'Ogapi' by using the ribosomal internal transcribed spacer (ITS) region. The ITS region has the highest possibility of effective and successful identification for the widest variety of molecular authentication. The ITS region was targeted for molecular analysis with Single nucleotide polymorphisms (SNPs) specific for morphologically similar to E. gracilistylus, E. senticosus, E. sessiliflorus from their adulterant, moreover, E. sieboldianus were detected within sequence data. Thus, based on these SNP sites, specific primers were designed and multiplex PCR analysis were conducted for molecular authentication of four plants (E. gracilistylus, E. senticosus, E. sessiliflorus, and E. sieboldianus). The findings of results indicated that ITS region might be established multiplex-PCR analysis systems and hence were proved to be an effective tools for molecular evaluation and comparison of 'Ogapi' with other plants.

• Journal of Microbiology and Biotechnology
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• 제31권2호
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• pp.280-289
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• 2021

Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

• The Plant Pathology Journal
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• 제38권6호
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• pp.629-636
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• 2022

Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 ㎍/ml of benomyl. The sequence analysis of the β-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.