• Research in Plant Disease
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• v.29 no.1
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• pp.94-99
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• 2023

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

• Microbiology and Biotechnology Letters
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• v.34 no.2
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• pp.101-108
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• 2006

YNB54 strain which shows inhibitory activities specific to the plant pathogenic Phytophthora sp. on potato dextrose agar medium was screened among lots of strains isolated from Korean soils. To identify taxonomy of the Phytophthora specific antagonistic bacteria YNB54, 165 rDNA sequence, MIDI fatty acid composition, DNA-DNA hybridization, GC content, and commercial multitest systems such as API 20E and Biolog GN were performed. Results of commercial kits including lots of biochemical and physiological reactions showed that this strain was closely related to taxa including Enterobacter cloacae and Enterobacter cancerogenus species than other genera(Citerobacter Klebsiella, Leclercia). Also, analysis of its MIDI, G+C contents, and DNA-DNA hybridization suggests that this strain was more similiar to the Genus Enterobacter than other genera (Citerobacter Klebsiella, Leclercia). This strain was potentially identified as Enterobacter sp. by these results. But our 16S ribosomal DNA sequences (rDNA) analysis confirmed that it was more closely related to the cluster of Citerobacter freundii ATCC 29935 than any other Enterobacter species. In the absence of defined phylogenetic critia for delineating genera, the results observed with Citrobacter and Enterobacter species suggest that further studies are needed to clarify their relationships. This investigation demonstrates that YNB54 strain is genetically diverse and potentially more taxonomically complex than hitherto realized. Further study is necessary to confirm their taxonomic positions.

• Mycobiology
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• v.47 no.2
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• pp.173-179
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• 2019

Cladophialophora is characterized by simple hyphomycetes with brown hyphae that give rise to branched chains of pale brown conidia and shows affinities with the Herpotrichiellaceae. A fungal strain belonging to the genus Cladophialophora was isolated from soil in Daegu, Korea. This strain produces numerous greenish to dark black lanose aerial mycelia with hair like structures. It is morphological similar to C. chaetospira, C. inabaensis, and C. multiseptata; however, the conidiophores and conidia sizes of the newly isolated strain (KNU16-032) are clearly different from them. The novelty of the strain was also confirmed based on phylogenetic analysis using the data sets of the internal transcribed spacer region of and the partial sequence of 28S ribosomal DNA region along with the cultural characteristics. Because morphological observations and phylogenetic analysis indicated that the strain is distinct from previously known Cladophialophora species, we propose this species as a new species Cladophialophora lanosa sp. nov., and provide the detailed descriptions in this study.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.47-53
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• 2007

The DY1 strain of Gram-positive, rod-shaped bacteria was isolated from the soil sample collected from Daeam mountain, Korea. The culture filtrate of DY1 strain showed a broad spectrum of antimicrobial activity on various pathogenic and food poisoning enteric bacterial species tested in vitro. It showed significant growth-inhibitory effect on Salmonella enterica sp., Shigella sp., pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahemolyticus, and Yersinia enterocolitica. For the identification of the DY1 strain, morphological, biochemical and molecular phylogenetic approaches were performed. The DY1 strain was found to be a member of the genus Paenibacillus on the basis of morphological and biochemical analyses. The 16S rDNA of DY1 showed the highest pairwise identity with Paenibacillus polymyxa with 99.79% (1,413 bp/1,416 bp). The antimicrobial entity from DY1 looked different from preciously reported ones and seems to have a great potential to be further studied as a candidate of new antibiotics to control multi-drug resistant pathogens.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1537-1543
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• 2006

A bacterial strain named HY-3 that produces a highly active extracellular protease was isolated from the digestive tract of a spider, Nephila clavata. The bacterium was a Gram-negative, oxidase-negative, catalase-positive, nonhalophilic, nitrate-reducing, facultative anaerobe. Transmission and scanning electron microscopies demonstrated that the isolate was non-spare-forming, straight, rod-shaped, and motile by peritrichous flagella. The G+C content of the DNA was 57.0 mol%. The isoprenoid quinone type was ubiquinone with 8 isoprene units (Q-8). The morphological and biochemical characteristics including the predominant fatty acid and phospholipids profiles placed the isolate HY-3 in the family Enterobacteriaceae. Further biochemical characterization and phylogenetic studies including determination of an almost complete 16S ribosomal DNA sequence suggested that the bacterium was closely related to the genus Serratia. DNA-DNA hybridization analysis revealed that this extracellular protease-producing strain belongs to Serratia proteamaculans, which is also known far its association with insects.

• ALGAE
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• v.29 no.2
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• pp.75-99
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• 2014

A small dinoflagellate, Ansanella granifera gen. et sp. nov., was isolated from estuarine and marine waters, and examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. In addition, the identity of the sequences (3,663-bp product) of the small subunit (SSU), internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), and D1-D3 large subunit (LSU) rDNA were determined. This newly isolated, thin-walled dinoflagellate has a type E eyespot and a single elongated apical vesicle, and it is closely related to species belonging to the family Suessiaceae. A. granifera has 10-14 horizontal rows of amphiesmal vesicles, comparable to Biecheleria spp. and Biecheleriopsis adriatica, but greater in number than in other species of the family Suessiaceae. Unlike Biecheleria spp. and B. adriatica, A. granifera has grana-like thylakoids. Further, A. granifera lacks a nuclear fibrous connective, which is present in B. adriatica. B. adriatica and A. granifera also show a morphological difference in the shape of the margin of the cingulum. In A. granifera, the cingular margin formed a zigzag line, and in B. adriatica a straight line, especially on the dorsal side of the cell. The episome is conical with a round apex, whereas the hyposome is trapezoidal. Cells growing photosynthetically are $10.0-15.0{\mu}m$ long and $8.5-12.4{\mu}m$ wide. The cingulum is descending, the two ends displaced about its own width. Cells of A. granifera contain 5-8 peripheral chloroplasts, stalked pyrenoids, and a pusule system, but lack nuclear envelope chambers, a nuclear fibrous connective, lamellar body, rhizocysts, and a peduncle. The main accessory pigment is peridinin. The SSU, ITS regions, and D1-D3 LSU rDNA sequences differ by 1.2-7.4%, >8.8%, and >2.5%, respectively, from those of the other known genera in the order Suessiales. Moreover, the SSU rDNA sequence differed by 1-2% from that of the three most closely related species, Polarella glacialis, Pelagodinium bei, and Protodinium simplex. In addition, the ITS1-5.8S-ITS2 rDNA sequence differed by 16-19% from that of the three most closely related species, Gymnodinium corii, Pr. simplex, and Pel. bei, and the LSU rDNA sequence differed by 3-4% from that of the three most closely related species, Protodinium sp. CCMP419, B. adriatica, and Gymnodinium sp. CCMP425. A. granifera had a 51-base pair fragment in domain D2 of the large subunit of ribosomal DNA, which is absent in the genus Biecheleria. In the phylogenetic tree based on the SSU and LSU sequences, A. granifera is located in the large clade of the family Suessiaceae, but it forms an independent clade.

• Microbiology and Biotechnology Letters
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• v.43 no.2
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• pp.97-104
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• 2015

Vinegar is a widely used acidic seasoning and can be manufactured using various methods and bases, including cereals, wheat, and fruits. Most studies on vinegar have been conducted to evaluate its antioxidant activity. In the present study, fermented dark vinegar (FDV) produced from unpolished rice was examined for its antimicrobial activity, biochemical content, including the amounts of sugar, total soluble sugar, organic acid, and free amino acids, and pH and physiological activity. The antimicrobial efficiency of FDV was assessed using the paper disc-agar diffusion method. FDV exhibited strong antimicrobial activity against the pathogenic bacteria and yeast strains that were tested. In fact, the activity of FDV was shown to be higher than that of the commercial antibiotics carbenicillin (50 µg/ml) and tetracycline (50 µg/ml) against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Yersinia enterocolitica, and Lodderomyces elongisporus. The antioxidant activity of FDV and ascorbic acid was evaluated. Using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, we found that FDV has the highest activity of the antioxidants. After spreading FDV onto tryptic soy broth and yeast extract-peptone-dextrose agar media, the microbial strains were isolated and characterized through physiological and biochemical analysis. Based on 16S ribosomal DNA sequence analysis, the isolated microorganisms exhibited a close similarity to Acetobacter papayae, Acetobacter pasteurianus, and Acetobacter peroxidans.

• Korean Journal of Clinical Laboratory Science
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• v.49 no.2
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• pp.135-140
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• 2017

Polymerase chain reaction (PCR) was used to detect cattle material from processed meat products. Seventy-eight different commercial processed meat products were purchased from several big food marts. Among them, 17 products contained cattle material (10 samples contained only cattle, 5 samples mixed with cattle and porcine, 2 samples mixed with cattle, porcine and chicken). The genomic DNA was extracted directly from the processed meat products, and strain-specific primer targeting the 16S ribosomal RNA mitochondrial gene was used. All PCR products were cloned into the pGEM-T easy vector and sequenced. Consequently, the PCR products were amplified from 10 processed meat products, which contained only cattle material in our conditions. Furthermore, PCR reactions showed the same results at mixed samples. The DNA sequence obtained from pGEM-T easy/PCR products showed more than 95% identity with Bos taurus 16S rRNA gene using homology analysis. In conclusion, we suggest that the method using PCR, as performed in this study, could be useful in detecting cattle material in processed meat products. Moreover, our system could be applicable in inspection procedures to improve the verification of correct labeling for import and export processed meat products.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.283-294
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• 2007

Conventional culture-based methods of enumerating rumen microorganisms (bacteria, archaea, protozoa, and fungi) are being rapidly replaced by nucleic acid-based techniques which can be used to characterise complex microbial communities without incubation. The foundation of these techniques is 16S/18S rDNA sequence analysis which has provided a phylogenetically based classification scheme for enumeration and identification of microbial community members. While these analyses are very informative for determining the composition of the microbial community and monitoring changes in population size, they can only infer function based on these observations. The next step in functional analysis of the ecosystem is to measure how specific and, or, predominant members of the ecosystem are operating and interacting with other groups. It is also apparent that techniques which optimise the analysis of complex microbial communities rather than the detection of single organisms will need to address the issues of high throughput analysis using many primers/probes in a single sample. Nearly all the molecular ecological techniques are dependant upon the efficient extraction of high quality DNA/RNA representing the diversity of ruminal microbial communities. Recent reviews and technical manuals written on the subject of molecular microbial ecology of animals provide a broad perspective of the variety of techniques available and their potential application in the field of animal science which is beyond the scope of this treatise. This paper will focus on nucleic acid based molecular methods which have recently been developed for studying major functional groups (cellulolytic bacteria, protozoa, fungi and methanogens) of microorganisms that are important in nutritional studies, as well as, novel methods for studying microbial diversity and function from a genomics perspective.

• The Korea Journal of Herbology
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• v.30 no.3
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• pp.41-47
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• 2015

Objectives : Due to the morphological similarity of the pericarp and description of multi-species in National Pharmacopoeia of Korea and China, the Zanthoxylum Pericarpium is difficult to authenticate adulterant in species levels. Therefore, we introduced the sequence analysis of DNA barcode and identification of single nucleotide polymorphism(SNP) to establish a reliable tool for the distinction of Zanthoxylum Pericarpium from its adulterants. Methods : To analyze DNA barcode region, genomic DNA was extracted from twenty-four specimens of authentic Zanthoxylum species and inauthentic adulterant and the individual internal transcribed spacer regions (rDNA-ITS and ITS2) of nuclear ribosomal RNA gene were amplified using ITS1, ITS2-S2F, and ITS4 primer. For identification of species-specific sequences, a comparative analysis was performed using entire DNA barcode sequences. Results : In comparison of four Zanthoxylum ITS2 sequences, we identified 16, 4, 6, and 4 distinct species-specific nucleotides enough to distinguish Z. schinifolium, Z. bungeanum, Z. piperitum, and Z. simulans, respectively. The sequence differences were available genetic marker to discriminate four species. Futhermore, phylogenetic relationship revealed a clear classification between different Zanthoxylum species showing 4 different clusters. These results indicated that comparative analysis of ITS2 DNA barcode was an useful genetic marker to authenticate Zanthoxylum Pericarpium in species levels. Conclusions : The marker nucleotides, enough to distinguish Z. schinifolium, Z. piperitum, Z. bungeanum, and Z. simulans, were obtained at 30 SNP marker nucleotides from ITS2 sequences. These differences could be used to authenticate official Zanthoxylum Pericarpium from its adulterants as well as discriminating each four species.