• Title/Summary/Keyword: Molecular sequence analyses

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Identification and Characterization of Colletotrichum Species Associated with Bitter Rot Disease of Apple in South Korea

  • Oo, May Moe;Yoon, Ha-Yeon;Jang, Hyun A;Oh, Sang-Keun
    • The Plant Pathology Journal
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    • v.34 no.6
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    • pp.480-489
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    • 2018
  • Bitter rot caused by Colletotrichum species is a common fruit rotting disease of apple and one of the economically important disease in worldwide. In 2015 and 2016, distinct symptoms of bitter rot disease were observed in apple orchards in five regions of South Korea. In the present study, infected apples from these regions were utilized to obtain eighteen isolates of Colletotrichum spp. These isolates were identified and characterized according to their morphological characteristics and nucleotide sequence data of internal transcribed spacer regions and glyceraldehyde-3-phosphate-dehydrogenase. Molecular analyses suggested that the isolates of Colletotrichum causing the bitter rot disease in South Korea belong to 4 species: C. siamense; C. fructicola; C. fioriniae and C. nymphaeae. C. siamense and C. fructicola belonged to Musae Clade of C. gloeosporioides complex species while C. fioriniae and C. nymphaeae belonged to the Clade 3 and Clade 2 of C. acutatum complex species, respectively. Additionally, we also found that the isolates of C. gloeosporioides species-complex were more aggressive than those in the C. acutatum species complex via pathogenicity tests. Taken together, our results suggest that accurate identification of Colletotrichum spp. within each species complex is required for management of bitter rot disease on apple fruit in South Korea.

Note on a Marine Algal Species, Cryptonemia lomation (Halymeniaceae) in Korea

  • Kang, Pil Joon;An, Jae Woo;Nam, Ki Wan
    • Korean Journal of Environmental Biology
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    • v.36 no.3
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    • pp.308-313
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    • 2018
  • During a survey of marine algal flora, a red algal species was collected from Giseong, Uljin located on the eastern coast of Korea. This species has the generic features of Cryptonemia belonging to Halymeniaceae, and is characterized by the presence of erect foliose thalli arising from a discoid holdfast, somewhat fan-shaped blade with an evanescent midrib at the base, narrow main axes with blade-like wings of slightly undulate margin, a perennial stalk, and entwined filamentous medulla with refractive stellate cells. In a phylogenetic tree based on rbcL sequence, the Korean alga nests in the same clade with C. lomation from France and C. seminervis from Spain. Genetic divergence among the sequences within the clade was not recognized thus suggesting that both the species are conspecific. The name C. lomation considered to be valid nomenclaturally is accepted for the entity. Based on the morphological and molecular analyses, the Korean alga is identified as C. lomation, originally described from Italy. This confirms the occurrence of C. lomation in Korea. The species appears to be distributed in the temperate region influenced more or less by the North Korea Cold Current.

Paramyrothecium eichhorniae sp. nov., Causing Leaf Blight Disease of Water Hyacinth from Thailand

  • Pinruan, Umpawa;Unartngam, Jintana;Unartngam, Arm;Piyaboon, Orawan;Sommai, Sujinda;Khamsuntorn, Phongsawat
    • Mycobiology
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    • v.50 no.1
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    • pp.12-19
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    • 2022
  • Paramyrothecium eichhorniae sp. nov. was observed and collected from Chiang Mai and Phetchaburi Provinces, Thailand. This new species is introduced based on morphological and molecular evidence. This fungus is characterized by its production of sporodochium conidiomata with a white setose fringe surrounding an olivaceous green to dark green slimy mass of conidia, penicillately branched conidiophores, and aseptate and cylindrical to ellipsoid conidia. Phylogenetic analyses of combined LSU rDNA, ITS rDNA, tef1, rpb2, tub2 and cmdA sequence data using maximum parsimony, maximum likelihood and Bayesian approaches placed the fungus in a strongly supported clade with other Paramyrothecium species in Stachybotryaceae (Hypocreales, Sordariomycetes). The descriptions of the species are accompanied by illustrations of morphological features, and a discussion of the related taxa is presented.

A new record of Ardisia×walkeri, a hybrid of A. japonica and A. pusilla, (Primulaceae) from Jeju Island, Korea

  • Goro Kokubugata;Satoshi Kakishima;Chan-ho Park;Takuro Ito;Atsushi Abe;Chikako Ishii;Gwan-Pil Song
    • Journal of Species Research
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    • v.12 no.3
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    • pp.258-265
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    • 2023
  • We conducted phylogenetic analyses using multiplexed inter-simple sequence repeat genotyping by sequencing and compared chloroplast DNA sequences among Ardisia japonica, A. pusilla, and morphologically intermediate plants found on Jeju Island, Korea. Our network analysis demonstrated that the intermediate plants were genetically positioned between A. japonica and A. pusilla. Our comparison of the intergenic spacer between the psbA and trnH genes in chloroplast DNA indicated that four nucleotide substitutions separate A. japonica and A. pusilla, whereas the intermediate plants exhibited the A. japonica haplotype. Our results suggest that the intermediate plants on Jeju Island represent a natural hybrid of A. japonica, as the maternal species, and A. pusilla, and that they are attributable to Ardisia×walkeri. This record constitutes the first documented occurrence of the hybrid taxon in Korea.

Phylogenetic Relationships Among Pleurotus species Inferred from Sequence Data of PCR Amplified ITS II Region in Ribosomal DNA (rDNA의 ITS II 부위의 염기서열분석에 의한 느타리버섯 종간의 근연관계)

  • Bae, Shin-Churl;Seong, Ki-Young;Lee, Shin-Woo;Go, Seung-Joo;Eun, Moo-Young;Rhee, In-Koo
    • The Korean Journal of Mycology
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    • v.24 no.2 s.77
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    • pp.155-165
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    • 1996
  • This study was carried out to identify the phylogenetic relationship among several isolates of Pleurotus species by comparing ITS II region of ribosomal DNA(rDNA) repeat unit. Two primers from ribosomal DNA sequences were chosen to amplify the specific internal transcribed spacer (ITS) II region of Pleurotus spp. The exact ITS II region with an unique band from six species of Pleurotus genus could be amplified using the two primers taken from at the 3'-end of 5.8S rDNA and 5'-end of 28S rDNA. Six representative species of the Pleurotus genus were easily characterized according to the length differences of ITS II region. Furthermore, within P. ostreatus species, different sizes of ITS II region could be observed in the isolates of ASI 2025 and ASI 2095 although they were classified as P. ostreatus by the conventional observation. The nucleotide sequence analyses of PCR-amplified ITS II region indicated that the isolates ASI 2025 and ASI 2095 were different from other Pleurotus spp. When the nucleotide sequences of six Pleurotus species were compared, three typical ITS II regions were highly variable especially at both ends of this region. The phylogenetic tree obtained by the Neighbor program of Felsenstein PHYLIP package with all the nucleotide sequence of Pleurotus spp. indicated that P. ostreatus, P. florida, P. sajor-caju and P. eryngii were closely related to one phylogenetic branch and P. cystidious was related to other branch with P. cornucopiae. The isolates ASI 2025 and 2038, however, were not closely related to any other Pleurotus spp. and formed their own individual branches.

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Cloning of Agarase Gene from Non-Marine Agarolytic Bacterium Cellvibrio sp.

  • Ariga, Osamu;Inoue, Takayoshi;Kubo, Hajime;Minami, Kimi;Nakamura, Mitsuteru;Iwai, Michi;Moriyama, Hironori;Yanagisawa, Mitsunori;Nakasaki, Kiyohiko
    • Journal of Microbiology and Biotechnology
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    • v.22 no.9
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    • pp.1237-1244
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    • 2012
  • Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

Molecular Identification and Chemical Analysis of Aconiti Kusnezoffii Tuber on the Domestic Markets (국내 시장에서 유통되는 초오의 DNA 감별과 화학적 분석)

  • Jang, Hyeri;Joe, Kyeong-Hwa;Song, Kwangho;Lee, Kyoung Jin;Park, Sait Byul;Lee, Chaemin;Ha, In Jin;Lee, Kyungjin;Suh, Youngbae;Kim, Yeong Shik
    • Korean Journal of Pharmacognosy
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    • v.49 no.2
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    • pp.145-154
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    • 2018
  • Aconiti Kusnezoffii Tuber has been traditionally used to treat the symptoms of rheumatoid arthritis and joint pain. The main constituents are diterpenoid alkaloids such as benzoylmesaconine, benzoylaconine, mesaconitine, aconitine, and hypaconitine. In Korea, Aconiti Kusnezoffii Tuber is officially defined as the tubers of Aconitum kusnezoffii Reichb., A. ciliare Decasisne, and A. triphyllum Nakai. On the other hand, only the tuber of A. kusnezoffii is to be used in China. In order to identify the botanical origin of Aconiti Kusnezoffii Tuber circulated in Korea, we analyzed 24 samples of Aconiti Kusnezoffii Tuber obtained from local markets for comparative DNA analysis. The sequence analysis of nrRNA ITS 1 was useful to distinguish Aconitum species and revealed that the roots of A. karakolicum were circulated in Korean markets without discretion. HPLC quantitative analysis showed that aconitine was detected at the highest amount in A. karakolicum. Authentic diterpenoid alkaloids were coinjected for quantification of aconitine-type ingredients. All data were statistically grouped by Principal Component Analysis (PCA). This study suggests that both molecular and chemical analyses should be utilized for the standardization and the quality control for Aconiti Kusnezoffii Tuber.

First record of a marine microalgal species, Chlorella gloriosa (Trebouxiophyceae) isolated from the Dokdo Islands, Korea

  • Kang, Nam Seon;Lee, Jung A;Jang, Hyeong Seok;Kim, Kyeong Mi;Kim, Eun Song;Yoon, Moongeun;Hong, Ji Won
    • Korean Journal of Environmental Biology
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    • v.37 no.4
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    • pp.526-534
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    • 2019
  • Chlorella gloriosa (Chlorellaceae, Trebouxiophyceae) was isolated from seawater off the coast of the Dokdo Islands in Korea. An axenic culture was established using the streak-plate method on f/2 agar media supplemented with antibiotics, allowing identification of the isolate by morphological, molecular, and physiological analyses. The morphological characteristics observed by light and electron microscopy revealed typical morphologies of C. gloriosa species. The molecular phylogenetic inference drawn from the small-subunit 18S rRNA sequence verified that the microalgal strain belongs to C. gloriosa. Additionally, gas chromatography-mass spectrometry analysis showed that the isolate was rich in nutritionally important omega-3 and -6 polyunsaturated fatty acids and high-performance liquid chromatography analysis revealed that the high-value antioxidants lutein and violaxanthin were biosynthesized as accessory pigments by this microalga, with arabinose, galactose, and glucose as the major monosaccharides. Therefore, in this study, a Korean marine C. gloriosa species was discovered, characterized, and described, and subsequently added to the national culture collection.

Transconjugation for Molecular Genetic Study of Streptomyces platensis Producing Transglutaminase (Transglutaminase를 생산하는 Streptomyces platensis의 분자생물학적인 연구를 위한 접합 전달법 확립)

  • Bae, Se-Joung;Jo, Yang-Ho;Choi, Sun-Uk
    • Journal of Life Science
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    • v.20 no.1
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    • pp.97-102
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    • 2010
  • Streptomyces platensis YK-2, newly isolated from forest soil, produces transglutaminase (TGase), which catalyses an acyl transfer reaction between the primary grade amine and protein or $\gamma$-carboxyamide group of peptide bound glutamine residues. For a molecular genetic study of S. platensis, an effective transformation method was established by using a conjugal transfer of DNA from Escherichia coli to spores of actinomycetes. The highest transconjugation frequency of S. platensis was obtained on an MS medium containing 50 mM $MgCl_2$, using $5{\times}10^7\;E$. coli as a DNA donor and $1{\times}10^8$ spores without heat treatment as a host. We also identified that S. platensis contains a single attB site within an ORF encoding a pirin-homolog, and that its attB site sequence shows high homology to that of S. logisporoflavus. In addition, it was confirmed by phenotypic analyses of exconjugants that the introduction of heterologous DNA into the attB site of the S. platensis chromosome does not affect its morphological differentiation and TGase production.

Molecular Cloning, Protein Expression, and Regulatory Mechanisms of the Chitinase Gene from Spodoptera littoralis Nucleopolyhedrovirus

  • Yasser, Norhan;Salem, Reda;Alkhazindar, Maha;Abdelhamid, Ismail A.;Ghozlan, Said A.S.;Elmenofy, Wael
    • Microbiology and Biotechnology Letters
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    • v.49 no.3
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    • pp.305-315
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    • 2021
  • The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.