Papers - YOKOTA Kenji
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Involvement of iturin and surfactin in inhibition of a post-harvest fungal pathogen on green bell pepper Reviewed International coauthorship International journal
Yuliar, Shun Tomita, Hinako Hishinuma, Kenji Yokota
Journal of Plant Protection Research 2025.03
Authorship：Last author Language：English Publishing type：Research paper (scientific journal)
This study aimed to investigate the potential of plant-associated bacteria as bio-control agents for the green bell pepper rot lesion caused by Colletotrichum scovillei 244830. A total of 378 bacteria strains isolated from stems and leaves of healthy red chili and tomato were tested for their antagonistic potential. Isolate TS001 associated with tomato stems was identified as Bacillus spp. It was found that TS001 showed remarkable inhibition to C. scovillei 244830 in in vitro and in vivo tests. TS001 significantly reduced rot lesions (P<0.05) of fresh green bell pepper fruits by 71.43%. Furthermore, the result of the LC-ESI-MS/MS showed that the culture broth of the strain Bacillus sp. TS001 contained iturin and surfactin homolog in No. 3S medium. TS001 exhibited the strongest antagonistic activity that effectively suppressed C. scovillei 244830 rot lesion. -
Characterization of NRPS operon homologue for surfactin synthesis distributed in Bacillus spp. Reviewed International journal
Kojiro Ito, Mana Adachi, Minenosuke Matsutani, Ryota Kataoka, Gen Enomoto, Akinobu Kajikawa, Kenji Yokota
Archives of Microbiology 2025
Authorship：Last author, Corresponding author Language：English Publishing type：Research paper (scientific journal)
Surfactin is a cyclic lipopeptide consists of a b-hydroxy fatty acid and a heptapeptide derived from Bacillus spp. The heptapeptide moiety of surfactin is synthesized by non-ribosomal peptide synthetases, and a variety of surfactin congeners which are varied by substitution of amino acid in the heptapeptide have been identified so far. Surfactin A, B and C are surfactin congeners which are substituted of Leu7 of surfactin A with Val for surfactin B or Ile for surfactin C. The operon encoding non-ribosomal peptide synthetases for surfactin A had been identified in B. subtilis JH 642. Here, we report that novel operons encoding non-ribosomal peptide synthetases for surfactin A and C biosynthesis, respectively.
The srfA operon for surfactin A biosynthesis in B. subtilis composed by 4-ORFs, srfAABCD. The novel operon for surfactin A biosynthesis was distinct from the srfA operon in B. subtilis JCM 1465 with 68.5, 69.2, 84.5 and 66.9 % homology of ORFs, respectively. The novel surfactin C showed 68.7, 69.3, 64.5 and 67.2 % homology with srfA operon in B. subtilis JCM 1465, respectively, and the adenylation domain to select Ile7 in surfactin C was identified by a domain substitution in srfA operon in a surfactin A-producing strain. Average Nucleotide Identity of genomic sequence of tested strains clearly showed that the novel operons for surfactin A and C biosynthesis are distributed in B. velezensis and B. amyloliquefaciens, respectively. Our results indicate surfactin congener production was strictly related to the species level of Bacillus and srf operon was suggested to be distributed by species. -
TAMAKI Mizuna, CHIKU Tsutomu, SUZUKI Shunya, MISAKI Aya, FUNAKUBO Asuka, MATSUSHIMA Yoshitaka, YOKOTA Kenji, IGIMI Shizunobu, KAJIKAWA Akinobu
Bioscience of Microbiota, Food and Health 44 ( 1 ) 70 - 79 2024.09
Language：English Publisher：BMFH Press
Although the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been extensively developed since its discovery for eukaryotic and prokaryotic genome editing and other genetic manipulations, there are still areas warranting improvement, especially regarding bacteria. In this study, BRD0539, a small-molecule inhibitor of Streptococcus pyogenes Cas9 (SpCas9), was used to suppress the activity of the nuclease during genetic modification of Lacticaseibacillus paracasei, as well as to regulate CRISPR interference (CRISPRi). First, we developed and validated a CRISPR-SpCas9 system targeting the sirA gene of L. paracasei. Then BRD0539 was used for CRISPR-dependent DNA cleavage in vivo. Our results suggested that the inhibitor worked partially in both Escherichia coli and L. paracasei. Next, we designed a CRISPRi system in a L. paracasei strain by inserting an inactive SpCas9 gene into the chromosome and introducing a plasmid encoding for a single guide RNA (sgRNA) targeting the sirA gene. Expression of sirA was successfully inhibited in the recombinant strains, and CRISPRi was abolished in an inhibitor-dependent manner. Our findings may help expand the CRISPR toolbox for research on lactic acid bacteria and other microbes.
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Heat-stable spores of carotenoid-producing Bacillus marisflavi and non-pigmented Bacillus subtilis cooperatively promote growth, quality, and gut microbiota of white-leg shrimp Reviewed International coauthorship International journal
T.T. Nguyen, A.T.P. Bui, N.T.H. Le, H.T. N. Vo, A.H. Nguyen, T.D. Pham, T. Hara, K. Yokota, M. Matsutani, Y. Takatsuka, A.T.V. Nguyen
Beneficial Microbes 2023.11
Language：English Publishing type：Research paper (scientific journal)
We evaluated the benefits of heat-stable carotenoid-producing Bacillus marisflavi SH8 spores individually and in combination with non-pigmented Bacillus subtilis SH23 spores on growth, colour change, nutritional content, innate immunity, and gut microbiota of white-leg shrimp. White-leg shrimp (Litopenaeus vannamei; n = 30 per tank; 2 tanks per group) were provided feed without (control group) or with SH8, SH23, or mixed spores (total, 1×106 cfu/g pellet) for 28 d. The SH8 and SH8-23 combination groups had significantly higher specific growth rates (9.6 and 11.0%), improved red-colour score (4 scores), astaxanthin concentration (1.8- and 2.3-fold), lipid contents (30 and 50%), and superoxidase dismutase activity (8.5 and 12.3%) than that of the control group. Analysis of shrimp’s gut microbiome using 16S rRNA metagenome sequencing revealed increased abundance of four useful species and reduced abundance of four harmful species in the combination group than in the control group. Heat-stable Bacillus spore combination improved growth parameters, nutrient content, red-colour score, live counts, and abundance of useful bacteria in the gut of L. vannamei. This is the first study to show the benefits of combining highly heat-stable pigmented and non-pigmented Bacillus spores and their possible mechanisms in a shrimp model.
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Negative chemotaxis of Ligilactobacillus agilis BKN88 against gut-derived substances Reviewed International journal
Shunya Suzuki, Kenji Yokota, Shizunobu Igimi, Akinobu Kajikawa
Scientific Reports 13 2023.09
Language：English Publishing type：Research paper (scientific journal)
Ligilactobacillus agilis is a motile lactic acid bacterium found in the gastrointestinal tracts of animals. The findings of our previous study suggest that the motility of L. agilis BKN88 enables gut colonization in murine models. However, the chemotactic abilities of motile lactobacilli remain unknown. This study aimed to identify the gut-derived chemoeffectors and their corresponding chemoreceptors in L. agilis BKN88. Chemotaxis assays with chemotactic and non-chemotactic (ΔcheA) L. agilis strains revealed that low pH, organic acids, and bile salts served as repellents. L. agilis BKN88 was more sensitive to bile and acid than the gut-derived non-motile lactobacilli, implying that L. agilis might utilize motility and chemotaxis instead of exhibiting stress tolerance/resistance. L. agilis BKN88 contains five putative chemoreceptor genes (mcp1–mcp5). Chemotaxis assays using a series of chemoreceptor mutants revealed that each of the five chemoreceptors could sense multiple chemoeffectors and that these chemoreceptors were functionally redundant. Mcp2 and Mcp3 sensed all tested chemoeffectors. This study provides further insights into the interactions between chemoreceptors and ligands of motile lactobacilli and the unique ecological and evolutionary features of motile lactobacilli, which may be distinct from those of non-motile lactobacilli.
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エノキタケ黒腐細菌病の生物的防除のためのPseudomonas sp. BM2-6株の効果的な処理方法 Reviewed
横山遼人, 鈴木千尋, 服部雄斗, 富田 駿, 横田健治, キムオッキョン, 岩波 徹, 篠原弘亮
東京農業大学農学集報 68 ( 1 ) 13 - 22 2023.06
Language：Japanese Publishing type：Research paper (scientific journal)
著者ら（2018）はエノキタケ黒腐細菌病（病原：Pseudomonas tolaasii）に対し発病抑制効果のある P. sp. BM2-6 株を見出した。本研究では BM2-6 株の有効な処理方法を検討し，発病抑制機構を考察した。 BM2-6 株細菌懸濁液のエノキタケ（Flammulina velutipes）幼子実体への噴霧処理または菌掻き時処理した ところ，防除価はいずれも高く，約 109 cfu/ml において 99.6，98.4 であった。約 106 cfu/ml において 83.3， 90.3 であった。約 1010 cfu/mL または約 106 cfu/mL の BM2-6 株細菌懸濁液処理および約 105 cfu/mL の P. tolaasii 814 株細菌懸濁液接種におけるエノキタケ菌床または子実体での両菌の各細菌数を調査した。これ らエノキタケに発病は認められず，菌床における BM2-6 株は各処理ともに約 108 cfu/g, P. tolaasii 814 株は それぞれ約 106 cfu/g, 約 107 cfu/g 定着していた。子実体における両菌は各処理ともそれぞれ約 107 cfu/g, 約 108 cfu/g 定着していた。BM2-6 株はトラシン解毒能も持たないことから，液体培地中で両菌を混合培養し， 両細菌数および腐敗の主因であるトラシンⅠの量を測定した。P. tolaasii 814 株の単独培養時，約 1010 cfu/ mL を超えるとトラシンⅠが最大 45.9 µg/mL 検出された。一方，混合培養では，両菌ともに約 1010 cfu/mL まで増殖したがトラシンⅠは非検出であったことから，BM2-6 株の発病抑制機構は P. tolaasii 814 株のトラ シン産生抑制であると判断した。以上，BM2-6 株は菌掻き時処理により本病を効果的に防除でき，P. tolaasii のトラシン産生を抑制することによって発病を抑制した。
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Strategies for Biological Control of Brown Blotch Disease
TOMITA Shun, SHINOHARA Hirosuke, YOKOTA Kenji
KAGAKU TO SEIBUTSU 61 ( 3 ) 116 - 124 2023.03
Language：Japanese Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Bacillus属細菌を用いた植物病害生物防除における環状リポペプチドの機能
横田 健治
日本農薬学会誌 47 ( 2 ) 124 - 125 2022.08
Language：Japanese Publisher：日本農薬学会
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Insights on suppression of bacterial leaf spot by Bacillus cyclic lipopeptides via induced resistance in Arabidopsis thaliana Reviewed
Carla Suzuki Altrão, Mana Kaneko, Shigeaki Shiina, Akinobu Kajikawa, Hirosuke Shinohara, Kenji Yokota
Journal of General Plant Pathology 2022.03
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
We evaluated disease suppression by surfactin and iturin A against Pseudomonas syringae pv. maculicola MAFF 302783 in Arabidopsis thaliana and its mutants to gain insight into resistance pathways induced. Root treatments revealed that salicylic acid signaling via the isochorismate pathway was the major signaling pathway elicited by both cyclic lipopeptides. Disease suppression was negated after treatment with higher levels of surfactin or iturin A, and this negation of disease suppression was not associated with an antagonistic effect by induction of jasmonate signaling pathway in host plants.
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Comparative study of disease suppression on various host plants by Bacillus cyclic lipopeptides Reviewed
Carla Suzuki Altrão, Takuya Kawashima, Minami Ohbu, Shuhei Matsuura, Misaki Higuchi, Yusuke Yanai, Yuki Hase, Hirosuke Shinohara, Kenji Yokota
Agricultural Sciences 13 2022.01
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
In biological controls using Bacillus spp., cyclic lipopeptides play a role as elicitors to induce disease resistance on various host plants. However, it is still unclear the specificity between cyclic lipopeptides and host plants to induce disease resistance. In this study, we aimed to clarify the specificity to induce disease resistance among cyclic lipopeptides on various host plants. Our data clearly showed both cyclic lipopeptides conferred disease suppression on most of host plants, but at different range of cyclic lipopeptide concen-tration. Our findings contribute to understanding the complex on the specificity of cy-clic lipopeptide derived induced disease resistance.
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Draft Genome Sequence of the Polychlorinated Biphenyl Degrader Comamonas testosteroni Strain YAZ2 Isolated from a Natural Landscape in the Tohoku Region of Japan Reviewed International journal
Tomijiro Hara, Yumiko Takatsuka, Yuh Shiwa, Kenji Yokota
Microbiology Resource Announcements 11 ( 1 ) e00806-21 2022.01
Language：English Publishing type：Research paper (scientific journal)
We report a draft genome sequence of Comamonas testosteroni strain YAZ2, a polychlorinated biphenyl (PCB) degrader that was isolated from a PCB-unpolluted
environment. The assembled genome contains a single 5.4-Mb chromosome and an 87-kb plasmid. The bph gene cluster, which is involved in PCB degradation, was
found on the chromosome. -
Insights into detoxification of tolaasins, the toxins behind mushroom bacterial blotch, by Microbacterium foliorum NBRC 103072T Reviewed International journal
Shun Tomita, Akinobu Kajikawa, Shizunobu Igimi, Hirosuke Shinohara, Kenji Yokota
PhytoFrontiers 2021.03
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of brown blotch disease of mushrooms, and are the toxins that cause the brown spots. We previously reported that Microbacterium foliorum NBRC 103072T is an effective tolaasin-detoxifying bacterium. In this study, we aimed to characterize the tolaasin-detoxification process of M. foliorum NBRC 103072T. The tolaasin-detoxification by M. foliorum NBRC 103072T was carried out by hydrolyzation of tolaasins at two specific sites in the peptide moiety of tolaasins by its cells, and the resulting fragments were released from bacterial cells. The tolaasin-hydrolyzing activity can be extracted by neutral detergent solution from M. foliorum NBRC 103072T cells. Moreover, tolaasin-adsorption to the bacterial cells occurred prior to hydrolyzation of tolaasins, which might contribute to the effective tolaasin-detoxification by M. foliorum NBRC 103072T. It is notable that the tolaasin-degradation process by M. foliorum NBRC 103072T is carried out by hydrolyzation at specific sites in the peptide moiety of lipopeptide by bacterial cells as a novel biological degradation process of cyclic lipopeptides.
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Ligilactobacillus agilis BKN88 possesses thermo-/acid-stable heteropolymeric flagellar filaments Reviewed
Naoto Eguchi, Shunya Suzuki, Kenji Yokota, Shizunobu Igimi, Akinobu Kajikawa
Microbiology 2021.01
Language：English Publishing type：Research paper (scientific journal)
Many flagellated bacteria possess multiple flagellins, but the roles and the compositions of each flagellin are diverse and poorly understood. In Ligilactobacillus agilis BKN88, there are two active flagellin gene paralogues but their function and composition in its flagellar filaments have not been described. The aim of this study is to find the function and composition of the flagellins by employing mutant strains each of which expresses a single flagellin or a modified flagellin. Two single flagellin-expressing strains were both flagellated while the number of flagella per cell in the single flagellin-expressing derivatives was lower than that in the wild type. Nonetheless, these derivative strains were apparently equally motile as the wild type. This indicates that either flagellin is sufficient for cell motility. The immunological activity via Toll-like receptor 5 of the single flagellin-expressing strains or purified single flagellins was readily detectable but mostly variably weaker than that of the wild type. The flagellar filaments of wild type L. agilis BKN88 were more acid-/thermo-stable than those of single flagellin-expressing derivatives. Using a combination of immunoprecipitation and flagellin-specific staining, wild type BKN88 appeared to possess heteropolymeric flagellar filaments consisting of both flagellins and each flagellin appeared to be equally distributed throughout the filaments. The results of this study suggest that the two flagellins together form a more robust filament than either alone and are thus functionally complementary.
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PCR-based screening, isolation, and partial characterization of motile lactobacilli from various animal feces Reviewed
Shunya Suzuki, Koki Fujita, Shintaro Maeno, Yuh Shiwa, Akihito Endo, Kenji Yokota, Shizunobu Igimi, Akinobu Kajikawa
BMC Microbiology 20 2020.06
Language：English Publishing type：Research paper (scientific journal)
Background
Most lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces.
Results
We applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species.
Conclusions
These results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche. -
Adsorption of tolaasins, the toxins behind mushroom bacterial blotch, by Microbacterium spp. is insufficient for its detoxification Reviewed
Shun Tomita, Anna Hirayasu, Akinobu Kajikawa, Shizunobu Igimi, Hirosuke Shinohara, Kenji Yokota
Current Microbiology 2020.01
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of bacterial blotch on several kinds of cultivated mushrooms. Our previous study reported on tolaasin detoxification by Microbacterium sp. K3-5 as a potential biocontrol of the disease. In this study, the tolaasin-detoxifying activities of various type strains of Microbacterium spp. were evaluated through chemical and biological assays. The bacterial cells of all tested strains of Microbacterium spp. showed tolaasin I-elimination from liquid phase. However, the toxin activities of tolaasins were still retained on the tolaasin-treated bacterial cells of all Microbacterium strains except M. foliorum NBRC 103072T. Furthermore, intact tolaasin I was recovered from the tolaasin-treated bacterial cells of all tested strains except M. foliorum NBRC 103072T. Our data reveal that Microbacterium spp. can be characterized as effective tolaasin I-eliminating bacteria through cell adsorption, but that this adsorption alone is insufficient for actual tolaasin detoxification. The biological degradation process must be needed to carry out the detoxification.
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Comparative analysis of immunological properties of S-layer proteins isolated from Lactobacillus strains Reviewed
Shunya Suzuki, Kenji Yokota, Shizunobu Igimi, Akinobu Kajikawa
Microbiology 165 188 - 196 2019.01
Language：English Publishing type：Research paper (scientific journal)
Previous studies have suggested that some Lactobacillus S-layer proteins could modulate immune responses. Primary structures of the S-layer proteins are variable, and their immunological differences are poorly understood. In this study, we evaluated the immunological properties of eight distinct S-layer proteins from different Lactobacillus species. We found that removal of the S-layer proteins from the cell surface reduced the immunological activities of Lactobacillus cells in THP-1 cells. Furthermore, the purified S-layer proteins induced the production of IL-12 p40, although their immunological activities varied between the different S-layer proteins. The production of IL-12 p40 was notably induced by the S-layer protein SLP(aly) from Lactobacillus amylolyticus NRIC 0558T. Multiple sequence alignment revealed that the percent identity of the S-layer proteins of the eight strains vary from 10 to 90 %. In particular, N-terminal regions showed high levels of diversity. To obtain more information about their structure and the immunogenicity, truncated and chimeric S-layer proteins were constructed in recombinant E. coli. Profiling of cytokine production in THP-1 cells by truncated and chimeric S-layer proteins suggested that the intact conformation of the N-terminal region of SLP(aly) contributes to high immunogenicity.
DOI： https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000766
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Disease suppression by the cyclic lipopeptides iturin A and surfactin from Bacillus spp. against Fusarium wilt of lettuce Reviewed
Shohei Fujita, Kenji Yokota
Journal of General Plant Pathology IF:0.741 85 ( 1 ) 44 - 48 2019.01
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
Iturin A and surfactin are antimicrobial cyclic lipopeptides secreted by antagonistic Bacillus strains, and both elicit defence gene expression in host plants. We previously showed that a soil amendment with either of the purified lipopeptides confers suppression against Fusarium yellows of tatsoi (Brassica rapa) caused by the soil-borne pathogen Fusarium oxysporum and that excess amounts of iturin A or surfactin do not confer suppression. Here, we evaluated the impacts of purified iturin A or surfactin against Fusarium wilt of lettuce (Lactuca sativa). As a soil amendment, iturin A and surfactin each conferred suppression of Fusarium wilt of lettuce on diverse lettuce cultivars similar to the results for tatsoi. However, higher concentrations of iturin A (1.88 mg/l of soil or higher) but not of surfactin (up to 7.5 mg/l of soil) negated the suppression. Growth of Fusarium in liquid cultures was suppressed by iturin A but not surfactin.
DOI： https://link.springer.com/article/10.1007/s10327-018-0816-1
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Effects of agriculture on the water quality of the Cisadane river system in West Java, Indonesia Reviewed International coauthorship
Luqman Arif Lubada, Kenji Yokota, Arief Hartono, Rie Miyaura
Tropical Agriculture and Development 62 ( 2 ) 94 - 103 2018.06
Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)
This study investigates the impact of agriculture on the water quality of the Cisadane River system in West Java, Indonesia by focusing on one stream flowing from an upland area through an intensive agricultural area before merging with the rest of the river system downstream. In total, 25 water samples were collected from this river system over a straight-line distance of ~70 km and an altitudinal difference of 750 m during the dry season of 2015 and the rainy season of 2016. The samples were analyzed to measure the pH, EC, and concentrations of NO3-N, NH4-N, and PO4-P. The average contamination in the water samples during the dry and rainy seasons was 0.61 and 0.45 mg NO3-N L-1, respectively, 0.56 and 0.28 mg NH4-N L-1, respectively, and 0.64 and 0.55 mg PO4-P L-1, respectively. The results revealed that water was contaminated by inorganic P even in the upper stream. Moreover, the spring water in the agriculture-intensive areas of a rural community, which is believed by the locals to be sufficiently clean for use, was found to more be contaminated by nitrogen than the water in the lower part of the river. As the flow of contaminated water can cause eutrophication in estuaries, it is important to characterize the contamination of water systems to encourage the implementation of sustainable water management strategies.
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Detoxification process of tolaasins, lipodepsipeptides, by Microbacterium sp. K3-5 Reviewed
Shun Tomita, Masayuki Sue, Akinobu Kajikawa, Shizunobu Igimi, Hirosuke Shinohara, Kenji Yokota
Bioscience, Biotechnology, and Biochemistry IF1.295 82 ( 8 ) 1455 - 1458 2018.04
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.
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Bacterial flora of fruiting bodies of Shiitake mushroom, isolation of tolaasin-detoxifing bacteria, and tolaasin-detoxification properties by Pseudoxanthomonas spp. Reviewed
Kenji Yokota, Takayuki Nanaumi, Shun Tomita, Ok-Kyung KIim Hiromitsu Negishi, Hirosuke Shinohara
Mushroom Science and Biotechnology 25 ( 4 ) 129 - 133 2018.02
Authorship：Lead author Language：Japanese Publishing type：Research paper (scientific journal)