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Development and evaluation of a SYBR Green-based RT-qPCR assay with a specific primer set for tomato seed testing against tomato brown rugose fruit virus

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JOURNAL OF GENERAL PLANT PATHOLOGY 2025年第3期91卷 160-170页

作者： Ota, Emi Shinosaka, Hibiki Ishibashi, Kazuhiro Takeyama, Sawana matsuyama, momoko Tomitaka, Yasuhiro Matsushita, Yosuke Osaki, Kohei Kubota, Kenji NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Ctr Seeds & Seedlings Dept DUS Test & Seed Inspect 2-2 Fujimoto Tsukuba Ibaraki 3050852 Japan NARO Inst Agrobiol Sci Div Plant Mol Regulat Res 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan NARO Inst Plant Protect Div Crop Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan

Since its first occurrence in Israel and Jordan in 2014 and 2015, the tomato brown rugose fruit virus (ToBRFV) has become one of the most concerning pathogens affecting tomatoes and other crops worldwide. Its rapid spread is believed to result from the international trade of contaminated seeds and its seed transmissibility, underscoring the critical importance of seed health testing for ToBRFV to prevent further dissemination of the virus. To this end, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols employing TaqMan probe chemistry have been widely adopted. However, the development of RT-qPCR protocols for ToBRFV seed testing using SYBR Green chemistry remains limited. The SYBR Green method offers the advantage of distinguishing ToBRFV from other tobamoviruses through melt curve analysis. In this study, we developed a SYBR Green-based RT-qPCR detection method using newly designed primer sets, which demonstrated high specificity for ToBRFV and sufficient sensitivity. While this protocol requires further optimization and validation for application in routine seed testing, it establishes a foundational approach for SYBR Green-based RT-qPCR seed testing. Additionally, this study raises an important question regarding the relationship between RT-qPCR results in seed tests and the likelihood of virus contamination or transmission via seeds.

关键词： ToBRFV Tobamoviruses SYBR green RT-qPCR Melt curve Tomato

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Molecular characterization and comparison of tomato zonate spot virus isolated in Japan and China

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VIRUS GENES 2024年第6期60卷 674-683页

作者： matsuyama, momoko Tomitaka, Yasuhiro Natl Agr & Food Res Org Inst Plant Protect Tsukuba Japan

The complete genome sequence of Orthotospovirus tomatozonae (tomato zonate spot virus, TZSV) isolated in Japan was determined and compared with that of Chinese isolates. The lengths of the S, M, and L segments of the RNA genomes of the Japanese isolate (TZSV-TZ1-3) were 3194, 4675, and 8916 nucleotides, respectively, which were similar to the Chinese isolates. Moreover, the eight motifs on the RNA-dependent RNA polymerase (RdRp) gene were conserved in both TZSV-TZ1-3 and Chinese TZSV isolates (TZSV-Bidens and TZSV-Tomato-YN). The nucleotide identity of the genes among the TZSV isolates was more than 94%, indicating low diversity among viruses. The phylogenetic analysis and the prediction of the cleavage sites in the glycoprotein showed that the TZSV-TZ1-3 isolate was closely related to TZSV-Tomato-YN isolated from China. However, there were unique frameshifts and deletions on the RdRp and glycoprotein genes of the TZSV-Tomato-YN isolate, suggesting that both isolates were genetically distinct. The findings of this study indicate that the TZSV-TZ1-3 isolate originated in China and show the sequence diversity among TZSV isolates.

关键词： Bunyaviricetes Orthotospovirus Tomato zonate spot virus Glycoprotein topology

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Tomato brown rugose fruit virus resistance generated by quadruple knockout of homologs of TOBAMOVIRUS MULTIPLICATION1 in tomato

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PLANT PHYSIOLOGY 2022年第2期189卷 679-686页

作者： Ishikawa, Masayuki Yoshida, Tetsuya matsuyama, momoko Kouzai, Yusuke Kano, Akihito Ishibashi, Kazuhiro NARO Inst Agrobiol Sci Div Plant Mol Regulat Res Crop Dis Res Grp 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan NARO Inst Agrobiol Sci Div Plant Mol Regulat Res Crop Stress Management Grp 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan Takii & Co Ltd Plant Breeding & Expt Stn Konan Shiga 5203231 Japan

Editing of host susceptibility genes in tomato confers strong resistance against an emerging virus capable of overcoming currently available resistance genes. Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-2(2) and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed. Here, we show that clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis of four tomato (Solanum lycopersicum) homologs of TOBAMOVIRUS MULTIPLICATION1 (TOM1), an Arabidopsis (Arabidopsis thaliana) gene essential for tobamovirus multiplication, confers resistance to ToBRFV in tomato plants. Quadruple-mutant plants did not show detectable ToBRFV coat protein (CP) accumulation or obvious defects in growth or fruit production. When any three of the four TOM1 homologs were disrupted, ToBRFV CP accumulation was detectable but greatly reduced. In the triple mutant, in which ToBRFV CP accumulation was most strongly suppressed, mutant viruses capable of more efficient multiplication in the mutant plants emerged. However, these mutant viruses did not infect the quadruple-mutant plants, suggesting that the resistance of the quadruple-mutant plants is highly durable. The quadruple-mutant plants also showed resistance to three other tobamovirus species. Therefore, tomato plants with strong resistance to tobamoviruses, including ToBRFV, can be generated by CRISPR/Cas9-mediated multiplexed genome editing. The genome-edited plants could facilitate ToBRFV-resistant tomato breeding.

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Inhibition of Fusarium trichothecene biosynthesis by yeast extract components extractable with ethyl acetate

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INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 2019年 289卷 24-29页

作者： Tanaka, Yuya Nakajima, Yuichi Maeda, Kazuyuki matsuyama, momoko Kanamaru, Kyoko Kobayashi, Tetsuo Ohsato, Shuichi Kimura, Makoto Nagoya Univ Grad Sch Bioagr Sci Chikusa Ku Furo Cho Nagoya Aichi 4648601 Japan Meiji Univ Grad Sch Agr 1-1-1 Higashi Mita Kawasaki Kanagawa 2148571 Japan

While Fusarium graminearum readily produces trichothecenes in complex media containing sucrose as the carbon source (YS_60), the amount of the mycotoxin is quite limited when other sugars, such as glucose and fructose, are used. We found that autoclaving of media containing fructose and yeast extract (YF_60) results in the formation of inhibitors of trichothecene biosynthesis by F. graminearum JCM 9873, a strain that produces 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Removal of the solvent fraction from the autoclaved media after ethyl acetate extraction attenuated the inhibitory activity against trichothecene production. In addition, extraction of the non-autoclaved complex media with ethyl acetate, followed by removal of the solvent fraction, similarly resulted in increased accumulation of the mycotoxin. Although the increase in trichothecene production differed considerably among fungal strains and yeast extract products, F. graminearum species complex generally responded to the medium treatments in the same way. These results suggest that some hydrophobic substances that arise during the drying and heating of yeast extract negatively affected trichothecene production in liquid culture. Modes of actions of inhibitory substances were partially characterized using strain JCM 9873, with focus on the transcriptional and functional analyses of Tri6, a key regulator gene in trichothecene biosynthesis. The presence of the ethyl acetate-extractable substances in autoclaved YF_60 media decreased the relative transcription level of Tri6, as well as that of a trichodiene synthase gene Tri5. Thus, the substances exerted their inhibitory action through suppression of Tri6 expression. By using a yeast extract lot that completely prevented trichothecene production by the wild-type strain in autoclaved YS_60 medium, we prepared YF_60 media and cultured a constitutive Tri6 overexpressor strain described by Maeda et al. (2018). Despite the high transcription level of Tr

关键词： Complex medium Fusarium graminearum Inhibitor Maillard reaction Trichothecene mycotoxin Yeast extract

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Elucidating the nature of seed-borne transmission of tomato brown rugose fruit virus in tomato, bell pepper, and eggplant

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JOURNAL OF GENERAL PLANT PATHOLOGY 2024年第1期90卷 23-34页

作者： Matsushita, Yosuke Takeyama, Sawana Tomitaka, Yasuhiro matsuyama, momoko Ishibashi, Kazuhiro Shinosaka, Hibiki Osaki, Kohei Kubota, Kenji NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Inst Plant Protect Div Crop Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Inst Agrobiol Sci Div Plant Mol Regulat Res 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan NARO Ctr Seeds & Seedlings Dept DUS Test & Seed Inspect 2-2 Fujimoto Tsukuba Ibaraki 3050852 Japan

Since the first report of the tobamovirus tomato brown rugose fruit virus (ToBRFV) in 2014, it has become globally distributed. Its rapid spread has been primarily attributed to seed-borne transmission. Here, the seed-borne nature of ToBRFV transmission was investigated in different cultivars of tomato, bell pepper, and eggplant. In situ hybridization to localize the virus in reproductive organs of ToBRFV-infected tomato plants revealed that the virus was not present in shoot apices, flower buds, or in ovules during flower opening, indicating the virus may be restricted to the outer integument and transported in the vascular bundles during seed development. However, during early fruit development, the virus was present in the integuments in the ovule. Seeds of tomato cultivars with or without tobamovirus resistance gene Tm-22 transmitted the virus to the progeny seedlings at rates that reflected the ineffectiveness of the gene against ToBRFV. Seeds of bell peppers transmitted ToBRFV at higher rates than tomato seeds, but a bell pepper cultivar that has resistance gene L3 was not systemically infected, and its seeds did not harbor the virus. Three eggplant cultivars were systemically infected with ToBRFV but without showing any obvious symptoms, and even though ToBRFV was present in their seeds, the seedlings were not infected. ToBRFV was detected in the seed coats of contaminated tomato and bell pepper seeds, but not in eggplant seed coats. These results indicate mechanistic differences in seed-borne transmission among the three Solanaceae crops.

关键词： Tobamovirus ToBRFV Seed transmission Localization Reproductive organ In situ hybridization

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Effectiveness of a fixed combination formula of ombitasvir/paritaprevir/ritonavir for hepatitis C virus infection in patients on maintenance haemodialysis

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NEPHROLOGY 2017年第7期22卷 562-565页

作者： Morisawa, Norihiko Koshima, Yohei Kuriyama, Satoru matsuyama, momoko Hayashi, Naomi Satoh, Jun-Ichi Amemiya, Morimasa Yokoo, Takashi Jikei Univ Div Nephrol & Hypertens Dept Internal Med Sch Med Tokyo Japan Saitama Red Cross Hosp Div Gastroenterol & Hepatol Dept Internal Med Saitama Japan Saitama Red Cross Hosp Div Nephrol Dept Internal Med Saitama Japan

A fixed-dose formula that combines Ombitasvir (OBV), Paritaprevir (PTV) and Ritonavir (RTV) has been launched into the field of anti-HCV therapy in Japan for patients infected with HCV genotypes 1 and 2 in 2015. However, little is yet known as to the efficacy and safety of this novel therapy in patients on maintenance haemodialysis (HD). The present report describes a preliminary experience in 10 patients (five males and five females) who underwent maintenance HD. All of them had HCV genotype 1b, without having the resistance-associated variants at Y93 or L31 in the nonstructural proteins 5A (NS5A) region. After the treatment, eight patients successfully achieved virus eradication and sustained a virological response at 12weeks (SVR12). In addition, mac-2 binding protein glycosylation isomer (M2BPGi), a biomarker for liver fibrosis, was reduced after the therapy. Two patients withdrew from the therapy due to the development of erythema multiforme and a strong drowsiness, respectively. These results suggest that triple therapy combining OBV, PTV and RTV is effective in achieving SVR12 in most of the HCV-infected patients on HD. In addition, this combination therapy contributed to retard the progression of liver fibrosis. However, we suggest that further trial will be required to establish its clinical efficacy and safety.

关键词： haemodialysis hepatitis C virus ombitasvir paritaprevir ritonavir

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An avoiding overlap method between acoustic shadow and organ for automated ultrasound diagnosis and treatment 11

An avoiding overlap method between acoustic shadow and organ...

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11th IEEE Global Conference on Consumer Electronics, GCCE 2022

作者： matsuyama, momoko Koizumi, Norihiro Nishiyama, Yu Tsumura, Ryosuke Tsukihara, Hiroyuki Numata, Kazushi The University of Electro-Communications Department of Mechanical and Intelligent Systems Engineering Chofu Japan The University of Electro-Communications Department of Computer and Network Engineering Chofu Japan Health and Medical Research Institute National Institute of Advanced Industrial Science and Technology Line 4 Tsukuba Japan The University of Tokyo Department of Cardiothoracic Surgery Bunnkyo-Ku Japan Yokohama City University Medical Center Gastroenterological Center Yokohama Japan

ISBN: (纸本)9781665492324

Ultrasound diagnosis and treatment have attracted attention because they are noninvasive and allow real-time observation of lesions. However, it is difficult to accurately estimate the location of the treatment area because of information loss due to acoustic shadows caused by the reflection of sound waves from hard tissues such as bone. In this study, we propose a probe manipulation method that uses deep learning to avoid overlap between organs and shadows for automated diagnosis. © 2022 IEEE.

关键词： Ultrasonics

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A novel complementation method of an acoustic shadow region utilizing a convolutional neural network for ultrasound-guided therapy

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INTERNATIONAL JOURNAL OF COMPUTER ASSISTED RADIOLOGY AND SURGERY 2022年第1期17卷 107-119页

作者： matsuyama, momoko Koizumi, Norihiro Otsuka, Akihide Kobayashi, Kento Yagasaki, Shiho Watanabe, Yusuke Zhou, Jiayi Nishiyama, Yu Matsumoto, Naoki Tsukihara, Hiroyuki Numata, Kazushi Univ Electrocommun 1-5-1 Chofugaoka Chofu Tokyo Japan Nihon Univ Chiyoda Ku 1-6 Kandasurugadai Tokyo Japan Univ Tokyo Bunkyo Ku 7-3-1 Hongo Tokyo Japan Yokohama City Univ Med Ctr Minami Ku 4-57 Urafune Cho Yokohama Kanagawa Japan

Purpose Noise-free ultrasound images are essential for organ monitoring during regional ultrasound-guided therapy. When the affected area is located under the ribs, however, acoustic shadow is caused by the reflection of sound from hard tissues such as bone, and the image is output with missing information in this region. Therefore, in the present study, we attempt to complement the image in the missing area. Methods The overall flow of the complementation method to generate a shadow-free composite image is as follows. First, we constructed a binary classification method for the presence or absence of acoustic shadow on a phantom kidney based on a convolutional neural network. Second, we created a composite shadow-free image by searching for a suitable image from a time-series database and superimposing the corresponding area without shadow onto the missing area of the target image. In addition, we constructed and verified an automatic kidney mask generation method utilizing U-Net. Results The complementation accuracy for kidney tracking could be enhanced by template matching. Zero-mean normalized cross-correlation (ZNCC) values after complementation were higher than that of before complementation under four different data generation conditions: (i) changing the position of the bed of the robotic ultrasound diagnostic system in the translational direction, (ii) changing the probe angle in the translational direction, (iii) with the addition of rotational motion of the probe to condition (ii). Although there was large variation in the shape of the kidney contour in condition (iii), the proposed method improved the ZNCC value from 0.5437 to 0.5807. Conclusions The effectiveness of the proposed method was demonstrated in phantom experiments. Verification of its effectiveness in real organs is necessary in future study.

关键词： Ultrasound image Acoustic shadow Synthesized Ultrasound-guided therapy

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Study on method of organ section retention and tracking through deep learning in automated diagnostic and therapeutic robotics

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INTERNATIONAL JOURNAL OF COMPUTER ASSISTED RADIOLOGY AND SURGERY 2023年第11期18卷 2101-2109页

作者： Fujibayashi, Takumi Koizumi, Norihiro Nishiyama, Yu Watanabe, Yusuke Zhou, Jiayi matsuyama, momoko Yamada, Miyu Tsumura, Ryosuke Yoshinaka, Kiyoshi Matsumoto, Naoki Tsukihara, Hiroyuki Numata, Kazushi Univ Electrocommun 1-5-1 Chofugaoka Chofu Tokyo Japan Natl Inst Adv Ind Sci & Technol 1-2-1 Namiki Tsukuba Japan Nihon Univ 1-6 KandasurugadaiChiyoda Ku Tokyo Japan Univ Tokyo 7-3-1 HongoBunkyo Ku Tokyo Japan Yokohama City Univ Med Ctr 4-57 Urafune ChoMinami Ku Yokohama Kanagawa Japan

PurposeIn high-intensity focused ultrasound (HIFU) treatment of the kidney and liver, tracking the organs is essential because respiratory motions make continuous cauterization of the affected area difficult and may cause damage to other parts of the body. In this study, we propose a tracking system for rotational scanning, and propose and evaluate a method for estimating the angles of organs in ultrasound *** proposed AEMA, AEMAD, and AEMAD++ as methods for estimating the angles of organs in ultrasound images, using RUDS and a phantom to acquire 90-degree images of a kidney from the long-axis image to the short-axis image as a data set. Six datasets were used, with five for preliminary preparation and one for testing, while the initial position was shifted by 2 mm in the contralateral axis direction. The test data set was evaluated by estimating the angle using each *** accuracy and processing speed of angle estimation for AEMA, AEMAD, and AEMAD++ were 23.8% and 0.33 FPS for AEMAD, 32.0% and 0.56 FPS for AEMAD, and 29.5% and 3.20 FPS for AEMAD++, with tolerance of +/- 2.5 degrees. AEMAD++ offered the best speed and *** the phantom experiment, AEMAD++ showed the effectiveness of tracking the long-axis image of the kidney in rotational scanning. In the future, we will add either the area of surrounding organs or the internal structure of the kidney as a new feature to validate the results.

关键词： Ultrasound image Robotic ultrasound HIFU Ultrasound-guided therapy

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GREEN TEA-CONTAINING BEVERAGE, METHOD FOR PREVENTING DISCOLORATION OF GREEN TEA-CONTAINING BEVERAGE, AND METHOD FOR MANUFACTURING GREEN TEA-CONTAINING BEVERAGE

GREEN TEA-CONTAINING BEVERAGE, METHOD FOR PREVENTING DISCOLO...

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作者： matsuyama momoko OSAKI Manabu

标准号: EP3861862(A4)

Provided is a green tea-containing beverage including: (A) green tea;(B) at least one selected from the group consisting of phytic acid, tartaric acid, and gluconic acid;and (C) at least one selected from the group consisting of ascorbic acid, a salt of ascorbic acid, erythorbic acid, and a salt of erythorbic acid, wherein the green tea-containing beverage has a copper content of 0.3 mg/L or more. Preferably, the concentration of the constituent (B) is 0.05-2.5 g/L. Preferably, the concentration of the constituent (C) is 0.2-5 g/L. Preferably the green tea-containing beverage further includes alcohol. Also provided is a method for preventing discoloration of a green tea-containing beverage, the method including: incorporating the constituents (B) and (C) in the green tea-containing beverage;and further incorporating copper so as to give the copper content of 0.3 mg/L or more.

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