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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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Delay Time Compensation for a Hybrid Simulator

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ADVANCED ROBOTICS 2010年第8-9期24卷 1081-1098页

作者： osaki, kohei Konno, Atsushi Uchiyama, Masaru Tohoku Univ Dept Aerosp Engn Sendai Miyagi 9808579 Japan

A hybrid simulation, which is also referred to as a hardware-in-the-loop (HIL) simulation, includes a hardware experiment within a numerical simulation loop. The hybrid simulation is an effective method to test space robotic operations providing a virtual microgravity environment on the ground. However, hybrid simulation generally suffers from the problem of energy increase when two hardwares in the loop collide with each other because of delay times. This paper presents a compensation method for the energy increase problem. A dead time model that represents various delay times of the system is introduced and it is clarified how the energy of the system is increased by the dead time. Based upon the dead time model, a compensation for the dead time is proposed. This compensation method realizes the desired energy consumption at a collision, which is represented by the coefficient of restitution in the hybrid simulation. Experiments of uniaxial collision are conducted to validate the proposed compensation. (C) Koninklijke Brill NV, Leiden and The Robotics Society of Japan, 2010

关键词： Space robot hybrid simulation HIL simulation delay time compensation coefficient of restitution

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Estimations of relative biological effectiveness of secondary fragments in carbon ion irradiation of water using CR-39 plastic detector and microdosimetric kinetic model

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MEDICAL PHYSICS 2020年第2期47卷 781-789页

作者： Hirano, Yoshiyuki Kodaira, Satoshi Souda, Hikaru osaki, kohei Torikoshi, Masami Gunma Univ Heavy Ion Med Ctr 3-39-22 Showa Machi Maebashi Gumma 3718511 Japan Natl Inst Quantum & Radiol Sci & Technol Natl Inst Radiol Sci Inage Ku 4-9-1 Anagawa Chiba 2638555 Japan

Purpose To estimate relative biological effectiveness (RBE) ascribed to secondary fragments in a lateral distribution of carbon ion irradiation. The RBE was estimated with the microdosimetric kinetic (MK) model and measured linear energy transfer (LET) obtained with CR-39 plastic detectors. Methods A water phantom was irradiated by a C-12 pencil beam with energy of 380 MeV/u at the Gunma University Heavy Ion Medical Center (GHMC), and CR-39 detectors were exposed to secondary fragments. Because CR-39 was insensitive to low LET, we conducted Monte Carlo simulations with Geant4 to calculate low LET particles. The spectra of low LET particles were combined with experimental spectra to calculate RBE. To estimate accuracy of RBE, we calculated RBE by changing yield of low LET particles by +/- 10% and +/- 40%. Results At a small angle, maximum RBE by secondary fragments was 1.3 for 10% survival fractions. RBE values of fragments gradually decreased as the angle became larger. The shape of the LET spectra in the simulation reproduced the experimental spectra, but there was a discrepancy between the simulation and experiment for the relative yield of fragments. When the yield of low LET particles was changed by +/- 40%, the change in RBE was smaller than 10%. Conclusions An RBE of 1.3 was expected for secondary fragments emitted at a small angle. Although, we observed a discrepancy in the relative yield of secondary fragments between simulation and experiment, precision of RBE was not so sensitive to the yield of low LET particles.

关键词： CR-39 heavy ion therapy microdosimetric kinetic model RBE secondary fragments

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Pathogenicity of Plasmodiophora brassicae populations from small, spheroid, resistant-type clubroot galls on roots of clubroot-resistant cultivars of Chinese cabbage (Brassica rapa L. subsp pekinensis)

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JOURNAL OF GENERAL PLANT PATHOLOGY 2008年第3期74卷 242-245页

作者： osaki, kohei Tanaka, Shuhei Ito, Shin-ichi Yamaguchi Univ Fac Agr Dept Biol & Environm Sci Yamaguchi 7538515 Japan Tottori Univ United Grad Sch Agr Sci Tottori 6808533 Japan

A resistant type of small, spheroid clubroot galls (SSGs) containing resting spores formed on the root surface of clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa L. subsp. pekinensis) inoculated with an avirulent population of Plasmodiophora brassicae. Populations isolated from the SSGs severely affected a common (susceptible) cultivar but did not have the typical pathogenicity on CR cultivars, indicating similarity in pathogenicity between SSG and original spore populaions. Populations pathogenic on CR cultivars were not detected from SSGs. Therefore, the ability of the avirulent population among the SSGs to form resting spores may need to be considered to achieve clubroot control in common cultivars of crucifers.

关键词： clubroot-resistant cultivars avirulent population Plasmodiophora brassicae resistant reaction resistant-type clubroot gall

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Relation between pathogenicity and genetic variation within Plasmodiophora brassicae

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JOURNAL OF GENERAL PLANT PATHOLOGY 2008年第4期74卷 281-288页

作者： osaki, kohei Fujiyama, Shozo Nakayama, Akiko Shimizu, Yoshiaki Ito, Shin-ichi Tanaka, Shuhei Yamaguchi Univ Fac Agr Dept Biol & Environm Sci Yamaguchi 7538515 Japan Tottori Univ United Grad Sch Agr Sci Tottori 6808533 Japan

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A-D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.

关键词： Cardamine flexuosa Chinese cabbage clubroot-resistant cultivars pathotype Plasmodiophora brassicae random amplified polymorphic DNA

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Seed transmission of 'Candidatus Liberibacter solanacearum' is unlikely in carrot

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JOURNAL OF GENERAL PLANT PATHOLOGY 2020年第4期86卷 266-273页

作者： Fujikawa, Takashi Yamamura, Kohji osaki, kohei Onozuka, Nobuya Taguchi, Mariko Sasaki, Akane Sato, Masatoshi Natl Agr & Food Res Org NARO Inst Fruit Tree & Tea Sci Fujimoto 2-1 Tsukuba Ibaraki 3058605 Japan NARO Inst Agroenvironm Sci Kannondai 3-1-3 Tsukuba Ibaraki 3058604 Japan NARO Ctr Seeds & Seedlings Fujimoto 2-2 Tsukuba Ibaraki 3050852 Japan

The gram-negative bacterium 'Candidatus Liberibacter solanacearum' (Lso) causes potato zebra chip disease and carrot Lso disease, which resembles a physiological disorder, and has raised concern on its impact on carrot production and whether the pathogen can be transmitted via carrot seeds. Here, we evaluated the possibility of carrot seed transmission of the bacterium using grow-out tests and probability analyses. On the basis of the tests, we concluded that transmission is unlikely in practice. Therefore, this finding will help to promote the international trade of carrot seeds.

关键词： Epidemiology Seed transmission Statistical analyses Proportion of transmission Poisson distribution Binomial distribution

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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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Mitogen-activated protein kinase 4-like carrying an MEY motif instead of a TXY motif is involved in ozone tolerance and regulation of stomatal closure in tobacco

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JOURNAL OF EXPERIMENTAL BOTANY 2016年第11期67卷 3471-3479页

作者： Yanagawa, Yuki Yoda, Hiroshi osaki, kohei Amano, Yuta Aono, Mitsuko Seo, Shigemi Kuchitsu, Kazuyuki Mitsuhara, Ichiro NARO Inst Agrobiol Sci 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan Tokyo Univ Sci Dept Appl Biol Sci Noda Chiba 2788510 Japan Natl Inst Environm Studies Div Environm Biol Tsukuba Ibaraki 3058506 Japan Tokyo Univ Sci Imaging Frontier Ctr Noda Chiba 2788510 Japan

Tobacco MPK4L is activated by wounding and ozone exposure in a pathway independent to that of MPK4 and plays a role in the control of stomatal *** mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKKEE), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation.

关键词： MAP kinase MPK4 Nicotiana tabacum ozone stomata TEY motif

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Linear energy transfer (LET) spectroscopy and relative biological effect estimation by SiC-based dosimeter at clinical carbon-beam cancer therapy field 25

Linear energy transfer (LET) spectroscopy and relative biolo...

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25th International Conference on Ion Beam Analysis and 17th International Conference on Particle Induce X-Ray Emission, IBA-PIXE 2021 and 23rd International Conference on Secondary Ion Mass Spectrometry, SIMS 2021

作者： Yamaguchi, kohei Yanagisawa, Rio osaki, kohei Ohki, Yuya Matsumura, Akihiko Sakai, Makoto Makino, Takahiro Ohshima, Takeshi Kada, Wataru Faculty of Science and Technology Gunma University Japan Japan Japan

A radiation-tolerant silicon carbide (SiC) based dosimeter was developed for the measurement of linear energy transfer (LET) distributions along the Bragg curve for pristine and spread-out Bragg peak (SOBP) clinical carbon beam. Since the conventional ionized chamber is not suitable and effective for LET measurement, several energy-dispersive dosimeters are being developed and tested in the clinical field. While most of the detector lacks radiation hardness against carbon beam, we have utilized wide-bandgap semiconductors of SiC with different epitaxial layers from 25, 69, and 170 μm as a dosimeter for the clinical carbon field at Gunma University Heavy Ion Medical Center (GHMC). LET spectra were obtained not only for pristine carbon beams with an energy of 290 MeV/n but also SOBP conditions. It was confirmed through the changes in LET distribution, primally carbons and fragments are identified, and those energy depositions are successfully obtained by the fabricated SiC dosimeter. The distribution of relative biological effectiveness (RBE) was estimated from LET through biological estimation models. Results suggested that SiC-based dosimeter is successful in being utilized in the detailed characterization of the clinical carbon therapy field. © Published under licence by IOP Publishing Ltd.

关键词： Silicon carbide

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The effects of thermo-mechanical processing on fatigue crack propagation in commercially pure titanium with a harmonic structure

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MATERIALS SCIENCE AND ENGINEERING A-STRUCTURAL MATERIALS PROPERTIES MICROSTRUCTURE AND PROCESSING 2020年第0期773卷 138892-000页

作者： osaki, kohei Kikuchi, Shoichi Nakai, Yoshikazu Kawabata, Mie Ota Ameyama, Kei Kobe Univ Grad Sch Engn Dept Mech Engn Nada Ku 1-1 Rokkodai Cho Kobe Hyogo 6578501 Japan Shizuoka Univ Fac Engn Dept Mech Engn Naka Ku 3-5-1 Johoku Hamamatsu Shizuoka 4328561 Japan Ritsumeikan Univ Coll Sci & Engn Dept Mech Engn 1-1-1 Noji Higashi Kusatsu Shiga 5258577 Japan

Commercially pure (CP) titanium having a bimodal harmonic structure, in which coarse grains are enveloped by a network consisting of fine grains, was obtained by consolidating mechanically milled powders to enhance both strength and ductility. We have examined the fatigue properties of materials having harmonic structures to achieve sufficient performance for practical applications in the engineering fields. In this study, thermomechanical processing (TMP) was performed for the CP titanium having a bimodal harmonic structure to improve its fatigue properties. Four-point bending fatigue tests and fatigue crack propagation tests were conducted under ambient conditions to investigate the fatigue crack initiation and propagation in CP titanium having a harmonic structure following TMP. Applying TMP to CP titanium having a harmonic structure increased its fatigue life due to greater resistance to the initiation of fatigue crack as a result of grain refinement. In contrast, CP titanium having a harmonic structure shows lower threshold stress intensity range, Delta K-th, values following TMP as a result of reduced the degree of crack closure and lower effective threshold stress intensity range, Delta K-eff,K-th, values due to grain refinement.

关键词： Fatigue Fracture mechanics Titanium Grain refinement Thermo-mechanical processing

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