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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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Seed transmission of potato spindle tuber viroid and its distribution in reproductive organs in Solanaceae weed species

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EUROPEAN JOURNAL OF PLANT PATHOLOGY 2023年第3期167卷 315-322页

作者： Matsushita, Yosuke kubota, kenji Natl Agr & Food Res Org NARO Inst Plant Protect Tsukuba Ibaraki 3058519 Japan

Potato spindle tuber viroid (PSTVd) is an important pathogen that severely affects tomato and potato production. Several Solanaceae species are naturally and experimentally susceptible to PSTVd, implying that Solanaceae weeds can be a reservoir of PSTVd through infected seeds in crop fields. To determine whether PSTVd is seed-transmissible in solanaceous weed species, we examined seed transmission rates and localization of PSTVd in the reproductive organs of Solanum nigrum and Physalis angulata. The results revealed that the seed transmission rate of PSTVd was 0%-50% and 89.7%-100% in S. nigrum and P. angulata, respectively, suggesting that these weed species act as reservoirs of PSTVd, and their seed seems to be an important source of PSTVd. While the seed transmission rate in S. nigrum was 0%-8% for PSTVd isolate VP35, it was 10.5%-50% for isolate VP72-1. In situ hybridization analysis showed that isolate VP72-1 (but not VP35) successfully invaded the ovule and embryo of S. nigrum, suggesting that the transmission rate in S. nigrum might depend on the distribution pattern of PSTVd in ovules and embryos. This is the first report to show the distribution patterns of a viroid in the reproductive organs of solanaceous weed species during their developmental stages.

关键词： Embryo Ovule Physalis angulata Physalis peruviana PSTVd Solanum nigrum

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Nucleotide sequence survey of perilla mosaic virus isolates in Japan unveils complex genetic structure and conserved RNA segments encoding homologous protein groups

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JOURNAL OF GENERAL PLANT PATHOLOGY 2024年第1期91卷 41-50页

作者： Chiaki, Yuya kubota, kenji NARO Inst Plant Protect Div Fruit Tree & Tea Pest Control Res 2-1 Fujimoto Tsukuba Ibaraki 3058605 Japan NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan

Perilla mosaic virus (PerMV) is a fimovirus that harbors ten RNA segments (RNAs 1, 2, 3a, 3b, 4, 5, 6a, 6b, 6c, and 7). The presence of PerMV was first reported in Kochi, followed by Ibaraki, Aichi, and Oita prefectures in Japan, and most recently from South Korea. To understand the genetic diversity of these PerMV isolates, partial nucleotide sequences of RNAs 1 to 4 from 21 Japanese isolates were determined. Phylogenetic analysis revealed that the segments of the isolates are divided into two clades: the Eastern clade (isolates in Ibaraki) and the Western clade (isolates in Kochi, Oita, and South Korea). Isolates in Aichi appeared to be reassortants of both;RNAs 1 and 3b belonged to the Eastern clade, while the others were of the Western clade. Isolates lacking RNA2, found in four prefectures, suggest RNA2 is not essential for plant infection. Near-complete nucleotide sequences from four isolates in Ibaraki, Aichi, and Oita were determined. They maintained ten RNA segments, including RNAs encoding proteins of P3s, P6s, and P7. This indicates that these proteins, encoded by an apparently redundant segment, are crucial for PerMV's infection and transmission cycles.

关键词： Fimovirus Emaravirus Genetic diversity Glycoprotein Homologous proteins

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Estimation of buried pipe depth in an artificial soil tank using ground-penetrating radar and moisture sensor

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JOURNAL OF APPLIED GEOPHYSICS 2024年 220卷

作者： Kanemitsu, Toshinori Morifuji, Yohei kubota, kenji Cent Res Inst Elect Power Ind 1646 Abiko Abiko Chiba 2701194 Japan

A method is required for accurate determination of the location (horizontal position and depth) of subsurface utilities with minimal digging. Ground-penetrating radars (GPRs) have been used at many sites as a nondestructive method for detecting underground structures. Previous studies have examined the accuracy and resolution of GPRs based on comparisons of exploration images and factors such as soil, pipe material, and environmental conditions. Although numerous studies have explored the influence of relative permittivity and water content on GPR surveys, only a limited number have conducted direct measurements of water content using a moisture sensor within a soil tank and applied this data to enhance the precision of depth estimation. In this study, the effects of pipe material, relative permittivity, and antenna frequency on the depth estimation accuracy were quantitatively investigated using an artificial soil tank made of silica sand with known physical properties. In addition to the GPR survey, relative permittivity measurements were conducted using time-domain reflectometry (TDR) sensors located at different water levels. The results show that the antenna frequency and pipe material affect the resolution but not the depth estimation accuracy. Furthermore, the GPR survey using the relative permittivity from the TDR sensors estimated the pipe depths with a relative error of < 7.33% relative to the actual burial depth under the following conditions: a volumetric water content of 2.6-38.2%, the relative permittivity of 2.9-23.2, and buried pipes of 0.1 m in diameter and 1.5 m in depth. The study findings show that the selection of an appropriate frequency and the estimation of soil permittivity using a moisture sensor can improve the depth estimation accuracy of buried pipes by GPR survey.

关键词： Ground-penetrating radar Relative permittivity Depth estimation Pipe material Subsurface visualization

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Detectability of 21-cm signal during the epoch of reionization with 21-cm-Lyman-α emitter cross-correlation - III. Model dependence

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MONTHLY NOTICES OF THE ROYAL ASTRONOMICAL SOCIETY 2020年第3期494卷 3131-3140页

作者： kubota, kenji Inoue, Akio K. Hasegawa, kenji Takahashi, Keitaro Kumamoto Univ Grad Sch Sci & Technol GSST 2-39-1 Kurokami Kumamoto 8608555 Japan Osaka Sangyo Univ Fac Design Technol Dept Environm Sci & Technol 3-1-1 Nakagaito Osaka 5748530 Japan Waseda Univ Sch Adv Sci & Engn Dept Phys Shinjuku Ku 3-4-1 Okubo Tokyo 1698555 Japan Waseda Res Inst Sci & Engn Shinjuku Ku 3-4-1 Okubo Tokyo 1698555 Japan Nagoya Univ Dept Phys & Astrophys Chikusa Ku Furo Cho Nagoya Aichi 4648602 Japan Kumamoto Univ Int Res Org Adv Sci & Technol 2-39-1 Kurokami Kumamoto 8608555 Japan

Detecting H121-cm line in the intergalactic medium during the epoch of reionization suffers from foreground contamination such as Galactic synchrotron and extragalactic radio sources. Cross-correlation between the 21-cm line and Lyman-alpha emitter (LAE) galaxies is a powerful tool to identify the 21-cm signal since the 21-cm line emission has correlation with LAEs, while the LAEs are statistically independent of the foregrounds. So far, the detectability of 21-cmLAE cross-power spectrum has been investigated with simple LAE models where the observed Ly alpha luminosity is proportional to the dark matter halo mass. However, the previous models were inconsistent with the latest observational data of LAEs obtained with Subaru/Hyper Suprime-Cam (HSC). Here, we revisit the detectability of 21-cm-LAE cross-power spectrum adopting a state-of-the-art LAE model consistent with all Subaru/HSC observations such as the Lya luminosity function, LAE angular autocorrelation, and the LAE fractions in the continuum selected galaxies. We find that resultant cross-power spectrum with the updated LAE model is reduced at small scales (k similar to 1 Mpc(-1)) compared to the simple models, while the amplitudes at large scales (k less than or similar to 0.2 Mpc(-1)) are not affected so much. We conclude that the large-scale signal would be detectable with Square Kilometre Array (SKA) and HSC LAE cross-correlation but detecting the small-scale signal would require an extended HSC LAE survey with an area of similar to 75 deg(2) or 3000 h observation time of 21-cm line with SKA.

关键词： instrumentation: interferometers methods: statistical galaxies: high-redshift dark ages, reionization, first stars

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Preparation of a new π-conjugated poly (p-phenylene) type polymer constituted of C₂ chiral repeating units and its solution and film properties

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REACTIVE & FUNCTIONAL POLYMERS 2006年第8期66卷 884-887页

作者： Yamamoto, Takakazu Iijima, Takayuki kubota, kenji Tokyo Inst Technol Chem Resources Lab Midori Ku Yokohama Kanagawa 2268503 Japan Gunma Univ Fac Engn Kiryu Gumma 3768515 Japan

Chiral poly [9, 10-dihydro-9,10-bis(tributylsilyloxy)phenanthrene-2,7-diyl] was prepared by dehalogenative polycondensation of the corresponding chiral dibromo monomer. Light scattering analysis revealed that the chiral polymer had a stronger intermolecular interaction than the corresponding racemic polymer. A cast film of the polymer showed a strong circular dichroism of about 6 degrees mu m(-1), which corresponds to a theta value of 3.7 x 10(6)degrees cm(2) dmol(-1). (C) 2005 Elsevier B.V. All rights reserved.

关键词： chiral conjugated polymers poly (p-phenylene) type polymer poly (9,10-dihydrophenthrene-2,7-diyl) strong circular dichroism in film state strong intermolecular interaction

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Expected constraints on models of the epoch of reionization with the variance and skewness in redshifted 21 cm-line fluctuations

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PUBLICATIONS OF THE ASTRONOMICAL SOCIETY OF JAPAN 2016年第4期68卷 1-9页

作者： kubota, kenji Yoshiura, Shintaro Shimabukuro, Hayato Takahashi, Keitaro Kumamoto Univ Dept Phys Kurokami 2-39-1 Kumamoto Kumamoto 8608555 Japan Nagoya Univ Dept Phys Chikusa Ku Furo Cho Nagoya Aichi 4648602 Japan

The redshifted 21 cm-line signal from neutral hydrogen in the intergalactic medium (IGM) gives a direct probe of the epoch of reionization (EoR). In this paper, we investigate the potential of the variance and skewness of the probability distribution function of the 21 cm brightness temperature for constraining EoR models. These statistical quantities are simple, easy to calculate from the observed visibility, and thus suitable for the early exploration of the EoR with current telescopes such as the Murchison Widefield Array (MWA) and LOw Frequency ARray (LOFAR). We show, by performing Fisher analysis, that the variance and skewness at z = 7-9 are complementary to each other to constrain the EoR model parameters such as the minimum virial temperature of halos which host luminous objects, ionizing efficiency, and mean free path of ionizing photons in the IGM. Quantitatively, the constraining power highly depends on the quality of the foreground subtraction and calibration. We give a best case estimate of the constraints on the parameters, neglecting the systematics other than the thermal noise.

关键词： cosmology: observations dark ages, reionization, first stars

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Production of antiserum and immunodetection of Cucurbit chlorotic yellows virus, a novel whitefly-transmitted crinivirus

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JOURNAL OF GENERAL PLANT PATHOLOGY 2011年第2期77卷 116-120页

作者： kubota, kenji Usugi, Tomio Tsuda, Shinya Natl Agr Res Ctr Tsukuba Ibaraki 3058666 Japan

Cucurbit chlorotic yellows virus (CCYV), a whitefly-transmitted virus, is a new member of the Crinivirus genus, that causes major economic losses in cucumber, melon and watermelon crops. To develop immunodiagnostic methods for CCYV, we produced an antiserum by immunizing rabbits with bacterially expressed recombinant coat protein of CCYV and detected CCYV from CCYV-infected plants with western blotting and immunoelectron microscopy. CCYV in extracts from infected plants was also readily detected by double antibody sandwich enzyme-linked immunosorbent assay with high sensitivity and specificity. A tissue blot immunoassay indicated that CCYV localizes in the phloem tissues of the petiole and is distributed in tiny spots within the leaf lamina.

关键词： Bemisia tabaci CCYV Cucumber DAS-ELISA Melon

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Investigation of chemical structure of nonprotein proteinase inhibitors from dried figs

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FOOD CHEMISTRY 2008年第1期107卷 305-311页

作者： Hatano, Ken-Ichi kubota, kenji Tanokura, Masaru Gunma Univ Fac Engn Dept Biol Sci Gunma 37658515 Japan Univ Tokyo Grad Sch Agr & Life Sci Dept Appl Biol Chem Bunkyo Ku Tokyo 1138657 Japan

Two types of the nonprotein proteinase inhibitors, P and B samples were previously isolated from dried figs by gel permeation and high-performance liquid chromatography. In the present study, we investigated the chemical structure of the B sample using both mass spectrometry (MS) and solid-state nuclear magnetic resonance. Fast atom bombardment and matrix-assisted laser desorption ionization-time of flight MS experiments showed losses of 16, 44, 176, and 338 Da. By electrospray ionization tandem MS, the product-ion spectra of m/z 712, 731, and 750 in the parent ions indicated that the fragmentation ion at m/z 175 was the repeated structure of the parent ions. We propose here that the 176- and 338-Da compounds might aggregate each other, probably by polymerizations, ether linkages, and hydrogen bonding, which would result in a mass variety of the B sample as observed in the MS analyses. (c) 2007 Elsevier Ltd. All rights reserved.

关键词： Ficus carica humic substances organic matter nonprotein chemical structure proteinase inhibitor

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Distal tuberosity osteotomy in open-wedge high tibial osteotomy does not exacerbate patellofemoral osteoarthritis on arthroscopic evaluation

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KNEE SURGERY SPORTS TRAUMATOLOGY ARTHROSCOPY 2020年第6期28卷 1750-1756页

作者： Horikawa, Tomohiro kubota, kenji Hara, Shintaro Akasaki, Yukio Omuta Tenryo Hosp Dept Orthopaed Surg 1-100 Tenryomachi Omuta 8368566 Japan Kyushu Univ Dept Orthopaed Surg Higashi Ku 3-1-1 Maidashi Fukuoka 8128582 Japan

Purpose The purpose of the present study was to use arthroscopy to evaluate the effect of distal tuberosity osteotomy (DTO) in open-wedge high tibial osteotomy (OW-HTO) on patellofemoral (PF) cartilage degradation. Methods Between 2012 and 2017, 46 knees underwent DTO in OW-HTO, and 65 knees underwent conventional OW-HTO (cOW-HTO). To assess changes in patellar height, the Blackburne-Peel (BP) ratio and the Caton-Deschamps (CD) index were measured. Arthroscopic evaluation on the PF joint was performed at the initial osteotomy and at the second-look procedure 1 year later. Statistical analyses were performed to compare difference between the DTO and the cOW-HTO group. Results In the cOW-HTO group, the mean BP ratio and CD index decreased significantly from 0.81 and 0.89 preoperatively, respectively, to 0.69 and 0.76 postoperatively, respectively (p < 0.001). In contrast, the DTO group maintained a consistent patellar height;the mean BP ratio and CD index were 0.77 and 0.83 preoperatively, respectively, and 0.73 and 0.80 postoperatively, respectively. Upon arthroscopic evaluation, 39 of 46 patients (84.8%) in the DTO group showed no progression of PF cartilage degradation at the second look;indeed, five of 46 patients (10.9%) even demonstrated improvement. In contrast, 21 of 65 patients (32.3%) in the cOW-HTO group exhibited increased PF cartilage degradation. There was a significant difference in progression of PF cartilage degradation between DTO and cOW-HTO (p < 0.001). Conclusion DTO in OW-HTO maintained the preoperative patellar height, which could help prevent progression of cartilage degeneration in the PF joint after surgery. In respect of the biplanar osteotomy direction in OW-HTO, the DTO, rather than cOWHTO, is the preferred technique for the treatment of varus knee osteoarthritis to avoid progression of PF cartilage degradation.

关键词： Osteoarthritis DTO Distal tuberosity osteotomy High tibial osteotomy OWHTO Patellar height Patellofemoral osteoarthritis

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