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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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Studies on the interaction between tomato spotted wilt tospovirus and thrips

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JOURNAL OF GENERAL PLANT PATHOLOGY 2019年第6期85卷 465-467页

作者： tomitaka, yasuhiro NARO Kyushu Okinawa Agr Res Ctr Koshi Kumamoto 8611192 Japan

Tomato spotted wilt tospovirus (TSWV), a type species of the genus Orthotospovirus, which belongs to the family Tospoviridae, causes necrotic yellow and necrosis diseases of vegetables including tomatoes and green peppers, and to flowers such as chrysanthemum and dahlia, and causes economic damage, yield loss, and loss of quality. TSWV has a spherical enveloped virion that contains three species of singlestrand RNA segments (L, M, and S). The genome encodes fives genes;a putative cell-to-cell movement protein (NSm), a non-structural protein (NSs), a nucleocapsid protein (N), an RNA-dependent RNA polymerase, and a precursor of surface glycoproteins (G(N)/G(C)). TSWV is transmitted in a circulative and propagative manner by thrips such as western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci). TSWV has been controlled using the virus resistance genes of the cultivars and by controlling the vector using insecticides. However, the emergence of TSWV that can no longer be controlled using the resistance gene of the cultivars and the development of resistance to insecticides by thrips is a serious problem worldwide. Hence, the interactions among TSWV, western flower thrips, and Arabisopsis thaliana were analysed to develop a novel way to control TSWV on the basis of the interaction mechanism.

关键词： Tospovirus Thrips Attractiveness

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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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A phylogeographical study of the Turnip mosaic virus population in East Asia reveals an 'emergent' lineage in Japan

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MOLECULAR ECOLOGY 2006年第14期15卷 4437-4457页

作者： tomitaka, yasuhiro Ohshima, Kazusato Saga Univ Fac Agr Lab Plant Virol Saga 8408502 Japan

The genetic structure of populations of Turnip mosaic virus (TuMV) in East Asia was assessed by making host range and gene sequence comparisons of 118 isolates utilizing a population genetic approach. Most, but not all, isolates collected from Brassica plants in China infected only Brassica plants, whereas those from Japan infected both Brassica and Raphanus (BR) plants. Analyses of the positions of recombination sites in five regions of the genomes (one third of the full sequence) of the many recombinant isolates were fully congruent with the results of phylogenetic analysis, and at least one recombination type pattern was shared between Chinese and Japanese populations. One lineage of nonrecombinant isolates from the basal-BR lineage was found in 2000 in Kyushu, Japan but none in China, and have since been found over the whole island. The sudden expansion of this basal-BR population was strongly supported by calculations showing the deviations from the neutral equilibrium model for the individual geographical lineages with overall lack of nucleotide diversity, and by analysis of mismatch distribution. Our study shows that the recent Chinese and Japanese TuMV isolates are part of the same population but are discrete lineages.

关键词： East Asia expansion phylogeography population recombination TuMV

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First report of tomato zonate spot virus on green pepper in Japan

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JOURNAL OF GENERAL PLANT PATHOLOGY 2023年第5期89卷 292-295页

作者： Shimada, Ryoko Okamoto, Masahiro tomitaka, yasuhiro Kanagawa Agr Technol Ctr 1617 Kamikichisawa Hiratsuka Kanagawa 2591204 Japan Natl Agr & Food Res Org Inst Plant Protect 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan

In 2021 in Kanagawa Prefecture, Japan, green pepper (Capsicum annuum L.) developed necrotic spots on leaves and necrosis of stems. The nucleotide sequence of the N gene of the virus, isolated through two-rounds of single-lesion isolation, shared 95% identity with that of tomato zonate spot virus (TZSV) reported in China. When green pepper plants were inoculated with the isolated virus, the original symptoms were reproduced, and RT-PCR confirmed the presence of the virus in the inoculated plants. This is the first report of TZSV in Japan.

关键词： Orthotospovirus Tomato zonate spot virus Green pepper

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Genetic structure of a population of Potato virus Y inducing potato tuber necrotic ringspot disease in Japan;: comparison with North American and European populations

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VIRUS RESEARCH 2008年第2期131卷 199-212页

作者： Ogawa, Tetsuji tomitaka, yasuhiro Nakagawa, Akio Ohshima, Kazusato Saga Univ Fac Agr Lab Plant Virol Saga 8408502 Japan Nagasaki Agr Forestry Stn Ainto Potato Branch Nagasaki 8540302 Japan

The structure of Potato virus Y (PVY) populations causing potato tuber necrotic ringspot disease (PTNRD) was analysed. The full-length sequences of the genomic RNAs of five geographically distinct isolates from Japan were determined. Recombination and phylogenefic analyses of European, North American and Japanese isolates of PVY showed that the world PVY population has three major lineages and two sublineages. Most recombinants were interlineage, and one isolate from Europe was identified as an intralineage recombinant. No recombinants were found among Japanese PTNRD isolates, which were most closely related to PTNRD isolates previously found in North America. Comparison of the within- and between population nucleotide diversities in the N lineage sequences from Japan, Europe and North America showed that Japanese population was distinct from the European and North American populations. The nucleotide sequences of the protein I and coat protein genes of a further 18 isolates were determined. One Japanese clade had radiated in a star burst as shown by its deviation from the neutral equilibrium model and its small nucleotide diversity. Our results suggest that PVY PTNRD was recently introduced-into Japan more than once, and has expanded throughout Japan from founder populations. (C) 2007 Elsevier B.V. All rights reserved.

关键词： recombination structure population PVY genome expansion potyvirus

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The genetic structure of populations of Turnip mosaic virus in Kyushu and central Honshu, Japan

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Journal of General Plant Pathology 2007年第3期73.0卷 197-208页

作者： tomitaka, yasuhiro Yamashita, Tohru Ohshima, Kazusato Laboratory of Plant Virology Faculty of Agriculture Saga University Saga 840-8502 1-banchi Honjo-machi Japan Nagano Agricultural Experiment Station Suzaka 382-0072 Nagano 492 Ogawara Japan

The genetic structure of the populations of Turnip mosaic virus in Kyushu and central Honshu, Japan was assessed. The host specificity of isolates was determined, and their gene sequences compared utilizing a population genetic approach. Phylogenetic analysis of partial sequences revealed that 32 of 49 Honshu isolates (65%) collected during 1997-2001 belonged to the basal-BR group as did 23 of 64 isolates from Kyushu. All these basal-BR isolates infected both Brassica and Raphanus plants. However, analyses of the positions of recombination sites in five regions of the genome (one third of the full sequence) showed that at least four intra-lineage recombinants were present in these populations. These analyses showed that Kyushu and Honshu shared none of these subpopulations, and genetically distinct basal-BR populations were present in the two districts. We conclude that different basal-BR subpopulations had expanded into those districts. © 2007 The Phytopathological Society of Japan and Springer.

关键词： Evolution Expansion Genetic structure Recombination Turnip mosaic virus

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First report of leaf curling and yellowing caused by ageratum yellow vein virus in Phaseolus vulgaris in Okinawa Prefecture, Japan

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JOURNAL OF GENERAL PLANT PATHOLOGY 2020年第3期86卷 227-232页

作者： tomitaka, yasuhiro Yamaguchi, Ayako Tanaka, Sayumi Kawano, Shinji Tamayose, Yuna Onuki, Masatoshi NARO Kyushu Okinawa Agr Res Ctr 2421 Suya Koshi Kumamoto 8611192 Japan Okinawa Prefectural Agr Res Ctr 820 Makabe Itoman Okinawa 9010336 Japan Okinawa Prefectural Plant Protect Ctr 123 Maji Naha City Okinawa 9020072 Japan

In November 2016, leaf curling and yellowing were observed in common bean plants on Ishigaki Island, Okinawa Prefecture, Japan. Those symptoms suggested a virus-like disease. PCR using specific primers for ageratum yellow vein virus (AYVV) yielded PCR products of the expected size from diseased plants. The complete nucleotide sequence of the DNA-A shared 99.9% identity with that of the AYVV-Ishigaki isolate. Further, the virus was transmitted by Bemisia tabaci Middle East-Asia Minor 1 and Mediterranean. This is the first report of the natural occurrence of AYVV in common bean plants.

关键词： Ageratum yellow vein virus Phaseolus vulgarism Bemisia tabaci

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Transmission of capsicum chlorosis virus by Thrips palmi (Thysanoptera: Thripidae)

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APPLIED ENTOMOLOGY AND ZOOLOGY 2020年第1期55卷 31-35页

作者： Chiaki, Yuya Kubota, Kenji tomitaka, yasuhiro Usugi, Tomio Sakurai, Tamito NARO Cent Reg Agr Res Ctr 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Kyushu Okinawa Agr Res Ctr 2421 Suya Kumamoto 3058666 Japan NARO Inst Fruit Tree & Tea Sci 301-2 Mitsu Higashihiroshima Hiroshima 7392494 Japan

Transmission efficiencies of capsicum chlorosis virus (CaCV) were investigated for four thrips species (Thysanoptera: Thripidae), Frankliniella occidentalis (Pergande), Frankliniella intonsa (Trybom), Thrips palmi (Karny), and Thrips tabaci Lindeman infesting green pepper in Japan. The first-instar larvae of the thrips were allowed to feed on CaCV-infected green pepper leaves for 24 h. The transmission of CaCV by thrips was examined using a petunia leaf disk assay. CaCV was transmitted by four male (8.70%) and four female (3.88%) T. palmi, but the virus was not transmitted by adults of any other species. In a second transmission assay, 10 adult T. palmi that had fed on infected leaves as larvae were released to healthy green pepper seedlings. After 21 days, the seedlings were examined and 50% of them showed symptoms of CaCV infection. RT-PCR confirmed that they were infected with the virus. The viruliferous rate of adult T. palmi was 60%, while CaCV was not detected in adults of the other three species. These results suggest that T. palmi is capable of being a new vector of CaCV.

关键词： Thrips palmi Capsicum chlorosis virus (CaCV) Transmission efficiency Green pepper

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Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate

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JOURNAL OF PLANT PHYSIOLOGY 2011年第10期168卷 1084-1097页

作者： Fujimoto, Taketo tomitaka, yasuhiro Abe, Hiroshi Tsuda, Shinya Futai, Kazuyoshi Mizukubo, Takayuki Natl Agr Res Ctr Tsukuba Ibaraki 3058666 Japan Kyoto Univ Grad Sch Agr Sakyo Ku Kyoto 6068502 Japan RIKEN Bioresource Ctr Dept Biol Syst Tsukuba Ibaraki 3050074 Japan

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0 mM at 1, 24, 48, and 72 h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5 mM or higher concentrations significantly reduced the infection of RKN implants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection. (C) 2010 Elsevier GmbH. All rights reserved.

关键词： Methyl jasmonate Multicystatin Proteinase inhibitors Root-knot nematode

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