研究者データベース
小松 健KOMATSU Kenコマツ ケン
| ■所属部署名 | 農学研究院 生物制御科学部門 | |||
| ■職名 | 教授 | |||
Last Updated :2025/04/07
業績情報
氏名・連絡先
氏名
コマツ ケン, 小松 健, KOMATSU Ken
主たる所属・職名
農学研究院 生物制御科学部門, 教授
その他の所属
学位
教育・研究活動状況
科学研究費助成事業
- 国際共同研究加速基金（国際共同研究強化(B)）
ベトナム国カントー地方の植物病原菌に潜伏感染するマイコウイルスの探索と性状解析
自 2023年, 至 2023年 - 挑戦的研究（萌芽）
ハチノスツヅリガを用いた病原性微生物のin vivo評価系の構築
自 2023年, 至 2023年 - 基盤研究（C）
脱分化ー再分化系を利用した園芸作物のウイルスフリー化技術の確立
自 2023年, 至 2023年 - 基盤研究(B)
ポテックスウイルスの複製複合体形成メカニズムの解明
自 2023年, 至 2025年 - 国際共同研究加速基金（国際共同研究強化(B)）
ベトナム国カントー地方の植物病原菌に潜伏感染するマイコウイルスの探索と性状解析
自 2022年, 至 2022年 - 基盤研究(B)
アグロフォレストリーはバナナの土壌病害を防止できるのか？
自 2022年, 至 2022年 - 基盤研究(C)
脱分化ー再分化系を利用した園芸作物のウイルスフリー化技術の確立
自 2022年, 至 2022年 - 国際共同研究加速基金（国際共同研究強化(B)）
ベトナム国カントー地方の植物病原菌に潜伏感染するマイコウイルスの探索と性状解析
自 2021年, 至 2021年 - 基盤研究(B)
アグロフォレストリーはバナナの土壌病害を防止できるのか？
自 2021年, 至 2021年 - 基盤研究(B)
アグロフォレストリーはバナナの土壌病害を防止できるのか？
自 2020年, 至 2020年 - 国際共同研究加速基金（国際共同研究強化(B)）
ベトナム国カントー地方の植物病原菌に潜伏感染するマイコウイルスの探索と性状解析
自 2020年, 至 2020年 - 基盤研究(C)
パンデミックウイルスのゆりかご：多年生野草から農作物へのホストジャンプ機構の解明
自 2019年, 至 2021年 - 基盤研究(A)
小型染色体の構造・機能解析によるフザリウム菌の病原性分化機構の解明
自 2018年, 至 2018年 - 基盤研究(A)
小型染色体の構造・機能解析によるフザリウム菌の病原性分化機構の解明
自 2017年, 至 2017年 - 基盤研究(A)
小型染色体の構造・機能解析によるフザリウム菌の病原性分化機構の解明
自 2016年, 至 2016年 - 若手研究(B)
植物RNAウイルスによるプログラム細胞死抑制機構の解明
自 2014年, 至 2015年
論文
- First report of southern blight on showy rattlepod (Crotalaria spectabilis) caused by Athelia rolfsii
Chen, Sarina; Kotera, Shunsuke; Saito, Hiroki; Komatsu, Ken; Arie, Tsutomu
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
In 2019-2020, showy rattlepod (Crotalaria spectabilis) in Tokyo developed symptoms including yellowing, wilt, and damping off. A fungus forming white mycelia and brown sclerotia on potato dextrose agar plate was isolated, which reproduced the original symptoms on showy rattlepod by inoculation assay. The fungus was identified as Athelia rolfsii by morphological and molecular characteristics. The host range of the fungus was examined which suggested a wide spectrum. This is the first report of showy rattlepod southern blight caused by A. rolfsii in Japan.
2023年11月, 研究論文（学術雑誌）, 共同, 89, 6, 1345-2630, DOI(公開)(r-map), 322, 327 - Successful full-length genomic cloning and characterization of site-specific nick structures of Phytophthora endornaviruses 2 and 3 in yeast, Saccharomyces cerevisiae
Sakuta, Kohei; Uchida, Keiko; Fukuhara, Toshiyuki; Komatsu, Ken; Okada, Ryo; Moriyama, Hiromitsu
FRONTIERS IN MICROBIOLOGY
FRONTIERS MEDIA SA
Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5 ' and 3 ' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5 ' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3. We have successfully constructed full-length cDNA clones of the entire RNA genomes of PEV2 and PEV3 using a homologous recombination system in the yeast, Saccharomyces cerevisiae. These full-length cDNA sequences were ligated downstream of a constitutive expression promoter (TDH3) or a galactose inducing promoter (GAL') in the shuttle vector to enable the production of the full-length RNA transcripts of PEV2 and PEV3 in yeast cells. Interestingly, a 1.0 kb RNA fragment from the PEV3 positive-strand transcript was also detected with a 5 '-region RNA probe, indicating that site-specific cleavage also occurred in yeast cells. Further, when PEV2 or PEV3 mRNA was overexpressed under the GAL' promoter, yeast cell growth was suppressed. A fusion protein combining EGFP to the N-terminus of the full-length PEV2 ORF or C-terminus of the full-length PEV3 ORF was expressed, and allowed PEV2 and PEV3 ORFs to be successfully visualized in yeast cells. Expression of the fusion protein also revealed presence of heterogeneous bodies in the cells.
2023年09月12日, 研究論文（学術雑誌）, 共同, 14, DOI(公開)(r-map) - Emergence of a deletion mutant of GFP-expressing plantago asiatica mosaic virus that has overcome acibenzolar-S-methyl-induced defense response against its long-distance movement
Sakugawa, Kagari; Novianti, Fawzia; Hamim, Islam; Arie, Tsutomu; Komatsu, Ken
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
The rapid emergence of new virus mutants that are adapted to host environments increases the threat to crop production. In a previous study, we reported that pretreatment of Nicotiana benthamiana (Nb) with a plant activator, acibenzolar-S-methyl (ASM), inhibited the infection of green fluorescent protein-expressing plantago asiatica mosaic virus (PlAMV-GFP) at two distinct stages: replication in inoculated leaves and the viral loading step for long-distance movement. However, whether the virus might mutate that will overcome host ASM-induced defenses at each of these stages is unknown. To address this question, we performed five serial passages of PlAMV-GFP in either ASM-treated or untreated Nb leaves. A virus lineage passaged in ASM-treated leaves, named the ASM lineage, had no dominant mutations and was unable to overcome ASM-mediated inhibition. The ASM lineage tended to have less genetic diversity than in the control lineage. In contrast, in a petiole of ASM-treated, inoculated leaves where viruses had accumulated after loading into the vascular tissue, we found a mutant with a 693-bp deletion in the GFP-coding region and an extension of 13 amino acids at the C-terminus of the movement protein TGBp3 that had less restricted accumulation in inoculated leaves and moved to the upper leaves faster than the wild-type PlAMV-GFP in ASM-treated plants. The emergence of this defense-evading mutant specifically from the loading stage suggests that the ASM-induced defense could impose selective pressure on viruses at the viral loading stage rather than at replication.
2023年09月, 研究論文（学術雑誌）, 共同, 89, 5, 1345-2630, DOI(公開)(r-map), 266, 276 - Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication
Shinji, Haruka; Sasaki, Nobumitsu; Hamim, Islam; Itoh, Yoshiyuki; Taku, Kazuo; Hayashi, Yuho; Minato, Nami; Moriyama, Hiromitsu; Arie, Tsutomu; Komatsu, Ken
VIRUS RESEARCH
ELSEVIER
Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the inter-action of NbDRP2 with the MET domain in PlAMV replication.
2023年07月02日, 研究論文（学術雑誌）, 共同, 331, 0168-1702, DOI(公開)(r-map) - Phylogenetic and population genetic analyses of plantago asiatica mosaic virus isolates reveal intraspecific diversification
Furuya, Minako; Tanai, Shino; Hamim, Islam; Yamamoto, Yuna; Abe, Haruki; Imai, Kazuho; Saito, Hiroki; Yamashita, Kazuo; Uehara-Ichiki, Tamaki; Hanada, Kaoru; Kon, Tatsuya; Inoue, Maki N.; Minato, Nami; Fuji, Shin-ichi; Komatsu, Ken
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
Plantago asiatica mosaic virus (PlAMV) infects ornamental lilies and causes severe necrotic symptoms. PlAMV has also been isolated from several perennial weeds, including Plantago asiatica. While multiple isolates from cultivated lilies have been identified and shown to share a high degree of sequence identity with one another, there have been few studies of PlAMV isolates from weeds. Furthermore, a comprehensive phylogenetic analysis of PIAMV isolates from both lilies and weeds has not been performed. Here, we performed phylogenetic and population genetic analyses of the full-length genome sequences of 36 PlAMV isolates, including 14 new isolates obtained in this study. These analyses suggested intraspecific diversification of PlAMV isolates into five clades, including a clade consisting of ornamental lily isolates (Lily clade) and four other clades consisting of isolates from weeds or medicinal plants. The specific clade membership identified by the phylogenetic analysis was mainly correlated with the geographical location of the members of each clade. All PlAMV isolates except those in the Lily clade had a high level of genetic diversity. The Lily clade had a higher level of genetic differentiation from the other clades. The neutrality test suggested that genetic drift contributed to shaping PlAMV isolates in the Lily clade. Infectivity tests with infectious clones of the representative PIAMV isolates to four plant species, including lilies and P. asiatica, showed that isolates in different phylogenetic clades had variation in infectivity to the subset of plant species.
2023年07月, 研究論文（学術雑誌）, 共同, 89, 4, 1345-2630, DOI(公開)(r-map), 224, 237 - Effects of soil types and fertility management practices on soil silicon availability and banana silicon uptake
Fujii, Kazumichi; Ichinose, Yuri; Arai, Kazuna; Komatsu, Ken; Hayakawa, Chie; Alvindia, Dionisio de Guzman; Watanabe, Kyoko; Hartono, Arief
SOIL SCIENCE AND PLANT NUTRITION
TAYLOR & FRANCIS LTD
Although silicon (Si) is one of soil abundant constituents and one of the key elements enhancing plant disease resistance, banana uptake of Si is limited by soil Si availability. It remains unclear what factors regulates soil Si availability and whether banana Si uptake increases with increasing soil Si availability in home garden soils. To test whether soil Si availability and banana leaf SiO2 contents are affected by soil types or fertility management practices, we measured (1) banana leaf SiO2 contents along the gradient of livestock inputs in Andisols of Tanzania and (2) water extractable Si, phosphate-buffer extractable Si, and banana leaf SiO2 in the home garden soils with different degree of volcanic ash admixing and with/without leguminous trees in Indonesia (East Java and East Kalimantan). Livestock dung application increased banana leaf SiO2 contents in the Tanzanian home gardens. Water extractable Si increased with increasing soil pH, but soil phosphate-buffer extractable Si and banana leaf SiO2 contents were strongly regulated by oxalate-extractable Si and andic properties in the Indonesian home gardens. Effects of companion trees (agroforestry) increased banana leaf SiO2 only in two of three Andisols (Java). These results suggest that banana leaf SiO2 contents could be primarily regulated by admixing degree of volcanic parent materials rich in weatherable minerals, but that banana leaf SiO2 contents could also be increased by inputs of livestock dung rich in Si (Tanzania) and agroforestry (Indoensia).
2023年05月04日, 研究論文（学術雑誌）, 共同, 69, 3, 0038-0768, DOI(公開)(r-map), 183, 189 - Viruses in agricultural systems: Interactions with plants, insect pollinators and fungi
Hamim, Islam; Komatsu, Ken; Prager, Sean Michael; Wu, Beilei
FRONTIERS IN MICROBIOLOGY
FRONTIERS MEDIA SA
2023年03月23日, 研究論文（学術雑誌）, 共同, 14, DOI(公開)(r-map) - Three species of Fusarium involved in the dry rot of taro (Colocasia esculenta) in Kagoshima Prefecture, Japan
Sireli, Masi; Kodama, Shinichirou; Ikezawa, Kazuhiro; Nishi, Yatsuka; Kawabe, Masato; Motohashi, Reiko; Komatsu, Ken; Arie, Tsutomu
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
In 2018 and 2019, taro (Colocasia esculenta) plants presenting symptoms including a reddish-brown dry rot of the corms and wilting of the leaves were found in several fields in Kagoshima, Japan. Fusarium solani, F. proliferatum and F. oxysporum were isolated and identified based on morphological and molecular characteristics. Among the three fungal species F. solani was the most frequently found. We inoculated taro cv. Dotare corms with representative isolates of each species, and all three isolates reproduced the original symptoms. This is the first report of F. proliferatum as a causal agent of taro dry rot in Japan.
2023年01月, 研究論文（学術雑誌）, 共同, 89, 1, 1345-2630, DOI(公開)(r-map), 16, 23 - Mating Types of Ustilago esculenta Infecting Zizania latifolia Cultivars in Japan Are Biased towards MAT-2 and MAT-3
Chigira, Yuka; Sasaki, Nobumitsu; Komatsu, Ken; Mashim, Kouji; Tanaka, Shigeyuki; Numamoto, Minori; Moriyama, Hiromitsu; Motobayashi, Takashi
MICROBES AND ENVIRONMENTS
JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE
Zizania latifolia cultivars infected by the endophytic fungus Ustilago esculenta develop an edible stem gall. Stem gall development varies among cultivars and individuals and may be affected by the strain of U. esculenta. To isolate haploids from two Z. latifolia cultivars in our paddy fields, Shirakawa and Ittenkou, we herein performed the sporadic isolation of U. esculenta strains from stem gall tissue, a PCR-based assessment of the mating type, and in vitro mating experiments. As a result, we obtained heterogametic strains of MAT-2 and MAT-3 as well as MAT-2, but not MAT-3, haploid strains. Another isolation method, in which we examined poorly growing small clusters of sporidia derived from teliospores, succeeded in isolating a MAT-3 haploid strain. We also identified the mating types of 10 U. esculenta strains collected as genetic resources from different areas in Japan. All strains, except for one MAT-1 haploid strain, were classified as MAT-2 haploid strains or heterogametic strains of MAT-2 and MAT-3. The isolated strains of MAT-1, MAT-2, and MAT-3 mated with each other to produce hyphae. Collectively, these results indicate that the mating types of U. esculenta infecting Z. latifolia cultivars in Japan are biased towards MAT-2 and MAT-3 and that U. esculenta populations in these Japanese cultivars may be characterized by the low isolation efficiency of the MAT-3 haploid.
2023年, 研究論文（学術雑誌）, 共同, 38, 3, 1342-6311, DOI(公開)(r-map) - How do emerging long-read sequencing technologies function in transforming the plant pathology research landscape?
Hamim, Islam; Sekine, Ken-Taro; Komatsu, Ken
PLANT MOLECULAR BIOLOGY
SPRINGER
Key message Long-read sequencing technologies are revolutionizing the sequencing and analysis of plant and pathogen genomes and transcriptomes, as well as contributing to emerging areas of interest in plant-pathogen interactions, disease management techniques, and the introduction of new plant varieties or cultivars. Long-read sequencing (LRS) technologies are progressively being implemented to study plants and pathogens of agricultural importance, which have substantial economic effects. The variability and complexity of the genome and transcriptome affect plant growth, development and pathogen responses. Overcoming the limitations of second-generation sequencing, LRS technology has significantly increased the length of a single contiguous read from a few hundred to millions of base pairs. Because of the longer read lengths, new analysis methods and tools have been developed for plant and pathogen genomics and transcriptomics. LRS technologies enable faster, more efficient, and high-throughput ultralong reads, allowing direct sequencing of genomes that would be impossible or difficult to investigate using short-read sequencing approaches. These benefits include genome assembly in repetitive areas, creating more comprehensive and exact genome determinations, assembling full-length transcripts, and detecting DNA and RNA alterations. Furthermore, these technologies allow for the identification of transcriptome diversity, significant structural variation analysis, and direct epigenetic mark detection in plant and pathogen genomic regions. LRS in plant pathology is found efficient for identifying and characterization of effectors in plants as well as known and unknown plant pathogens. In this review, we investigate how these technologies are transforming the landscape of determination and characterization of plant and pathogen genomes and transcriptomes efficiently and accurately. Moreover, we highlight potential areas of interest offered by LRS technologies for future study into plant-pathogen interactions, disease control strategies, and the development of new plant varieties or cultivars.
2022年12月, 研究論文（学術雑誌）, 共同, 110, 6, 0167-4412, DOI(公開)(r-map), 469, 484 - First report of brown stripe on perennial ryegrass (Lolium perenne L.) caused by Acidovorax avenae
Netsu, Osamu; Komatsu, Ken; Yoshimura, Takuma; Takikawa, Yuichi; Matsuda, Kazunori; Tanaka, Akemi
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
In summer 2018, brown stripe symptoms were observed on leaf blades of perennial ryegrass (Lolium perenne L.) used as a turfgrass in an athletic field in Saitama Prefecture, Japan. Bacterial ooze exuded from lesions, from which a pathogenic bacterium was isolated. Inoculation of blades of perennial ryegrass with the isolates reproduced the same symptoms. Pathogenicity and bacteriological properties of the isolates were almost identical to those of Acidovorax avenae, and the identification was supported by phylogenetic analysis. This is the first report of the brown stripe on perennial ryegrass caused by A. avenae (= A. avenae subsp. avenae) in Japan.
2022年11月, 研究論文（学術雑誌）, 共同, 88, 6, 1345-2630, DOI(公開)(r-map), 399, 404 - Discovery, Genomic Sequence Characterization and Phylogenetic Analysis of Novel RNA Viruses in the Turfgrass Pathogenic Colletotrichum spp. in Japan
Hamim, Islam; Urayama, Syun-ichi; Netsu, Osamu; Tanaka, Akemi; Arie, Tsutomu; Moriyama, Hiromitsu; Komatsu, Ken
VIRUSES-BASEL
MDPI
Turfgrass used in various areas of the golf course has been found to present anthracnose disease, which is caused by Colletotrichum spp. To obtain potential biological agents, we identified four novel RNA viruses and obtained full-length viral genomes from turfgrass pathogenic Colletotrichum spp. in Japan. We characterized two novel dsRNA partitiviruses: Colletotrichum associated partitivirus 1 (CaPV1) and Colletotrichum associated partitivirus 2 (CaPV2), as well as two negative single-stranded (ss) RNA viruses: Colletotrichum associated negative-stranded RNA virus 1 (CaNSRV1) and Colletotrichum associated negative-stranded RNA virus 2 (CaNSRV2). Using specific RT-PCR assays, we confirmed the presence of CaPV1, CaPV2 and CaNSRV1 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-264, as well as CaNSRV2 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-288. This is the first time mycoviruses have been discovered in turfgrass pathogenic Colletotrichum spp. in Japan. CaPV1 and CaPV2 are new members of the newly proposed genus Zetapartitivirus and genus Alphapartitivirus, respectively, in the family Partitiviridae, according to genomic characterization and phylogenetic analysis. Negative sense ssRNA viruses CaNSRV1 and CaNSRV2, on the other hand, are new members of the family Phenuiviridae and the proposed family Mycoaspirividae, respectively. These findings reveal previously unknown RNA virus diversity and evolution in turfgrass pathogenic Colletotrichum spp.
2022年11月, 研究論文（学術雑誌）, 共同, 14, 11, DOI(公開)(r-map) - Plantago asiatica mosaic virus: An emerging plant virus causing necrosis in lilies and a new model RNA virus for molecular research
Komatsu, Ken; Hammond, John
MOLECULAR PLANT PATHOLOGY
WILEY
Taxonomy Plantago asiatica mosaic virus belongs to the genus Potexvirus in the family Alphaflexiviridae of the order Tymovirales. Virion and genome properties Plantago asiatica mosaic virus (PlAMV) has flexuous virions of approximately 490-530 nm in length and 10-15 nm in width. The genome of PlAMV consists of a single-stranded, positive-sense RNA of approximately 6.13 kb. It contains five open reading frames (ORFs 1-5), encoding a putative viral polymerase (RdRp), movement proteins (triple gene block proteins, TGBp1-3), and coat protein (CP), respectively. Host range PlAMV has an exceptionally wide host range and has been isolated from various wild plants, including Plantago asiatica, Nandina domestica, Rehmannia glutinosa, and other weed plants. Experimentally PlAMV can infect many plant species including Nicotiana benthamiana and Arabidopsis thaliana. It also infects ornamental lilies and frequently causes severe necrotic symptoms. However, host range varies depending on isolates, which show significant biological diversity within the species. Genome diversity PlAMV can be separated into five clades based on phylogenetic analyses; nucleotide identities are significantly low between isolates in the different clades. Transmission PlAMV is not reported to be transmitted by biological vectors. Virions of PlAMV are quite stable and it can be transmitted efficiently by mechanical contact. Disease symptoms PlAMV causes red-rusted systemic necrosis in ornamental lilies, but it shows much weaker, if any, symptoms in wild plants such as P. asiatica. Control Control of the disease caused by PlAMV is based mainly on rapid diagnosis and elimination of the infected bulbs or plants.
2022年10月, 研究論文（学術雑誌）, 共同, 23, 10, 1464-6722, DOI(公開)(r-map), 1401, 1414 - Ophiosphaerellaagrostidis causes leaf-sheath rot of Zingibermioga
Oki, Tomoka; Yano, Kazutaka; Komatsu, Ken; Shimomoto, Yoshifumi; Arie, Tsutomu; Morita, Yasuaki
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
In 2014 and 2018, brown rot appeared on the leaf sheaths of mioga (Zingibermioga) plants growing in Kochi, Japan. A filamentous fungus that formed pale red colonies on potato dextrose agar was isolated from the lesions and reproduced the symptoms observed on mioga plants in inoculation tests. On the basis of microscopic characters and phylogenetic analysis, the fungus was identified as Ophiosphaerellaagrostidis. This is the first report of O.agrostidis as a causal agent of a disease on mioga plants. We propose to name the new disease leaf sheath rot and yosho fuhai byo in Japanese.
2022年05月, 研究論文（学術雑誌）, 共同, 88, 3, 1345-2630, DOI(公開)(r-map), 173, 177 - Differentiation of the Pea Wilt Pathogen Fusarium oxysporum f. sp. pisi from Other Isolates of Fusarium Species by PCR
Kotera, Shunsuke; Hishiike, Masashi; Saito, Hiroki; Komatsu, Ken; Arie, Tsutomu
MICROBES AND ENVIRONMENTS
JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE
Pea wilt disease, caused by the soilborne and seedborne fungal pathogen Fusarium oxysporum f. sp. pisi (Fop), first appeared in Japan in 2002. We herein investigated the molecular characteristics of 16 Fop isolates sampled from multiple locations and at different times in Japan. The 16 isolates were divided into three clades in molecular phylogenic analyses based on both the TEF1 alpha gene and the rDNA-IGS region. All of the Fop isolates harbored a PDA1 gene, which encodes the cytochrome P450 pisatin demethylase (Pda1), and also carried one or both of the SIX6 and SIX13 genes, which encode secreted in xylem (Six) proteins. Other forms of F. oxysporum and other species of Fusarium did not carry these sets of genes. Based on these results, a PCR method was developed to identify Fop and differentiate it from other forms and non-pathogenic isolates of Fusarium spp. We also demonstrated that the PCR method effectively detected Fop in infected pea plants and infested soils.
2022年, 研究論文（学術雑誌）, 共同, 37, 1, 1342-6311, DOI(公開)(r-map) - Fusariosis in rubber tree: pathogenic, morphological, and molecular characterization of the causal agent
Pizetta, Marilia; Pierozzi, Caroline Geraldi; Ayukawa, Yu; Kashiwa, Takeshi; Komatsu, Ken; Teraoka, Tohru; Arie, Tsutomu; Furtado, Edson Luiz
EUROPEAN JOURNAL OF PLANT PATHOLOGY
SPRINGER
Fusariosis, one of the phytosanitary problems found in rubber producing areas in the northwest of the state of Sao Paulo, is a disease that affects the bark of the adult plants, affecting the exploitation of latex. The typical symptoms appear as cracks in the bark that expand from the rootstock towards the tapping panel, causing a drying of the latex flux in the injured region, impeding latex tapping. Due to the recent incidence of this disease in rubber plantations, the goal of this study was to characterize the Fusarium associated with symptomatic rubber tissue in three different locations in the state of Sao Paulo. In order to identify Fusarium species, pathogenicity, morphological, cultural and molecular studies were carried out. A total of 51 isolates were obtained and separated into three groups based on macroconidium morphology, presence or absence of sporodochia, types of chlamydospores, formation of phialides and conidiogenesis of microconidia and mesoconidia, mycelial growth rate and coloring of the colonies. These groups were corroborated using DNA sequence information for five different genetic loci, and were subsequently recognized as Fusarium oxysporum, F. incarnatum and F. decemcellulare. Our results further showed that all 51 of the Fusarium isolates recovered were pathogenic to rubber tree seedlings of RRIM 600 standard clone. This study also reports for the presence of F. oxysporum and F. incarnatum in rubber plantations in the state of Sao Paulo and in Brazil.
2021年12月, 研究論文（学術雑誌）, 共同, 161, 4, 0929-1873, DOI(公開)(r-map), 769, 782 - Acibenzolar-S-methyl-mediated restriction of loading of plantago asiatica mosaic virus into vascular tissues of Nicotiana benthamiana
Novianti, Fawzia; Sasaki, Nobumitsu; Arie, Tsutomu; Komatsu, Ken
VIRUS RESEARCH
ELSEVIER
Long-distance movement via vascular tissues is an essential step for systemic infection by plant viruses. We previously reported that pre-treatment of Nicotiana benthamiana with acibenzolar-S-methyl (ASM) both suppressed the accumulation of plantago asiatica mosaic virus (PlAMV) in inoculated leaves and delayed the longdistance movement to uninoculated upper leaves. These two effects occurred independently of each other. However, it remained unclear where and when the viral long-distance movement is inhibited upon ASM treatment. In this study, we found that ASM treatment restricted the loading of GFP-expressing PlAMV (PlAMV-GFP) into vascular tissues in the inoculated leaves. This led to delays in viral translocation to the petiole and the main stem, and to untreated upper leaves. We used cryohistological fluorescence imaging to show that ASM treatment affected the viral localization and reduced its accumulation in the phloem, xylem, and mesophyll tissues. A stem girdling experiment, which blocked viral movement downward through phloem tissues, demonstrated that ASM treatment could inhibit viral systemic infection to upper leaves, which occurred even with viral downward movement restricted. Taken together, our results showed that ASM treatment affects the loading of PlAMV-GFP into the vascular system in the inoculated leaf, and that this plays a key role in the ASM-mediated delay of viral long-distance movement.
2021年12月, 研究論文（学術雑誌）, 共同, 306, 0168-1702, DOI(公開)(r-map) - Unique Terminal Regions and Specific Deletions of the Segmented Double-Stranded RNA Genome of Alternaria Alternata Virus 1, in the Proposed Family Alternaviridae
Wu, Chien-Fu; Aoki, Nanako; Takeshita, Naoki; Fukuhara, Toshiyuki; Chiura, Hiroshi X.; Arie, Tsutomu; Kotta-Loizou, Ioly; Okada, Ryo; Komatsu, Ken; Moriyama, Hiromitsu
FRONTIERS IN MICROBIOLOGY
FRONTIERS MEDIA SA
Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35-193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1-4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36-50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35-193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.
2021年10月22日, 研究論文（学術雑誌）, 共同, 12, DOI(公開)(r-map) - Identification of a Proline-Kinked Amphipathic alpha-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation
Komatsu, Ken; Sasaki, Nobumitsu; Yoshida, Tetsuya; Suzuki, Katsuhiro; Masujima, Yuki; Hashimoto, Masayoshi; Watanabe, Satoru; Tochio, Naoya; Kigawa, Takanori; Yamaji, Yasuyuki; Oshima, Kenro; Namba, Shigetou; Nelson, Richard S.; Arie, Tsutomu
JOURNAL OF VIROLOGY
AMER SOC MICROBIOLOGY
Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic alpha-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic alpha-helical configuration and unveiled a kink caused by a highly conserved proline residue in the alpha-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic alpha-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the alpha-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic alpha-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic alpha-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.
2021年10月, 研究論文（学術雑誌）, 共同, 95, 20, 0022-538X, DOI(公開)(r-map) - A pair of effectors encoded on a conditionally dispensable chromosome of Fusarium oxysporum suppress host-specific immunity
Ayukawa, Yu; Asai, Shuta; Gan, Pamela; Tsushima, Ayako; Ichihashi, Yasunori; Shibata, Arisa; Komatsu, Ken; Houterman, Petra M.; Rep, Martijn; Shirasu, Ken; Arie, Tsutomu
COMMUNICATIONS BIOLOGY
NATURE RESEARCH
Yu Ayukawa, Shuta Asai, et al. report the genome sequence of a Fusarium oxysporum isolate and demonstrate that it contains different conditionally dispensable chromosomes which are important to confer virulence on specific hosts, like Arabidopsis thaliana or cabbage. Altogether, these results provide further insight into the mechanisms underlying F. oxysporum pathogenicity. Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis (Arabidopsis thaliana) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.
2021年06月09日, 研究論文（学術雑誌）, 共同, 4, 1, DOI(公開)(r-map) - Cell-death-independent antiviral response mediated by N resistance factor in Nicotiana benthamiana involves inhibited localization of tobamovirus movement protein to plasmodesmata
Sasaki, Nobumitsu; Murakami, Tomoya; Yoshimoto, Nanae; Komatsu, Ken; Matsushita, Yasuhiko; Nyunoya, Hiroshi
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
The tobacco N gene encodes the N resistance factor that recognizes a tobacco mosaic virus elicitor called p50 to induce a cell-death-associated hypersensitive reaction, which confines virus infections to local areas. Coexpression of N and p50 cDNAs in Nicotiana benthamiana induces resistance against virus infection without visible cell death. In this study, we examined how this N-mediated resistance in the heterologous plant inhibits virus infection. After inoculation with GFP-encoding tomato mosaic virus (ToMV), plants coexpressing N and p50 had fewer and smaller infection sites as the levels of coexpressed proteins of N and p50 increased. This antiviral response was associated with production of reactive oxygen species but not ion leakage or expression of PR1a (encoding a salicylic acid signaling marker). In addition, it was likely that GFP-tagged movement protein (MP) of ToMV seemed to have failed to localize at plasmodesmata during virus infection in leaves coexpressing N and p50. These findings indicate that the cell death-independent, N-mediated antiviral response in N. benthamiana may involve interference with the intracellular transport function of MP.
2021年05月, 研究論文（学術雑誌）, 共同, 87, 3, 1345-2630, DOI(公開)(r-map), 170, 177 - Resistance to demethylation inhibitors in Cercospora beticola, a pathogen of sugar beet in Japan, and development of unique cross-resistance patterns
Kayamori, Miyuki; Zakharycheva, Alisa; Saito, Hiroki; Komatsu, Ken
EUROPEAN JOURNAL OF PLANT PATHOLOGY
SPRINGER
Cercospora beticola Sacc. is the most destructive pathogen of sugar beet (Beta vulgaris L.) and causes Cercospora leaf spot (CLS). Since 1986, fungicides that function as demethylation inhibitors (DMIs) have been used to control CLS in Hokkaido, which is the only area in Japan where sugar beet is grown. Reduced sensitivity of C. beticola to DMI fungicides, based on the half maximal effective concentration (EC50), was first reported in Hokkaido in 1999, however the fungicides continued to be used effectively until 2014. In a field experiment in 2016, we found that the efficacy of difenoconazole against the field population of C. beticola was greatly reduced. We subsequently tested over 600 isolates collected throughout the sugar beet-growing region of Hokkaido and revealed that the mean resistance factor of four DMI fungicides (difenoconazole, fenbuconazole, tebuconazole, and tetraconazole) were high, which indicates that DMI-resistant isolates were distributed throughout the beet cultivation area. Moreover, we identified three types of isolates that have unique cross-resistance patterns between difenoconazole and fenbuconazole, with their EC50 rate (= difenoconazole EC50/ fenbuconazole EC50) converged to 31, 4.0, and 0.40, respectively, which appeared to be affected by the local history of fungicide usage. The F144L substitution in CbCYP51 was only found in the group whose EC50 rate was 0.40. This is the first report of DMI resistance in C. beticola in Japan, and the findings in this study could contribute to our understanding of the mechanism of DMI resistance.
2021年05月, 研究論文（学術雑誌）, 共同, 160, 1, 0929-1873, DOI(公開)(r-map), 39, 52 - Two Novel Endornaviruses Co-infecting a Phytophthora Pathogen of Asparagus officinalis Modulate the Developmental Stages and Fungicide Sensitivities of the Host Oomycete
Uchida, Keiko; Sakuta, Kohei; Ito, Aori; Takahashi, Yumi; Katayama, Yukie; Omatsu, Tsutomu; Mizutani, Tetsuya; Arie, Tsutomu; Komatsu, Ken; Fukuhara, Toshiyuki; Uematsu, Seiji; Okada, Ryo; Moriyama, Hiromitsu
FRONTIERS IN MICROBIOLOGY
FRONTIERS MEDIA SA
Two novel endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3) were found in isolates of a Phytophthora pathogen of asparagus collected in Japan. A molecular phylogenetic analysis indicated that PEV2 and PEV3 belong to the genus Alphaendornavirus. The PEV2 and PEV3 genomes consist of 14,345 and 13,810 bp, and they contain single open reading frames of 4,640 and 4,603 codons, respectively. Their polyproteins contain the conserved domains of an RNA helicase, a UDP-glycosyltransferase, and an RNA-dependent RNA polymerase, which are conserved in other alphaendornaviruses. PEV2 is closely related to Brown algae endornavirus 2, whereas PEV3 is closely related to Phytophthora endornavirus 1 (PEV1), which infects a Phytophthora sp. specific to Douglas fir. PEV2 and PEV3 were detected at high titers in two original Phytophthora sp. isolates, and we found a sub-isolate with low titers of the viruses during subculture. We used the high- and low-titer isolates to evaluate the effects of the viruses on the growth, development, and fungicide sensitivities of the Phytophthora sp. host. The high-titer isolates produced smaller mycelial colonies and much higher numbers of zoosporangia than the low-titer isolate. These results suggest that PEV2 and PEV3 inhibited hyphal growth and stimulated zoosporangium formation. The high-titer isolates were more sensitive than the low-titer isolate to the fungicides benthiavalicarb-isopropyl, famoxadone, and chlorothalonil. In contrast, the high-titer isolates displayed lower sensitivity to the fungicide metalaxyl (an inhibitor of RNA polymerase I) when compared with the low-titer isolate. These results indicate that persistent infection with PEV2 and PEV3 may potentially affect the fungicide sensitivities of the host oomycete.
2021年02月03日, 研究論文（学術雑誌）, 共同, 12, DOI(公開)(r-map) - Antifungal activity of bacteria isolated from Japanese frog skin against plant pathogenic fungi
Susilawati, Lela; Iwai, Noriko; Komatsu, Ken; Arie, Tsutomu
BIOLOGICAL CONTROL
ACADEMIC PRESS INC ELSEVIER SCIENCE
Frogs carry bacterial communities on their skin (Harris et al., 2009) that benefit the host frogs by preventing harmful pathogen infections. Our study aimed to collect culturable bacteria from the skin of wild frogs sampled in Japan and evaluate the antagonistic activity of the bacteria toward plant pathogenic fungi. A collection of 106 bacterial isolates was obtained from three species of frogs, namely Hyla japonica, Pelophylax porosus porosus and Buergeria burgeri. Using a dual-culture method, three isolates, HjD52, HjD92 and B341, were selected based on their ability to significantly inhibit the growth of Colletotrichum orbiculare, the causal fungus of cucumber anthracnose disease. These three bacterial isolates also showed a broad-spectrum of antagonistic activity against plant pathogenic fungi. Furthermore, spray treatment with the bacterial suspensions (109 cfu/ml) effectively reduced the number of anthracnose lesions in greenhouse-grown, potted cucumber plants. Based on the 16S rDNA sequence analysis and similarity search, isolates HjD57, HjD92 and B341 were identified as Paenibacillus sp., Raoultella sp. and Citrobacter sp., respectively. This is the first report showing the potential of Paenibacillus sp., Raoultella sp. and Citrobacter sp. from frog skin to serve as potent biocontrol agents against plant diseases.
2021年02月, 研究論文（学術雑誌）, 共同, 153, 1049-9644, DOI(公開)(r-map) - Spray Application of Nonpathogenic Fusaria onto Rice Flowers Controls Bakanae Disease (Caused by Fusarium fujikuroi) in the Next Plant Generation
Saito, Hiroki; Sasaki, Mai; Nonaka, Yoko; Tanaka, Jun; Tokunaga, Tomomi; Kato, Akihiro; Tran Thi Thu Thuy; Le Van Vang; Le Minh Tuong; Kanematsu, Seiji; Suzukie, Tomotaka; Kurauchi, Kenichi; Fujitaa, Naoko; Teraoka, Tohru; Komatsu, Ken; Arie, Tsutomu
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
AMER SOC MICROBIOLOGY
Bakanae disease, caused by Fusarium fujikuroi, is an economically important seed-borne disease of rice. F. fujikuroi is horizontally transmitted to rice flowers and vertically transmitted to the next generation via seeds. The fungus induces typical symptoms such as abnormal tissue elongation and etiolation. Sanitation of seed farms and seed disinfection are the only effective means to control bakanae disease at present; however, the efficacy of these methods is often insufficient. Therefore, alternative and innovative control methods are necessary. We developed a novel method for applying nonpathogenic fusaria as biocontrol agents by spraying spore suspensions onto rice flowers to reduce the incidence of seed-borne bakanae. We visualized the interaction between Fusarium commune W5, a nonpathogenic fusarium, and Fusarium fujikuroi using transformants expressing two different fluorescent proteins on/in rice plants. W5 inhibited hyphal extension of F. fujikuroi on/in rice flowers and seedlings, possibly by competing with the pathogen, and survived on/in rice seeds for at least 6 months. IMPORTANCE We demonstrated that a spray treatment of rice flowers with the spores of nonpathogenic fusaria mimicked the disease cycle of the seed-borne bakanae pathogen Fusarium fujikuroi and effectively suppressed the disease. Spray treatment of nonpathogenic fusaria reduced the degree of pathogen invasion of rice flowers and vertical transmission of the pathogen to the next plant generation via seeds, thereby controlling the bakanae disease. The most promising isolate, F. commune W5, colonized seeds and seedlings via treated flowers and successfully inhibited pathogen invasion, suggesting that competition with the pathogen was the mode of action. Seed-borne diseases are often controlled by seed treatment with chemical fungicides. Establishing an alternative method is a pressing issue from the perspectives of limiting fungicide resistance and increasing food security. This work provides a potential solution to these issues using a novel application technique to treat rice flowers with biocontrol agents.
2021年01月, 研究論文（学術雑誌）, 共同, 87, 2, 0099-2240, DOI(公開)(r-map) - Mutations Found in the Asc1 Gene That Confer Susceptibility to the AAL-Toxin in Ancestral Tomatoes from Peru and Mexico
Tsuzuki, Rin; Cabrera Pintado, Rosa Maria; Biondi Thorndike, Jorge Andres; Gutierrez Reynoso, Dina Lida; Amasifuen Guerra, Carlos Alberto; Guerrero Abad, Juan Carlos; Aragon Caballero, Liliana Maria; Huarhua Zaquinaula, Medali Heidi; Ureta Sierra, Cledy; Alberca Cruz, Olenka Ines; Elespuru Suna, Milca Gianira; Blas Sevillano, Raul Humberto; Torres Arias, Ines Carolina; Flores Ticona, Joel; de Baldarrago, Fatima Caceres; Perez, Enrique Rodoriguez; Hozum, Takuo; Saito, Hiroki; Kotera, Shunsuke; Akagi, Yasunori; Kodama, Motoichiro; Komatsu, Ken; Arie, Tsutomu
PLANTS-BASEL
MDPI
Tomato susceptibility/resistance to stem canker disease caused by Alternaria alternata f. sp. lycopersici and its pathogenic factor AAL-toxin is determined by the presence of the Asc1 gene. Several cultivars of commercial tomato (Solanum lycopersicum var. lycopersicum, SLL) are reported to have a mutation in Asc1, resulting in their susceptibility to AAL-toxin. We evaluated 119 ancestral tomato accessions including S. pimpinellifolium (SP), S. lycopersicum var. cerasiforme (SLC) and S. lycopersicum var. lycopersicum jitomate criollo (SLJ) for AAL-toxin susceptibility. Three accessions, SP PER018805, SLC PER018894, and SLJ M5-3, were susceptible to AAL-toxin. SLC PER018894 and SLJ M5-3 had a two-nucleotide deletion (nt 854_855del) in Asc1 identical to that found in SLL cv. Aichi-first. Another mutation (nt 931_932insT) that may confer AAL-toxin susceptibility was identified in SP PER018805. In the phylogenetic tree based on the 18 COSII sequences, a clade (S3) is composed of SP, including the AAL-toxin susceptible PER018805, and SLC. AAL-toxin susceptible SLC PER018894 and SLJ M5-3 were in Clade S2 with SLL cultivars. As SLC is thought to be the ancestor of SLL, and SLJ is an intermediate tomato between SLC and SLL, Asc1s with/without the mutation seem to have been inherited throughout the history of tomato domestication and breeding.
2021年01月, 研究論文（学術雑誌）, 共同, 10, 1, DOI(公開)(r-map) - Direct and sensitive detection of a microsporidian parasite of bumblebees using loop-mediated isothermal amplification (LAMP)
Kato, Yuto; Yanagisawa, Takahiro; Nakai, Madoka; Komatsu, Ken; Inoue, Maki N.
SCIENTIFIC REPORTS
NATURE PUBLISHING GROUP
The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction. Although the decline of bee populations affects both wild plants and human food supply, the effects of Nosema spp. infections are not known because it is difficult to obtain infective spores from wild bees due to their low prevalence. Microscopical observation of fecal samples or midgut homogenates and/or PCR are generally used for N. bombi detection. However, the germination rate of microsporidian spore declines if they are kept at 4 degrees C for a long time or frozen. It is therefore crucial to minimize the diagnosis and isolation time of infective spores from field-collected samples. Therefore, we performed a loop-mediated isothermal amplification (LAMP) assay for the direct detection of N. bombi in bumblebee midgut homogenates. Using this method, we could detect N. bombi from individuals from which it was visible under the microscope and directly from wild individuals.
2020年01月24日, 研究論文（学術雑誌）, 共同, 10, 1, 2045-2322, DOI(公開)(r-map) - Acibenzolar-S-Methyl Restricts Infection of Nicotiana benthamiana by Plantago Asiatica Mosaic Virus at Two Distinct Stages
Matsuo, Yuki; Novianti, Fawzia; Takehara, Miki; Fukuhara, Toshiyuki; Arie, Tsutomu; Komatsu, Ken
MOLECULAR PLANT-MICROBE INTERACTIONS
AMER PHYTOPATHOLOGICAL SOC
Plant activators, including acibenzolar-S-methyl (ASM), are chemical compounds that stimulate plant defense responses to pathogens. ASM treatment inhibits infection by a variety of plant viruses, however, the mechanisms of this broad-spectrum and strong effect remain poorly understood. We employed green fluorescent protein (GFP)-expressing viruses and Nicotiana benthamiana plants to identify the infection stages that are restricted by ASM. ASM suppressed infection by three viral species, plantago asiatica mosaic virus (PlAMV), potato virus X (PVX), and turnip mosaic virus (TuMV), in inoculated cells. Furthermore, ASM delayed the long-distance movement of PlAMV and PVX, and the cell-to-cell (short range) movement of TuMV. The ASM-mediated delay of long-distance movement of PlAMV was not due to the suppression of viral accumulation in the inoculated leaves, indicating that ASM restricts PIAMV infection in at least two independent steps. We used Arabidopsis thaliana mutants to show that the ASM-mediated restriction of PlAMV infection requires the NPR1 gene but was independent of the dicer-like genes essential for RNA silencing. Furthermore, experiments using protoplasts showed that ASM treatment inhibited PlAMV replication without cell death. Our approach, using GFP-expressing viruses, will be useful for the analysis of mechanisms underlying plant activator-mediated virus restriction.
2019年11月, 研究論文（学術雑誌）, 共同, 32, 11, 0894-0282, DOI(公開)(r-map), 1475, 1486 - Magnaporthe oryzae chrysovirus 1 strain D confers growth inhibition to the host fungus and exhibits multiform viral structural proteins
Higashiura, Tomoya; Katoh, Yu; Urayama, Syun-ichi; Hayashi, Osamu; Aihara, Mitsuhiro; Fukuhara, Toshiyuki; Fuji, Shin-ichi; Kobayashi, Takashi; Hase, Shu; Arie, Tsutomu; Teraoka, Tohru; Komatsu, Ken; Moriyama, Hiromitsu
VIROLOGY
ACADEMIC PRESS INC ELSEVIER SCIENCE
A Japanese isolate of Magnaporthe oryzae is infected by Magnaporthe oryzae chrysovirus 1-D (MoCV1-D), which is classified in cluster II of the family Chrysoviridae. The genome of MoCV1-D consists of five dsRNAs. dsRNAs 1-4 show high identity with those of related MoCV1 viruses, whereas dsRNA5 shows relatively low identity and is sometimes deleted during virus propagation. MoCV1-D causes growth inhibition of its host fungus, and the protein encoded by its dsRNA4 impairs cell growth when expressed in yeast cells. It also causes abnormal pigmentation and colony albinization, and we showed that these phenotypes are associated with reduced accumulation of the melanin biosynthesis intermediate scylatone. MoCV1-D exhibits multiform viral structural proteins during prolonged culture. The original host isolate is co-infected with MoCV1-D, a victorivirus, and a partitivirus, and these mycoviruses are detected in cell-free supernatant fractions after prolonged liquid culturing. Hyphal fusion experiments demonstrated that MoCV1-D is transmissible via anastomosis.
2019年09月, 研究論文（学術雑誌）, 共同, 535, 0042-6822, DOI(公開)(r-map), 241, 254 - Development of the VIGS System in the Dioecious Plant Silene latifolia
Fujita, Naoko; Kazama, Yusuke; Yamagishi, Noriko; Watanabe, Kyoko; Ando, Saki; Tsuji, Hiroyuki; Kawano, Shigeyuki; Yoshikawa, Nobuyuki; Komatsu, Ken
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
MDPI
(1) Background: Silene latifolia is a dioecious plant, whose sex is determined by XY-type sex chromosomes. Microbotryum lychnidis-dioicae is a smut fungus that infects S. latifolia plants and causes masculinization in female flowers, as if Microbotryum were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in S. latifolia plants, we selected Apple latent spherical virus (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, S. latifolia plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in S. latifolia plants. Successful VIGS (virus-induced gene silencing) in S. latifolia plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in S. latifolia plants, which can unveil gene functions and networks of S. latifolia plants, such as the mechanisms of sex determination and fungal-induced masculinization.
2019年03月01日, 研究論文（学術雑誌）, 共同, 20, 5, 1422-0067, DOI(公開)(r-map) - Differences in infectivity and pathogenicity of two Plantago asiatica mosaic virus isolates in lilies
Tanaka, Masashi; Verbeek, Martin; Takehara, Miki; Pham, Khanh; Lemmers, Miriam; Slootweg, Casper; Arie, Tsutomu; Komatsu, Ken
EUROPEAN JOURNAL OF PLANT PATHOLOGY
SPRINGER
Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus in the family Alphaflexiviridae and has been isolated from a variety of host plants. In particular, PlAMV isolates from ornamental lilies (Lilium spp.) cause necrotic symptoms in these plants, which significantly reduces their commercial value. However, it has not been clear whether PlAMV isolates from other host plants differ in their infectivity and/or pathogenicity to ornamental lilies, and whether growth conditions affect infectivity and pathogenicity. In this study, we inoculated an edible lily species (Lilium leichtlinii) and seven varieties of ornamental lilies with two PlAMV isolates, an isolate from ornamental lily (PlAMV-OL) and an isolate from edible lily (PlAMV-Li1). We found that PlAMV-OL showed higher infection rates and exhibited necrotic symptoms more frequently in lilies than PlAMV-Li1. Moreover, we observed higher infection rates of PlAMV-OL in open field than in greenhouse, and higher rates of necrotic symptoms in autumn test than in spring test, suggesting that growth conditions and season affect infectivity and pathogenicity of PlAMV in lilies. Our study would provide important information for estimating the risk of necrotic disease caused by PlAMV, as well as for cultivation management preventing the occurrence of the disease.
2019年03月, 研究論文（学術雑誌）, 共同, 153, 3, 0929-1873, DOI(公開)(r-map), 813, 823 - The Plant Noncanonical Antiviral Resistance Protein JAX1 Inhibits Potexviral Replication by Targeting the Viral RNA-Dependent RNA Polymerase
Yoshida, Tetsuya; Shiraishi, Takuya; Hagiwara-Komoda, Yuka; Komatsu, Ken; Maejima, Kensaku; Okano, Yukari; Fujimoto, Yuji; Yusa, Akira; Yamaji, Yasuyuki; Namba, Shigetou
JOURNAL OF VIROLOGY
AMER SOC MICROBIOLOGY
Understanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole-and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electro-phoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication. IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits de novo potexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.
2019年02月, 研究論文（学術雑誌）, 共同, 93, 3, 0022-538X, DOI(公開)(r-map) - Induction of resistance to diseases in plant by aerial ultrasound irradiation
Kawakami, Aichi; Yoshida, Takanobu; Kanemaru, Yutaro; Zaquinaula, Medali Heidi Huarhua; MizuKami, Tomomichi; Arimoto, Michiko; Shibata, Takahiro; Goto, Akihiro; Enami, Yoshinari; Amano, Hiroshi; Teraoka, Tohru; Komatsu, Ken; Arie, Tsutomu
JOURNAL OF PESTICIDE SCIENCE
PESTICIDE SCI SOC JAPAN
Ultrasound, which refers to frequencies above the audible limit of human hearing, is a candidate for inducing resistance to pathogens in plants. We revealed that aerial ultrasound of 40.5 kHz could induce disease resistance in tomatoes and rice when the plants were irradiated with ultrasound of ca. 100 dB for 2 weeks during nursery season and reduced the incidence of Fusarium wilt and blast diseases, respectively, when plants were inoculated with pathogen 0 or 1 week after terminating irradiation. Disease control efficacy was also observed with ultrasound at frequencies of 19.8 and 28.9 kHz. However, cabbage yellows and powdery mildew on lettuce were not suppressed by ultrasound irradiation. No significant positive or negative effect on growth was observed in tomato and rice plants. RT-qPCR showed that the expression of PRla involved in the salicylic acid (SA) signaling pathway was upregulated in the ultrasound-irradiated tomato. (C) Pesticide Science Society of Japan
2019年, 研究論文（学術雑誌）, 共同, 44, 20190102, 1348-589X, DOI(公開)(r-map), 41, 47 - Chrysoviruses in Magnaporthe oryzae
Moriyama, Hiromitsu; Urayama, Syun-ichi; Higashiura, Tomoya; Tuong Minh Le; Komatsu, Ken
VIRUSES-BASEL
MDPI
Magnaporthe oryzae, the fungus that causes rice blast, is the most destructive pathogen of rice worldwide. A number of M. oryzae mycoviruses have been identified. These include Magnaporthe oryzae. viruses 1, 2, and 3 (MoV1, MoV2, and MoV3) belonging to the genus, Victorivirus, in the family, Totiviridae; Magnaporthe oryzae. partitivirus 1 (MoPV1) in the family, Partitiviridae; Magnaporthe oryzae. chrysovirus 1 strains A and B (MoCV1-A and MoCV1-B) belonging to cluster II of the family, Chrysoviridae; a mycovirus related to plant viruses of the family, Tombusviridae (Magnaporthe oryzae. virus A); and a (+)ssRNA mycovirus closely related to the ourmia-like viruses (Magnaporthe oryzae. ourmia-like virus 1). Among these, MoCV1-A and MoCV1-B were the first reported mycoviruses that cause hypovirulence traits in their host fungus, such as impaired growth, altered colony morphology, and reduced pigmentation. Recently we reported that, although MoCV1-A infection generally confers hypovirulence to fungi, it is also a driving force behind the development of physiological diversity, including pathogenic races. Another example of modulated pathogenicity caused by mycovirus infection is that of Alternaria alternata chrysovirus 1 (AaCV1), which is closely related to MoCV1-A. AaCV1 exhibits two contrasting effects: Impaired growth of the host fungus while rendering the host hypervirulent to the plant, through increased production of the host-specific AK-toxin. It is inferred that these mycoviruses might be epigenetic factors that cause changes in the pathogenicity of phytopathogenic fungi.
2018年12月, 研究論文（学術雑誌）, 共同, 10, 12, 1999-4915, DOI(公開)(r-map) - A putative RNA silencing component protein FoQde-2 is involved in virulence of the tomato wilt fungus Fusarium oxysporum f. sp lycopersici
Jo, Seong-Mi; Ayukawa, Yu; Yun, Sung-Hwan; Komatsu, Ken; Arie, Tsutomu
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
RNA silencing pathways in filamentous fungi are composed of multiple component proteins and known to be involved in vegetative growth, virulence or sexual reproduction. We found that the tomato wilt fungus, Fusarium oxysporum f. sp. lycopersici (Fol), carries four homologues genes of Qde-2, an argonaute protein gene and one of the main component protein genes in Neurospora crassa. Gene targeting revealed that FoQde-2, one of the Qde-2 homologues in Fol, is involved in virulence to tomato but not in vegetative growth.
2018年11月, 研究論文（学術雑誌）, 共同, 84, 6, 1345-2630, DOI(公開)(r-map), 395, 398 - Infectivity and pathogenicity of two different Plantago asiatica mosaic virus isolates in lilies
Komatsu, K.; Tanaka, M.; Verbeek, M.; Arie, T.
PHYTOPATHOLOGY
AMER PHYTOPATHOLOGICAL SOC
2018年10月, 研究論文（学術雑誌）, 共同, 108, 10, 0031-949X, DOI(公開)(r-map) - Cytological karyotyping of Fusarium oxysporum by the germ tube burst method (GTBM)
Ayukawa, Yu; Komatsu, Ken; Taga, Masatoki; Arie, Tsutomu
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
Fusarium oxysporum is an ascomycete fungus including plant pathogenic and nonpathogenic strains. Genome analyses have indicated that the karyotype of F. oxysporum is diverse among isolates. Here we used the germ tube burst method (GTBM), a more reliable method than conventional cytology or pulsed field gel electrophoretis, to karyotype isolates of F. oxysporum ff. spp. lycopersici and conglutinans and nonpathogenic F. oxysporum. In this first application of GTBM for F. oxysporum, pathogenic isolates were found to have more chromosomes than in nonpathogenic isolates. We also used a ribosomal DNA probe and fluorescence in situ hybridization (FISH) to analyze chromosome structure.
2018年07月, 研究論文（学術雑誌）, 共同, 84, 4, 1345-2630, DOI(公開)(r-map), 254, 261 - Molecular characterization of a novel mycovirus in Alternaria alternata manifesting two-sided effects: Down-regulation of host growth and up-regulation of host plant pathogenicity
Okada, Ryo; Ichinose, Shun; Takeshita, Kana; Urayama, Syun-ichi; Fukuhara, Toshiyuki; Komatsu, Ken; Arie, Tsutomu; Ishihara, Atsushi; Egusa, Mayumi; Kodama, Motoichiro; Moriyama, Hiromitsu
VIROLOGY
ACADEMIC PRESS INC ELSEVIER SCIENCE
A double-stranded RNA (dsRNA) mycovirus was detected in a strain of Alternaria altemata showing impaired growth phenotypes. The A. altemata strain is the Japanese pear pathotype, which produces a host-specific AK-toxin. Sequence analysis of the viral genome dsRNAs revealed that this mycovirus consists of five dsRNAs and is evolutionarily related to members of the family Chrysoviridae; the virus was named Alternaria altemata chrysovirus 1 (AaCV1). AaCV1-ORF2 protein accumulated in dsRNA-high-titer sub-isolates with severely impaired phenotypes; heterologous AaCV1-ORF2 overexpression in Saccharomyces cerevisiae caused growth inhibition. In contrast to this yeast growth inhibition phenomenon, the dsRNA-high-titer isolates displayed enhanced pathogenicity against Japanese pear plants, in accordance with a 13-fold increase in AK-toxin level in one such isolate. These findings indicated that AaCV1 is a novel mycovirus that exhibits two contrasting effects, impairing growth of the host fungus while rendering the host 'hypervirulene to the plant.
2018年06月, 研究論文（学術雑誌）, 共同, 519, 0042-6822, DOI(公開)(r-map), 23, 32 - Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, Magnaporthe oryzae
Aihara, Mitsuhiro; Urayama, Syun-ichi; Minh Tuong Le; Katoh, Yu; Higashiura, Tomoya; Fukuhara, Toshiyuki; Arie, Tsutomu; Teraoka, Tohru; Komatsu, Ken; Moriyama, Hiromitsu
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.
2018年03月, 研究論文（学術雑誌）, 共同, 84, 2, 1345-2630, DOI(公開)(r-map), 92, 103 - Panama disease of banana occurred in Miyakojima Island, Okinawa, Japan
Nitani, Takao; Akai, Kotaro; Hasegawa, Ryo; Ayukawa, Yu; Romero Garcia, Ricardo; Chitose, Atsushi; Komatsu, Ken; Kikuno, Hidehiko; Natsuaki, Keiko T.; Arie, Tsutomu
JOURNAL OF GENERAL PLANT PATHOLOGY
SPRINGER JAPAN KK
In 2016, in Miyakojima, Okinawa, Japan, banana plants (Musa x paradisiaca) 'Shima-banana' developed yellowing and wilt associated with vascular discoloration of the pseudostems. Fusarium oxysporum, identified based on morphological characters, was frequently isolated from the vascular tissue of the infected plant and reproduced the original symptoms on 'Shima-banana' after drench inoculation with a spore suspension. Thereby, we determined that the disease is Panama disease caused by F. oxysporum. This is the first official report of Panama disease (Panama-byo in Japanese) of banana in Japan.
2018年03月, 研究論文（学術雑誌）, 共同, 84, 2, 1345-2630, DOI(公開)(r-map), 165, 168 - N-terminal region of cysteine-rich protein (CRP) in carlaviruses is involved in the determination of symptom types
Fujita, Naoko; Komatsu, Ken; Ayukawa, Yu; Matsuo, Yuki; Hashimoto, Masayoshi; Netsu, Osamu; Teraoka, Tohru; Yamaji, Yasuyuki; Namba, Shigetou; Arie, Tsutomu
MOLECULAR PLANT PATHOLOGY
WILEY
Plant viruses in the genus Carlavirus include more than 65 members. Plants infected with carlaviruses exhibit various symptoms, including leaf malformation and plant stunting. Cysteine-rich protein (CRP) encoded by carlaviruses has been reported to be a pathogenicity determinant. Carlavirus CRPs contain two motifs in their central part: a nuclear localization signal (NLS) and a zinc finger motif (ZF). In addition to these two conserved motifs, carlavirus CRPs possess highly divergent, N-terminal, 34 amino acid residues with unknown function. In this study, to analyse the role of these distinct domains, we tested six carlavirus CRPs for their RNA silencing suppressor activity, ability to enhance the pathogenicity of a heterologous virus and effects on virus accumulation levels. Although all six tested carlavirus CRPs showed RNA silencing suppressor activity at similar levels, symptoms induced by the Potato virus X (PVX) heterogeneous system exhibited two different patterns: leaf malformation and whole-plant stunting. The expression of each carlavirus CRP enhanced PVX accumulation levels, which were not correlated with symptom patterns. PVX-expressing CRP with mutations in either NLS or ZF did not induce symptoms, suggesting that both motifs play critical roles in symptom expression. Further analysis using chimeric CRPs, in which the N-terminal region was replaced with the corresponding region of another CRP, suggested that the N-terminal region of carlavirus CRPs determined the exhibited symptom types. The up-regulation of a plant gene upp-L, which has been reported in a previous study, was also observed in this study; however, the expression level was not responsible for symptom types.
2018年01月, 研究論文（学術雑誌）, 共同, 19, 1, 1464-6722, DOI(公開)(r-map), 180, 190 - Novel loop-mediated isothermal amplification (LAMP) assay with a universal QProbe can detect SNPs determining races in plant pathogenic fungi
Ayukawa, Yu; Hanyuda, Saeri; Fujita, Naoko; Komatsu, Ken; Arie, Tsutomu
SCIENTIFIC REPORTS
NATURE PUBLISHING GROUP
Tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici (Fol) is grouped into three races based on their pathogenicity to different host cultivars. Rapid detection and discrimination of Fol races in field soils is important to prevent tomato wilt disease. Although five types of point mutations in secreted in xylem 3 (SIX3) gene, which are characteristic of race 3, have been reported as a molecular marker for the race, detection of these point mutations is laborious. The aim of this study is to develop a rapid and accurate method for the detection of point mutations in SIX3 of Fol. Loop-mediated isothermal amplification (LAMP) of SIX3 gene with the universal QProbe as well as two joint DNAs followed by annealing curve analysis allowed us to specifically detect Fol and discriminate race 3 among other races in about one hour. Our developed method is applicable for detection of races of other plant pathogenic fungi as well as their pesticide-resistant mutants that arise through point mutations in a particular gene.
2017年06月, 研究論文（学術雑誌）, 共同, 7, 2045-2322, DOI(公開)(r-map) - Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus
Komatsu, Ken; Yamashita, Kazuo; Sugawara, Kota; Verbeek, Martin; Fujita, Naoko; Hanada, Kaoru; Uehara-Ichiki, Tamaki; Fuji, Shin-ichi
ARCHIVES OF VIROLOGY
SPRINGER WIEN
Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and the other from the eudicot shrub Nandina domestica Thunb. (PlAMV-NJ). Their genomes contain five open reading frames (ORFs), which is characteristic of potexviruses. Surprisingly, the isolates showed only 76.0-78.0 % sequence identity with each other and with other PlAMV isolates, including isolates from Japanese lily and American nandina. Amino acid alignments of the replicase coding region encoded by ORF1 showed that the regions between the methyltransferase and helicase domains were less conserved than other regions, with several insertions and/or deletions. Phylogenetic analyses of the full-length nucleotide sequences revealed a moderate correlation between phylogenetic clustering and the original host plants of the PlAMV isolates. This study revealed the presence of two highly divergent PlAMV isolates in Japan.
2017年02月, 研究論文（学術雑誌）, 共同, 162, 2, 0304-8608, DOI(公開)(r-map), 581, 584 - Rapid sex identification method of spinach (Spinacia oleracea L.) in the vegetative stage using loop-mediated isothermal amplification
Fujita, Naoko; Ayukawa, Yu; Fuke, Mitsutoshi; Teraoka, Tohru; Watanabe, Kyoko; Arie, Tsutomu; Komatsu, Ken
PLANTA
SPRINGER
A LAMP-mediated, simple and rapid method for sex identification in spinach was developed. Nutrient compositional analysis showed a higher iron content in male than female plants. Spinach (Spinacia oleracea L.) is a dioecious plant with its sex determined by the XY system. Male and female floral organs differ morphologically, but plants do not differ in the vegetative stage before flowering. PCR with Y chromosome markers has been used to determine the sex of dioecious plants before flowering. In this study, we developed a genotype-specific loop-mediated isothermal amplification (LAMP) for sex identification of individual vegetative-stage spinach plants, using primers designed for the genomic region flanked by male-specific markers. LAMP could specifically detect spinach males. The method was further modified to omit DNA purification and use just an aliquot of crude leaf extract homogenized in water. We compared the nutrient composition of males and females, finding higher amounts of iron in the males. Our method could therefore be used for rapidly discriminating male plants in the field, which is useful for efficient hybrid breeding.
2017年01月, 研究論文（学術雑誌）, 共同, 245, 1, 0032-0935, DOI(公開)(r-map), 221, 226 - Sequencing of individual chromosomes of plant pathogenic Fusarium oxysporum
Kashiwa, Takeshi; Kozaki, Toshinori; Ishii, Kazuo; Turgeon, B. Gillian; Teraoka, Tohru; Komatsu, Ken; Arie, Tsutomu
FUNGAL GENETICS AND BIOLOGY
ACADEMIC PRESS INC ELSEVIER SCIENCE
A small chromosome in reference isolate 4287 of F. oxysporum f. sp. lycopersici (Fol) has been designated as a 'pathogenicity chromosome' because it carries several pathogenicity related genes such as the Secreted In Xylem (SIX) genes. Sequence assembly of small chromosomes in other isolates, based on a reference genome template, is difficult because of karyotype variation among isolates and a high number of sequences associated with transposable elements. These factors often result in misassembly of sequences, making it unclear whether other isolates possess the same pathogenicity chromosome harboring SIX genes as in the reference isolate. To overcome this difficulty, single chromosome sequencing after Contour-clamped Homogeneous Electric Field (CHEF) separation of chromosomes was performed, followed by de novo assembly of sequences. The assembled sequences of individual chromosomes were consistent with results of probing gels of CHEF separated chromosomes with SIX genes. Individual chromosome sequencing revealed that several SIX genes are located on a single small chromosome in two pathogenic forms of F. oxysporum, beyond the reference isolate 4287, and in the cabbage yellows fungus F. oxysporum f. sp. conglutinans. The particular combination of SIX genes on each small chromosome varied. Moreover, not all SIX genes were found on small chromosomes; depending on the isolate, some were on big chromosomes. This suggests that recombination of chromosomes and/or translocation of SIX genes may occur frequently. Our method improves sequence comparison of small chromosomes among isolates. (C) 2016 Elsevier Inc. All rights reserved.
2017年01月, 研究論文（学術雑誌）, 共同, 98, 1087-1845, DOI(公開)(r-map), 46, 51