First Report of Chinese wheat mosaic virus Infecting Barley in Japan
First Report of Chinese wheat mosaic virus Infecting Barley in Japan
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日本首次报道中国小麦花叶病毒感染大麦
DOI：
10.1094/pdis-12-21-2803-pdn
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发表时间：
2022
期刊：
影响因子：
4.5
通讯作者：
Ohki Takehiro
中科院分区：
文献类型：
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作者：
Kondo Hideki;Masejima Hidekazu;Maruyama Kazuyuki;Fujita Miki;Ohki Takehiro
中国小麦花叶病毒（CWMV）是Virgaviridae科Furovirus属的成员（亚当斯等人，2017），具有正义RNA基因组，由禾多粘菌传播。CWMV是中国冬小麦黄花叶病的病原体（Guo et al. 2019）。在日本北方有限地区（长野县和岩手县）的小麦植株中也检测到了CWMV（Fuji等人，2022年; Maejima等人，2010年; Shirako和Maejima，2008年）。在使用针对CWMV衣壳蛋白的抗血清的Western印迹的初步测试中（由Dr. Y. Shirako，东京大学），我们在大麦育种系（‘Tozan Kawa 111，[Hordeum vulgare L.]，收集于2012年4月）在长野县的CWMV和小麦黄花叶病毒（Bymovirus属）侵染的实验田中生长。为了调查大麦植物感染禾谷类植物相关土传病毒的情况，我们在2016年4月（2015/16生长季节）收集了表现出黄色花叶症状但没有明显枯萎或发育不良的Tozan Kawa 111株（10株）的叶片样品。用TaKaRa RNAiso试剂（TaKaRa Bio）从有症状的叶样品中提取总RNA，并进行逆转录聚合酶链反应（RT-PCR）以检测病毒因子。使用具有随机六聚体的Moloney鼠白血病病毒逆转录（Thermo Fisher Scientific）进行cDNA合成后，使用QuickTaqHS染料混合物（Toyobo Co.）使用对已知感染日本小麦和大麦植物的两种糠病毒和三种黄病毒特异的引物组进行（Tamada和Kondo 2013）。RT-PCR分析检测到的感染与CWMV的叶片样品的Tozan Kawa 111植物，但没有与其他土传病毒测试。扩增的PCR产物（CWMV RNA 1和RNA 2分别为752和718 bp）通过Wizard SV Gel和PCR Clean-Up System（Promega）纯化，并进行桑格测序以确认它们的核苷酸序列。来自PCR扩增子的病毒序列以登录号LC 657081（RNA 1）和LC 657082（RNA 2）保藏在GenBank/DDBJ/ENA中。BLASTn分析表明，该序列与CWMV日本北方分离物（accession no.AB299271）的RNA 1和CWMV长野A分离物（AB 935554）的RNA 2的核苷酸序列同源性分别为98.7%和98.8%。相同的序列也被发现在症状小麦叶样品（品种Fukuho Komugi）从同一领域获得。通过透射电子显微镜（Hitachi H-7650）在有症状的Tozan Kawa 111叶片样品中观察到棒状颗粒。使用逆转录环介导等温扩增（Fukuta et al. 2013），在2015/16至2017/18生长季节，在同一田地的Tozan Kawa 111株植物中检测到CWMV，但在2018/19至2020/21生长季节未检测到。在2015/16至2017/18生长季节，在大麦植物（合并五个植物样本）栽培品种Kashimamugi中检测到CWMV，其表现出类似的黄色花叶症状，但在田间种植的大多数其他大麦变种中没有检测到。据我们所知，这是第一次在小麦以外的植物中报道CWMV田间感染（Kühne 2009）。进一步广泛的田间病毒筛选和病毒接种实验是必要的，以了解CWMV在大麦和可能在其他谷类作物的病理。
Chinese wheat mosaic virus (CWMV), a member of the genus Furovirus in the family Virgaviridae (Adams et al. 2017), has a positive-sense RNA genome and is transmitted by Polymyxa graminis. CWMV is a causal agent of yellow mosaic disease in winter wheat in China (Guo et al. 2019). CWMV has also been detected in wheat plants in limited areas of northern Japan (Nagano and Iwate Prefectures)(Fuji et al. 2022; Maejima et al. 2010; Shirako and Maejima 2008). In preliminary tests using Western blotting with an antiserum raised against CWMV capsid protein (by Dr. Y. Shirako, Tokyo University), we detected a furovirus in a breeding line of barley (‘Tozan Kawa 111,[Hordeum vulgare L.], collected in April 2012) grown in an experimental field infested with CWMV and wheat yellow mosaic virus (genus Bymovirus) in Nagano Prefecture. To investigate the infection of barley plants with cereal plant-associated soil-borne viruses, we collected leaf samples of Tozan Kawa 111 plants (10 plants) showing yellow mosaic symptoms but with no apparent wilting or stunting in April 2016 (2015/16 growing season). Total RNA was extracted from symptomatic leaf samples with TaKaRa RNAiso reagent (TaKaRa Bio) and subjected to reverse transcription polymerase chain reaction (RT-PCR) to detect virus agents. After cDNA synthesis using Moloney murine leukemia virus reverse transcription (Thermo Fisher Scientific) with random hexamers, PCR amplification with QuickTaqHS Dye Mix (Toyobo Co.) was conducted using primer sets specific to two furoviruses and three bymoviruses known to infect wheat and barley plants in Japan (Tamada and Kondo 2013). RT-PCR analysis detected infection with CWMV in the leaf samples of Tozan Kawa 111 plants, but not with the other soil-borne viruses tested. The amplified PCR products (752 and 718 bp for CWMV RNA1 and RNA2, respectively) were purified by Wizard SV Gel and PCR Clean-Up System (Promega) and subjected to Sanger sequencing to confirm their nucleotide sequences. The virus sequences from PCR amplicons were deposited in GenBank/DDBJ/ENA with accession numbers LC657081 (RNA1) and LC657082 (RNA2). Nucleotide Basic Local Alignment Search Tool (BLASTn) analysis showed that the sequences have 98.7 and 98.8% nucleotide sequence identity with RNA1 of CWMV Japanese northern isolate (accession no. AB299271) and RNA2 of CWMV Nagano-A isolate (AB935554), respectively. Identical sequences were also found in symptomatic wheat leaf samples (cultivar Fukuho Komugi) obtained from the same field. Rod-shaped particles were observed by transmission electron microscopy (Hitachi H-7650) in symptomatic Tozan Kawa 111 leaf samples. Using reverse transcription loop-mediated isothermal amplification (Fukuta et al. 2013), CWMV was detected in Tozan Kawa 111 plants in the same field in the 2015/16 to 2017/18 growing seasons but not in the 2018/19 to 2020/21 growing seasons. In the 2015/16 to 2017/18 growing seasons, CWMV was detected in the barley plants (pooled five plant samples) cultivar Kashimamugi, which showed similar yellow mosaic symptoms but not in most of the other barley variants planted in the field. To our knowledge, this is the first report of CWMV field infection in plants other than wheat (Kühne 2009). Further extensive virus screening in fields and virus inoculation experiments are necessary to understand the pathology of CWMV in barley and possibly in other cereal crops.