云南辣椒正番茄斑萎病毒属病毒的分子鉴定

辣椒是云南省主要经济作物之一,近年来病毒病尤其是正番茄斑萎病毒属病毒发病严重,影响了辣椒产量和品质。利用RT-PCR技术对从云南辣椒主产区采集的疑似感染正番茄斑萎病毒属病毒的25份辣椒样品进行分子鉴定,结果显示,12份样品检测出正番茄斑萎病毒属病毒,检出率为48.0%,其中6份是番茄斑萎病毒tomato spotted wilt orthotospovirus (TSWV),检出率为24.0%;5份是番茄环纹斑点病毒tomato zonate spot orthotospovirus (TZSV),检出率为20.0%;有1份是TSWV和TZSV复合侵染,检出率为4.0%,这是在云南辣椒生产上首次发现TSWV和TZSV的复合侵染。通过鉴定,初步了解正番茄斑萎病毒属病毒在云南辣椒生产中的发生情况和种类,为制定云南地区该属病毒的防治策略提供理论依据。     

露地栽培番茄斑萎病毒病发生流行规律调查及TSWV重要寄主植物监测

为明确云南省昆明市露地栽培条件下番茄斑萎病毒病的发生流行特征,于2014—2015年采用病害系统调查法结合病毒ELISA及RT-PCR检测方法研究露地栽培条件下由番茄斑萎病毒(tomato spotted wilt virus,TSWV)引起的病毒病发生规律及其重要寄主种类,并研究利用防虫网隔离蓟马对番茄斑萎病毒病的防控效果。结果表明:番茄斑萎病毒病在露地番茄主要种植期3—10月普遍发生,番茄苗期和移栽初期是该病毒病防控的关键期,带毒种苗调运是该病毒病的主要传播途径;田间多种茄科和菊科植物是TSWV的重要中间寄主。在田间,菊科寄主植物油麦菜、莴苣、鬼针草、牛膝菊上TSWV的检出率均较高,在42.53%～81.63%之间;茄科寄主植物中辣椒上TSWV的检出率最高,为41.99%,其次为马铃薯,TSWV检出率为27.78%,在番茄上TSWV的检出率为19.02%,因此生产中应对这些TSWV重要中间寄主给予更多关注和防控。应用防虫网能有效隔离蓟马,使番茄斑萎病毒病发病率和病情指数较对照分别降低了6.44个百分点和5.31,可有效降低番茄苗期及定植期斑萎病毒病的发生。     

云南省红河地区传播番茄斑萎病毒属病毒的蓟马及其寄主植物种类调查

2011年7月至2012年8月对云南省红河州传播番茄斑萎病毒属(Tospovirus)病毒的蓟马种类及其寄主植物进行了系统调查。明确了西花蓟马、花蓟马、烟蓟马和棕榈蓟马4种为传毒蓟马。其寄主植物主要有辣子草、粉花月见草、茴茴蒜等30种以上田间杂草和白菜、蚕豆、番茄等超过20种作物。     

西花蓟马传播番茄斑萎病毒研究进展

西花蓟马Frankliniella occidentalis是世界性重要检疫性害虫之一,不仅直接取食危害作物而且传播病毒,从而造成极为严重的经济损失。由于西花蓟马在我国具有广泛的适生范围,随其入侵我国并随之传播的番茄斑萎病毒(Tomato spotted wilt virus)已在我国不同地域发现,对经济作物已形成严重威胁。本文综述了西花蓟马对番茄斑萎病毒的获取、携带和传播扩散过程及其病毒在蓟马体内的循环过程和机理,总结了影响西花蓟马传播番茄斑萎病毒效率的因素,并评述了西花蓟马-病毒-植物这一互作系统及其对西花蓟马生长发育适合度的影响,以期为我国西花蓟马传播番茄斑萎病毒的基础研究和防控提供理论依据与指导。     

西藏昌都市卡若区蔬菜主要病毒检测及番茄斑萎病毒鉴定

为明确西藏昌都市卡若区高原条件下的蔬菜主要病毒种类, 对温室、大棚和露地栽培的主要蔬菜进行了病毒病调查, 采集典型病毒病症状样品进行ELISA检测, 明确病毒种类; 并利用电子显微镜观察, RT-PCR扩增克隆与测序分析对主要病毒进行鉴定分析。ELISA检测结果表明, 西藏昌都市卡若区温室及大棚栽培的番茄、辣椒和莴苣上的主要病毒有番茄斑萎病毒(tomato spotted wilt virus, TSWV)、马铃薯Y病毒(potato virus Y, PVY)及凤果花叶病毒(pepino mosaic virus, PepMV)。其中TSWV检出率最高, 为45%。进一步对检出TSWV的蔬菜样品进行电子显微镜观察, 发现其中含有典型的正番茄斑萎病毒属Orthotospovirus病毒粒体, 应用TSWV-N基因特异性引物进行RT-PCR扩增克隆和序列分析, 发现西藏昌都市卡若区蔬菜感染的TSWV与云南TSWV分离株亲缘关系最近。本研究结果明确了西藏昌都市卡若区蔬菜的主要病毒种类。综合抗体检测、病毒粒体形态观察与分类相关基因的克隆测序结果, 明确了西藏昌都市卡若区蔬菜感染的主要病毒为TSWV。这也是TSWV在西藏的首次报道, 为了解TSWV的发生分布及其防控提供了依据。     

北京地区发现番茄斑萎病毒

通过采用特异快速试纸条以及进一步的RT-PCR和序列测定证实,在北京局部地区的辣椒和茄子上已发生番茄斑萎病毒,这两种作物上同时发生的蓟马种类主要为西花蓟马,并且携带该病毒。番茄斑萎病毒病是极其危险的一种病害,可对作物造成毁灭性灾害。建议有关部门立即采取相应的预防与管理措施,防止其扩散蔓延。     

黑龙江地区番茄斑萎病毒的鉴定及其部分生物学特征分析

近年来,番茄斑萎病毒Tomato spotted wilt tospovirus (TSWV)在我国多个地区发生严重危害。本研究采用小RNA深度测序和RT-PCR相结合的方法对采自黑龙江的番茄病毒病样品中的病毒进行了鉴定。提取番茄样品的RNA并构建小RNA文库进行测序,数据分析发现比对至TSWV基因组的reads数占比对到病毒核酸总reads数的92.64%,表明侵染此番茄样品的病原物可能是TSWV。利用RT-PCR方法进一步确定侵染此番茄样品的病毒为TSWV,将其命名为TSWV-HLJ1。对TSWV-HLJ1的核苷酸序列进行分析并构建系统进化树,结果表明TSWV-HLJ1与TSWV云南甜椒分离物(TSWV-YNgp)的亲缘关系最近。介体传毒试验表明,西花蓟马可将TSWV-HLJ1传播感染健康寄主植物。摩擦接种试验表明,TSWV黑龙江分离物能够侵染本氏烟、辣椒和番茄。这是我国首次报道在黑龙江地区发现TSWV的危害。     

番茄斑萎病毒系统侵染我国大蒜

番茄斑萎病毒Tomato spotted wilt tospovirus(TSWV)是严重危害世界经济作物的一种病毒,寄主范围广泛。我们在研究中发现番茄斑萎病毒能侵染我国大蒜Allium sativum L.。利用摩擦接种法将TSWV接种到健康大蒜上,结果显示:接种14 d后大蒜新生叶片出现褪绿和白色斑点症状。ELISA检测显示大蒜叶片汁液与TSWV的单克隆抗体产生血清学反应,采用TSWV N基因的引物对大蒜叶片总RNA进行RT-PCR,结果扩增出约800 bp的条带,在NCBI上BLAST显示与TSWV YN5576的同源性最高,为98.52%。这些数据表明番茄斑萎病毒系统侵染我国大蒜。     

番茄环纹斑点病毒核壳体蛋白抗血清制备及其血清学特性分析

 番茄环纹斑点病毒(Tomato zonate spot virus, TZSV)是番茄斑萎病毒属的一个新种,对云南番茄、辣椒生产造成严重危害。用RT-PCR 从感病番茄中扩增得到长度为837 bp TZSV 的核壳体蛋白基因(N 基因),将其克隆到原核表达载体pET-28a( + ),获得重组原核表达载体pET-TZSV-N,在大肠杆菌BL21 中表达,SDS-PAGE 分析表明,该载体高效表达33kDa 的融合蛋白;以纯化的融合蛋白作为抗原免疫家兔制备TZSV 核壳体蛋白(N 蛋白)的多克隆抗体,间接酶联免疫测定(ID-ELISA)表明效价为1 / 6 000;Western blot 分析表明,该抗血清能与西瓜银色斑驳病毒(WSMoV)血清组的辣椒褪绿病毒(CaCV)反应,而与番茄斑萎病毒(TSWV)、凤仙花坏死斑病毒(INSV)无血清交叉反应,说明获得的抗血清能用于WS-MoV 血清组成员的检测,TZSV 属于WSMoV 血清组成员。     

西花蓟马传播病毒病的研究进展

西花蓟马［Frankliniella occidentalis (Pergande)］是一种世界性的重要农业害虫,目前在69个国家和地区已有报道。西花蓟马能以持久性的方式传播番茄斑萎病毒属(Tospovirus)的病毒,所传播病毒造成的经济损失远远大于其本身所造成的损失。因此,许多学者对西花蓟马及其传播的番茄斑萎病毒属病毒进行了大量研究。本文主要综述了近年来西花蓟马传播病毒的种类、番茄斑萎病毒属病毒的结构以及西花蓟马的传毒机制等方面的研究进展。     

Characterization of two Tospoviruses in Italy: tomato spotted wilt and impatiens necrotic spot

Tospovirus serogroups I and III have recently been designated as species, tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), while the species status of serogroup II isolates remains undefined. Fifteen Tospovirus isolates from ornamental and vegetable crops in Liguria, Italy, were found to belong either to TSWV (seven isolates) or to INSV (eight isolates) on the basis of test-plant reactions, serological techniques using DAS ELISA kits raised against the nucleoproteins of the type members of the two species, and cytopathology. None of them could be assigned to serogroup II using DAS ELISA kits raised against nucleoproteins of this serogroup. Italian isolates representative of the two species reacted in indirect ELISA using a polyclonal antiserum against the entire particle of a TSWV isolate, but with higher intensity for our TSWV isolates than for the INSV isolates. Western blots and dot immunobinding assays confirmed that the nucleoproteins of the two species are unrelated whereas the glycoproteins are related. The cytopathology was similar for two isolates representative of TSWV and INSV, except that the type of filaments encountered was different, and appeared to be characteristic of the species.     

Purification and serology of virions of impatiens necrotic spot tospovirus

Impatiens necrotic spot tospovirus (INSV) virions were purified using a procedure devised for tomato spotted wilt tospovirus (TSWV) from systemically infectedNicotiana benthamiana plants grown at 33 °C day/26 °C night and a photoperiod of 14 hours. With plants grown at 24/18 ° C purification was unsuccessful. In SDS-PAGE the protein pattern of INSV was similar to that reported for TSWV, except the appearance of a single G2 protein band. A polyclonal antiserum, prepared against virions, reacted in Western blots with INSV nucleoprotein and glycoproteins but only with TSWV glycoproteins. In DAS ELISA the antiserum reacted with both INSV and TSWV infected plant sap and, after absorption with TSWV, only with INSV. In TAS ELISA the antiserum trapped both INSV and TSWV nucleoproteins and glycoproteins as detected by specific monoclonal antibodies, and, after absorption with TSWV, only the homologous proteins. This appears to be the first report of the purification of INSV virions and the production of an antiserum reacting with both nucleoprotein and glycoprotein antigens.     

侵染四川辣椒的番茄斑萎病毒和辣椒轻斑驳病毒的小RNA测序检测及鉴定

 调查发现四川省汶川县当地辣椒的病毒病严重且症状多样,病样粗提液摩擦接种辣椒、矮牵牛和三生烟,出现辣椒系统性花叶焦枯和茎尖坏死,指示植物表现局部枯斑。对3个不同症状的病果进行sRNA深度测序鉴定,发现均含有番茄斑萎病毒(Tomato spotted wilt virus,TSWV)和辣椒轻斑驳病毒(Pepper mild mottle virus,PMMoV)。通过RT-PCR技术进行验证,结果显示所有病样的果皮和部分新鲜种子以及回接寄主的病叶均检测到TSWV和PMMoV,表明该地辣椒病毒病是由TSWV和PMMoV复合侵染引起。这是TSWV侵染四川辣椒的首次报道。分别基于TSWV N基因序列和PMMoV CP基因序列构建系统发育树,汶川分离物TSWV-WC(MK468469)与贵州分离物(KP684518)亲缘关系最近,PMMoV-WC(MK408614)与北京分离物(AY859497)亲缘关系最近。推测该地辣椒病毒病可能与品种引进有关。     

云南西瓜银灰斑驳病毒病害的鉴定

 应用DAS-ELISA和RT-PCR方法从褪绿和银色斑驳的西瓜叶片中检测到病毒分离物（WSMoV-YN）,感病样品能与WSMoV/GBNV复合抗血清（Agdia）呈阳性反应。获得WSMoV N蛋白的多克隆抗体,抗体能与WSMoV血清组成员CaCV和TZSV反应,但不能与INSV、TSWV、HCRV和GYSV反应。为明确引起该病害的病毒种类,采用Tospovirus通用引物对样品的总RNA进行RT-PCR扩增,获得长度为3 554 nt的S RNA全序列,经Blastn比对分析与WSMoV中国台湾分离物同源性最高,为95.8%,其N和NSs蛋白氨基酸序列同源性分别为99%和97.6%。构建系统进化树发现,西瓜银灰斑驳病毒云南分离物（WSMoV-YN）与其他WSMoV聚为一支。确定引起云南西瓜病害的病毒为WSMoV。     

云南怒江番茄种植新区番茄病毒检测及病毒种传特性

病毒病对番茄生产造成严重危害, 近年来在番茄种植新区发生严重, 疑似为种子带毒传播。本研究通过对云南省怒江州番茄种植新区的番茄病毒病样品采用RNA-seq高通量测序, RT-PCR验证的方法检测病毒种类;对番茄病果种子进行超薄切片制样透射电子显微镜观察, 将病果种子播种后对种苗进行RT-PCR带毒检测。结果表明, RNA-seq高通量测序及RT-PCR检测到的病毒有番茄环纹斑点病毒(tomato zonate spot orthotospovirus, TZSV)、番茄黄斑驳相关病毒(tomato yellow mottle-associated virus, TYMaV)、辣椒脉斑驳病毒(chili veinal mottle virus, ChiVMV)、南方番茄病毒(southern tomato virus, STV)。透射电镜观察到种胚细胞及胚乳细胞中分布典型的正番茄斑萎病毒属Orthotospovirus病毒粒体。病果种子播种28 d后的种苗具有病毒病症状, 通过RT-PCR检出TZSV、ChiVMV、STV, 检出率分别为60%、100%、80%。上述研究结果为TZSV通过种子传播提供了有利的证据, 并为源头防控番茄病毒病提供了依据。     

Ornamental plants and thrips populations associated with tomato spotted wilt virus in Greece

A survey was conducted in order to record the ornamental plants that are hosts of tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) in Greece. Polyclonal antibodies prepared against the N protein of a Greek isolate of TSWV fromGerbera jamesonii (GR-34) were used. Leaf samples were taken from plants showing typical symptoms of tospovirus infection such as chlorotic and necrotic rings on the leaves and malformation and necrosis of the flowers. The samples were tested by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal antibodies to the N proteins of TSWV and INSV (NL-07). ELIS A-positive samples were mechanically transmitted to plants ofPetunia hybrida, Nicotiana rustica andN. benthamiana to confirm infection. Although none of the samples was found infected with INSV, TSWV presence was recorded in 42 botanical species that belong to 40 genera in 27 families. Among them the speciesBeloperone guttata, Coleus barbatus, Impatiens petersiana andLilium auratum are reported for the first time as hosts of TSWV, whereasBegonia sp.,Catharanthus roseus Celosia cristata, Dianthus chinensis, Fuchsia hybrida andStephanotis floribunda are found as new hosts of the virus in Greece. Thrips collected from TSWV-infected plants were in most cases identified asFrankliniella occidentalis, except from plants ofDendranthema sp. andDianthus caryophyllus whereThrips tabaci individuals were also identified. Different percentages of transmitters were noticed when the thrips populations collected from TSWV-infected ornamental hosts were tested for transmission of TSWV.     

Comparison of ambisense m RNA of watermelon silver mottle virus with other tospoviruses

ABSTRACT Double-stranded genomic RNAs (dsRNAs) extracted from Chenopodium quinoa infected with watermelon silver mottle virus (WSMV) were similar to those of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), except that the S dsRNA of WSMV is 0.75 and 0.6 kbp longer than those of TSWV and INSV, respectively. The complete nucleotide sequence of the genomic M RNA of WSMV was determined from cDNA clones generated from separated M dsRNA. The M RNA is 4,880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The M RNA-encoded nonstructural (NSm) ORF located on the viral strand encodes a protein of 312 amino acids (35 kDa), and the G1/G2 ORF located on the viral complementary strand encodes a protein of 1,121 amino acids (127.6 kDa). The RNA probe corresponding to the NSm or G1/G2 ORF of WSMV failed to hybridize with the M dsRNAs of TSWV and INSV. Comparison of M and S RNAs of WSMV, TSWV, INSV, and peanut bud necrosis virus (PBNV, serogroup IV) revealed a consensus sequence of eight nucleotides of 5'-AGAGCAAU...-3' at their 5' ends and 5'-...AUUGCUCU-3' at their 3' ends. The low overall nucleotide identities (56.4 to 56.9%) of the M RNA and the low amino acid identities of the NSm and G1/G2 proteins (30.5 to 40.9%) with those of TSWV and INSV indicate that WSMV belongs to the Tospovirus genus but is phylogenetically distinct from viruses in serogroups I and III. The M RNA of WSMV shares a nucleotide identity of 79.6% with that of PBNV, and the two viruses share 83.4 and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full-length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 and 3.4 kb, respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana. The 1.0- and 3.4-kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid protein.     

Serological and Molecular Characterization of Peanut chlorotic fan-spot virus, a New Species of the Genus Tospovirus

ABSTRACT To clarify the serological relationship of Peanut chlorotic fan-spot virus (PCFV) with other tospoviruses, antisera were produced against the nucleocapsid (N) proteins of this virus and tospoviruses from four serogroups including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Groundnut ringspot virus (GRSV), and Watermelon silver mottle virus (WSMoV). In immunodiffusion tests, the antisera only reacted with their homologous antigens. Similar results were noticed in indirect enzyme-linked immunosorbent assay and immunoblot tests, with the exception that strong cross-reactions were observed in heterologous combinations between TSWV and GRSV. The results indicated that the N protein of PCFV is not serologically related to those of the tospoviruses from the four serogroups. To further characterize the virus, viral S double-stranded RNA was extracted from PCFV-infected Chenopodium quinoa and used for cDNA cloning and sequencing. The full-length viral strand of the S RNA was determined to be 2,833 nucleotides, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA is identical to those of other tospoviruses, indicating that PCFV belongs to the genus Tospovirus. The N and the NSs proteins of PCFV share low amino acid identities (22.3 to 67.5% and 19.3 to 54.2%) with those of reported tospoviruses, respectively. The phylogenetic dendrogram of the N gene of PCFV compared with those of other tospoviruses indicates that PCFV is distinct from other tospoviruses. In hybridization analyses, an N gene cDNA probe of PCFV did not react with viral RNAs of TSWV, GRSV, INSV, and WSMoV, and vice versa. Thus, based on these results, we conclude that PCFV is a new tospovirus species.     

青蒿中番茄斑萎病毒和烟草花叶病毒的分子鉴定及相关序列分析

番茄斑萎病毒(tomato spotted wilt virus, TSWV)和烟草花叶病毒(tobacco mosaic virus, TMV)是2种重要的植物病原病毒, 对多种经济作物的产量和品质均造成严重影响。2021年-2022年, 在云南省丽江市烟草种植区不同烟区采集叶片黄化、皱缩以及无症状的青蒿Artemisia caruifolia样品共计14份, 利用免疫金标速测卡和RT-PCR对其病原病毒进行检测。利用免疫金标速测卡检测结果显示, 在所检样品中有9份样品检测出TSWV, 检出率为64.28%, 有3份样品检测出TMV, 检出率为21.43%, 2种病毒复合侵染的检出率同样为21.43%;利用RT-PCR对复合侵染的3份样品进行分子检测, 结果显示, 在3份复合侵染青蒿样品中获得3条TSWV N基因序列、3条TMV cp基因序列和2条TMV RdRp部分序列。TSWV青蒿分离物与分离自云南的TSWV-2分离物相似性最高, 为99.6%;TMV青蒿分离物与分离自辽宁的TMV-Shenyang分离物和分离自云南的TMV-Yongren-1相似性最高, 均大于99.4%。这是首次发现TSWV和TMV 2种不同属病毒复合侵染青蒿。