番茄斑萎病毒属6种病毒的多重RT-PCR检测

根据番茄斑萎病毒属(Tospovirus)中6种病毒S RNA上的N基因序列设计特异性引物,建立可同时检测这6种Tospovirus病毒的多重PCR体系。多重PCR扩增结果显示,番茄环纹斑点病毒(776 bp)、甜瓜黄斑病毒(505 bp)、鸢尾黄斑病毒(296 bp)、凤仙花坏死斑病毒(221 bp)、番茄斑萎病毒(175 bp)和番茄褪绿斑病毒(110 bp)均出现清晰的目标条带,各病毒的特异性引物不会对其他病毒产生非特异性扩增。本研究建立的6种Tospovirus病毒的多重PCR检测方法,有助于提高目标病毒的检测效率。     

番茄斑萎病毒NASBA检测方法的建立

为建立一种快速检测番茄斑萎病毒(Tomato spotted wilt virus,TSWV)的方法,以TSWV-CP1/TSWV-CP2为引物对TSWV的N基因进行PCR扩增及序列测定,在TSWV N基因的高度保守区设计特异性扩增引物NA-P1/NA-P2进行核酸序列依赖性扩增(NASBA)反应,并对NASBA方法的特异性和灵敏度进行验证。结果表明,建立的NASBA方法最佳反应时间为1.5 h。该方法特异性较好,只有TSWV阳性样品中出现了预期大小为235 bp的扩增产物,与烟草环斑病毒(Tobacco ring spot virus,TRSV)、番茄黑环病毒(Tomato black ring virus,TBRV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)、番茄黄化曲叶病毒(Tomato yellow curl virus,ToYCLV)无交叉反应。灵敏度验证中,实时荧光RT-PCR的灵敏度最高,为1.56×10~(-5)ng/μL感病植物RNA模板,NASBA次之,为1.56×10~(-4)ng/μL,普通RT-PCR最低,为1.56×10~(-3)ng/μL。在对9份实际样品检测中,NASBA的阳性检出率与实时荧光RT-PCR、普通RT-PCR相同,均为33%,高于ELISA检测的22%。表明NASBA方法适用于实际样品检测,可对TSWV进行快速检测。     

由美国进境生菜种子上番茄斑萎病毒的检测鉴定

采用DAS-ELISA检测方法,从美国进境的生菜种子中筛选出携带有番茄斑萎病毒(Tomato spotted wilt virus,TSWV)的疑似阳性样品,通过建立的RT-PCR方法对酶联免疫吸附测定阳性样品进行复核鉴定,并进一步测序。测序结果与GenBank中已有的番茄斑萎病毒分离物KT160282、AY744474、GU369726、KP330470的序列同源性达到96%。结果表明,TSWV可通过种子携带入境。     

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。     

番茄斑萎病毒的检疫技术

番茄斑萎病毒为布尼亚病毒科（Bunyaviridae）番茄斑萎病毒属（Tospovirus）的代表成员，其寄主范围很广，可引起许多重要的园艺植物和农作物的严重病害，造成严重的经济损失。该病毒病在许多国家的局部地区有发生，为减轻其危害，各国对该病毒的发生和流行特点及控制措施等进行了较深入的研究，本文概述了该病毒的相关研究进展。     

从云南蝴蝶兰上检测到番茄斑萎病毒属病毒

 应用电镜观察、DAS-ELISA以及RT-PCR检测,从症状表现黄化、环斑的云南蝴蝶兰(Phalaenopsis amabilis)病样中分离得到的一个病毒分离物Tospo-Pha。该分离物粒子近球形、具包膜、直径约90nm,与番茄斑萎病毒属(Tospovirus)的西瓜银色斑驳病毒(Watermelon silver mosaic virus,WSMoV)/花生芽坏死病毒(Groundnut bud necrosis virus,GBNV)复合抗血清呈强阳性反应,分子检测发现该分离物SRNA5'末端序列与CaCV大岩桐分离物(CaCV-Gloxinia)同源性最高(91.0%),在系统进化树中与CaCV聚于同一分支。上述结果表明,从云南蝴蝶兰中分离到的Tospo-Pha属于Tospovirus病毒。     

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。     

北京辣椒和番茄上番茄斑萎病毒的鉴定

北京大兴区某蔬菜种植基地的辣椒和番茄发现疑似病毒感染,造成较大经济损失.经症状分析和RT-PCR检测,证实其感染番茄斑萎病毒(tomato spotted wilt virus,TSWV).基于TSWV序列特征对该地区TSWV进化及来源进行分析,结果显示其与北京顺义区已经报道TSWV毒株进化关系较近,但较国内其他地区株...     

深度测序技术分析番茄斑萎病毒病害寄主及介体中病毒种类

 正番茄斑萎病毒属病毒(orthotospoviruses)是严重危害云南蔬菜等重要农业经济作物的病毒病原之一。采用血清学检测、小RNA深度测序以及RT-PCR验证相结合的方法,从云南省昆明市晋宁区的主要作物寄主(番茄、辣椒、油麦菜)、重要中间寄主(鬼针草)和传毒介体(蓟马)中鉴定到TSWV、TZSV、PCSV和INSV 4种病毒,其中TSWV为该地区的主要优势病毒,而PCSV则是首次报道侵染鬼针草。通过对云南番茄斑萎病毒病害重病区作物寄主、中间寄主及蓟马三者进行病毒种类分析研究,明确TSWV为引起云南省昆明市晋宁区作物的主要病毒,TZSV、PCSV和INSV零星发生于不同寄主中。     

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。     

番茄斑萎病毒对云南莴苣类蔬菜的侵染为害

莴苣类蔬菜是常见的食用蔬菜。近年在云南发现生菜、莴笋、油麦菜有症状表现为叶片黄化、褪绿、坏死斑的病害流行危害,为确定其病原,通过电子显微镜观察、回接试验、ELISA检测、病毒基因分析,在典型症状病样组织粗汁液和超薄切片中均观察到有类似番茄斑萎病毒属病毒的粒体,该病毒粒体为球形,直径80～85 nm; ELISA检测和病毒基因分析显示,侵染莴苣类蔬菜的病毒为番茄斑萎病毒。     

番茄斑萎病毒核衣壳蛋白基因的克隆与分析

从云南表现褪绿黄化症状的番茄上分离到番茄斑萎病毒(Tomato spotted wilt virus)YN-1株系,利用RT-PCR方法克隆了该株系的核衣壳蛋白基因,并测定了该基因的序列,全长为777 bp,编码258个氨基酸。对该基因的氨基酸序列分析结果表明,YN-1 N基因与韩国3个株系的氨基酸序列相似性均高达97%。     

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。     

Biological and molecular characterization of tomato spotted wilt virus (TSWV) resistance-breaking isolates from Argentina

Tomato spotted wilt virus (TSWV) has been present in Argentina since 1938 and had limited sweet pepper and tomato production until the introduction of resistant cultivars bearing Tsw and Sw-5b genes. However, the wide use of TSWV-resistant pepper plants in La Plata Horticultural Belt (LPHB) triggered the emergence of resistance-breaking isolates (RB), increasing the economic impact of TSWV in pepper. This work characterized 11 natural RB pepper isolates from LPHB that have overcome the Tsw resistance gene in Capsicum sp. but are unable to break the Sw-5b-mediated resistance in tomato. Phylogenetic analysis of the N gene showed that the LPHB isolates are most closely related to isolates from Asia, indicating that Argentine TSWV isolates might have emerged from the Asian continent. The NSs sequence analysis reinforces the hypothesis that the appearance of an RB phenotype is a consequence of a number of different single amino acid substitutions spread along the NSs gene that lead to multiple independent evolutionary events. These results provide information on the current situation of the tospovirus–pepper/tomato pathosystems in LPHB, which represents a fundamental prerequisite to include these RB isolates in future screening programmes in order to select new and durable sources of resistance to TSWV in pepper.     

Identification and characterization of a new class of Tomato spotted wilt virus isolates that break Tsw-based resistance in a temperature-dependent manner

The single dominant Tsw resistance gene from Capsicum chinense against the Tomato spotted wilt orthotospovirus (TSWV) is temperature sensitive, i.e. the resistance fails to function at or above 32 °C. This study describes a new class of temperature-sensitive resistance-breaking TSWV isolates that induce Tsw-mediated resistance at T < 28 °C but at T ≥ 28 °C break this resistance. The NSs genes from these isolates were cloned and expressed to be analysed for RNA silencing suppressor (RSS) activity and the ability to induce a Tsw-mediated hypersensitive response (HR) in C. chinense and Capsicum annuum (Tsw+). Unlike in viral infection, transient expression of some of the NSs proteins at standard temperatures (22 °C) did not induce Tsw-mediated HR, although varying degrees of RSS activity were observed. Attempts to express and test the NSs proteins for functionality at an elevated temperature through agroinfiltration remained unsuccessful. The NSs proteins of some TSWV resistance-breaking (RB) isolates were analysed and found to lack amino acid residues that were previously shown to be important for RNA silencing suppression and avirulence. This study describes a new class of resistance-breaking TSWV isolates that may be of importance for breeders and growers and for which the underlying mechanism still remains unknown.     

Biological and molecular characterization of Tomato spotted wilt Virus in Israel

Received April 24, 1997; received in final form June 29, 1997. Symptoms resembling tomato spotted wilt virus (TSWV) infections were documented among ornamental and vegetable crops in commercial greenhouses and open fields in Israel. Plants exhibiting these symptoms were collected from January 1992 to December 1996. Among cultivated plants analyzed for TSWV by enzyme-linked immunosorbent assay (ELISA), 19 species representing five families were found to be infected; natural infection was also recorded in six plant species of weeds. Virus identity was characterized by host range, serology and electron microscopy. Serological reaction with the isolates, found in Israel, using antisera from different sources as well as the sequence analysis of the nucleocapsid gene, demonstrated that the Israeli isolates of TSWV are a member of tospovirus serogroup I, type I (BR-01 strain). No virus transmission was found in seeds collected from virus-infected vegetable and ornamental crops. A non-radioactive molecular probe derived from the cloned nucleocapsid isolate enables specific detection of the virus in crude sap from infected plants. The detection of TSWV in Israel constitutes a severe potential threat to the ornamental and vegetable industry.     

Verbena as a trap crop to suppress thrips-transmitted Tomato spotted wilt virus in chrysanthemums

The effect of verbena as a trap crop on the occurrence of western flower thrips, Frankliniella occidentalis, and the incidence of Tomato spotted wilt virus (TSWV) in chrysanthemums were investigated. Verbena cvs. Pink Parfait and/or Fancy Parfait were cultivated alongside chrysanthemum cv. Jimba in a greenhouse in the proportion of 17%–25% of the chrysanthemum plants. Verbena plants attracted vector thrips, reducing western flower thrips colonization of chrysanthemum until flower bud initiation, and markedly suppressing TSWV incidence on chrysanthemums until flowering. Significant quantities of linalool oxide pyran were produced by the flower of cv. Fancy Parfait; and the ratio of cis-linalool oxide pyran, an attractant for vector thrips, to the trans-type was approximately 1 : 5. Our results suggest that cultivation of verbena as a trap crop may be useful in integrated pest management programs as a control for thrips-transmitted TSWV in chrysanthemums.