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。     

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。     

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。     

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。     

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。     

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...     

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。     

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。     

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。     

蝴蝶兰软腐病中一种新致病菌的分离与鉴定

 Soft rot disease often affects Phalaenopsis amabilis during the growing season. However, the pathogen of the disease is remaining poorly studied. In this study, bacterial strain R1 was isolated from soft rot tissues in Wuhan. The pathogenic, morphological, physiological, biochemical tests and 16S rDNA sequence analysis were carried out. The homology of 16S rDNA sequence between strain R1 and Pseudomonas grimontii was 99.72%, and its physiological and biochemical properties were also similar to those of Pseudomonas grimontiis. All these evidences indicated that strain R1 could be identified as a novel strain of Pseudomonas grimontii. The pathogenicity of the novel isolate was proved according to the Koch's postulates. This is the first report that Pseudomonas grimontii can cause soft rot disease of Phalaenopsis amabilis.     

Artichoke yellow ringspot virus infecting vetch (Vicia sativa) in Greece

A seed-transmitted virus was isolated from vetch seedlings showing severe stunting, reduced lamina and leaf mottling. Identification was based on host range, seed transmission, coat protein and nucleic acid analysis, electron microscopy and serology. This is the first record of vetch as a natural host of artichoke yellow ringspot virus (AYRSV). http://www.phytoparasitica.org posting Jan. 13, 2002.     

葡萄的两种检疫性病毒的多重RT-PCR检测

 本研究利用多重RT-PCR技术,对葡萄的检疫性病毒中番茄环斑病毒(ToRSV)和烟草环斑病毒(TRSV)进行检测,并研究了反应体系中各个参数对多重RT-PCR的影响。结果表明:可以在同一反应体系中实现对ToRSV和TRSV的RT-PCR检测,但模板浓度、引物浓度、循环数等3个因素对检测结果的准确性有较大的影响,3个参数的增加都会导致非特异性扩增的增多,而dNTP以及Taq酶的浓度对实验结果并无较大影响。     

金钱松实时荧光PCR鉴定方法的建立

本研究根据金钱松核糖体基因的内转录间隔区(ITS)设计特异性引物与荧光探针,建立了金钱松的实时荧光PCR检测方法.利用该方法检测4个不同来源的金钱松样本,均能够得到典型的扩增曲线,与53个松柏科其他树种如日本五针松、罗汉松、湿地松样本无交叉反应,表明了该方法的特异性.灵敏度可达1.6×10-5ng/μL.该方法可快速、...     

广东西瓜上检测到甜瓜黄斑病毒

在广东发现了可能被番茄斑萎病毒属(Tospovirus)病毒侵染的西瓜,采用ELISA和RT-PCR法对该西瓜病样进行了检测,西瓜病叶粗汁液不与番茄斑萎病毒(Tomato spotted wilt virus,TSWV)和西瓜银斑驳病毒(Watermelon silver mottle virus,WSMoV)的血清发生反应;利用引物J13/UHP通过RT-PCR可以扩增出约1400 bp的基因片段,该片段包括一个840 bp的核衣壳蛋白ORF,其推导的氨基酸序列与已报道的Melon yellow spot virus(MYSV)NP基因氨基酸序列的同源率都为99%,进化树分析表明侵染广东西瓜的病毒(命名为MYSV-GZ)属于Tospovirus的MYSV血清组。     

广东番茄上检测到Tospovirus病毒

 Some tomato samples possibly infected by tospovirus in Guangdong were detected with indirect ELISA and RT-PCR. The results showed that the virus infected tomato did not react with the antiserum of Tomato spotted wilt virus (TSWV), but about 500 bp fragment of RT-PCR shared 83%-84% nucleotide identities with N gene of those reported tospoviruses. The phylogenetic tree of the N gene fragment compared with those of other tospoviruses indicated that the virus infected tomato was belonged to Tospovirus.     

侵染云南花魔芋的马铃薯Y病毒属病毒分离物的检测及分子鉴定

 YN80 was isolated from Amorphophallus rivieri Durieu showing mosaic and crinkle symptoms in Songming, Yunnan province. Flexuous filamentous particles were found in diseased leave sap and pinwheel inclusion bodies were found in the leave tissue. YN80 had positive reaction to universal antibody of Potyvirus by DAS-ELISA. 3'-terminal sequence of YN80 was cloned and sequenced. cp gene of YN80 consisted of 987 nt, encoded 328 aa (36.1 kDa). Sequence analysis showed that YN80 shared the highest identity (97.0%)with CP amino acid sequence of Dasheen mosaic virus (DsMV). These data indicated that YN80 was an isolate of DsMV. This is the first molecular identification of A. rivieri Durieu isolate of DsMV in China.     

Diversity of the coat protein-coding region among Ilarvirus isolates infecting hop in Australia

Coat protein (CP) sequences of 17 Ilarvirus isolates were obtained from hops at three farms in Tasmania, Australia. Phylogenetic analysis of these sequences and additional database sequences indicated several Apple mosaic virus (ApMV) isolate clusters distinct from Prunus necrotic ringspot virus (PNRSV): one containing isolates from apple; one containing a single isolate from almond; a third containing Australian hop isolates of the 'apple' serotype and a German isolate of unknown origin; and a fourth containing Australian hop isolates of the 'intermediate' serotype. Isolates from hop, pear and prune from the Czech Republic either formed a fifth grouping, or were divergent members of the 'intermediate' serotype group. Deduced amino acid (aa) residue differences between the coat proteins of the two hop isolate serotype groups were highlighted as possible regions of serological differentiation. No evidence for coinfection of plants with both serotypes was found. Tests of ApMV-infected hop buds using the Shirofugen flowering cherry assay revealed a possible differentiation of the two strains based on hypersensitivity. Because of serological similarities to PNRSV, these viruses have commonly been reported as strains of PNRSV. However, this study shows ilarviruses from Australian hops are strains of ApMV, but distinct from those infecting Malus spp.     

长蒴母草黄脉病毒——一个双生病毒的新种

 从采集于海南儋州地区表现黄脉症状的长蒴母草(Lindernia anagallis)上分离到病毒分离物L2, DNA-A全序列分析结果表明, 全长2739个核苷酸(nt)(GenBank登录号:AY795900), 共编码6个ORF, 其中病毒链编码AV1(CP)、AV2, 互补链编码AC1、AC2、AC3、AC4。利用BLAST程序对DNA-A进行分析表明, 与L2 DNA-A有同源关系的病毒均为双生病毒科(Geminiviridae)菜豆金色黄花叶病毒属(Begomovirus)成员。进一步比较发现, L2 DNA-A与我国广东报道的广东番茄曲叶病毒(Tomato leaf curl Guangdong virus, ToLCGuV)(AY602165)全基因组核苷酸序列的同源性最近, 仅为77.0%, 说明L2为Begomovirus中的一个新种, 命名为长蒴母草黄脉病毒(Lindernia anagallis yellow vein virus, LAYVV)。与L2的IR区及各基因编码的氨基酸序列有最高同源性的病毒均来源于亚洲。利用DNA-B特异引物和DNA-β的特异引物, 均未检测到DNA-B和卫星DNA-β的存在。     

Tomato necrotic ring virus (TNRV), a recently described tospovirus species infecting tomato and pepper in Thailand

Two tospovirus isolates collected from tomato and bell pepper in Thailand were studied. The isolates induced severe necrotic mottling and/or necrotic spots and rings on the leaves and fruits of the respective plants as confirmed by back-inoculation. A polyclonal antiserum raised against its nucleocapsid (N) protein reacted only with an extract from plants infected with the homologous virus. Analysis of the nucleocapsid (N) gene sequence and its deduced amino acid sequence (Mw ∼31 kDa) showed 99% amino acid sequence homology with that of Tomato necrotic ring virus (TNRV). The nucleotide sequence of the 5΄ untranslated region and intergenic region flanking the N gene revealed typical features of the S RNA segment of tospoviruses. Mechanical inoculation of the virus on some plant species showed that most of the tested solanaceous species were susceptible to this virus. The biological, serological and molecular data presented here indicate that both isolates are identical to TNRV, a recently described tospovirus species in Thailand.