陕西省番茄黄化曲叶病毒基因间隔区缺失突变体的鉴定与不同分离物间群体进化研究

为明确番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)基因组的变异情况, 分别从陕西省杨凌区?泾阳县?大荔县和阎良区采集了表现为番茄黄化曲叶病症状的抗病品种番茄样品20份以及感病品种番茄样品4份?经TYLCV特异性引物检测, 24个样品均为阳性?通过全长扩增测序获得了15个TYLCV分离物的基因组序列?序列分析显示, 其中7个分离物基因组大小为正常的2 781 bp, 而有8个TYLCV分离物基因组大小为2 768 bp?与正常基因组相比, 这8个分离物在基因间隔区(intergenic region, IR)的TATA-box与保守9核苷酸序列之间发生了13个核苷酸的缺失?对我国不同地区TYLCV分离物全基因组序列进行变异分析, 发现IR区域是发生核苷酸变异最大的区域?系统进化分析表明, 本研究分离到的15个TYLCV分离物均属于TYLCV-Israel进化分支下的中国亚群, 其中8个TYLCV IR缺失突变体与国内已报道的IR缺失突变体BJ04和BJ06亲缘关系较远, 而与本研究中分离得到的正常TYLCV分离物亲缘关系最近, 疑似为陕西地区出现的一类新TYLCV IR缺失突变体?本研究首次对我国不同地区多个TYLCV分离物出现IR区缺失现象进行了报道?     

北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定

 番茄黄化曲叶病毒病是番茄生产中的一种毁灭性病毒病害,2009年传入北京。利用烟粉虱传双生病毒简并引物PA/PB对2010年~2011年采集自北京市5个区县的53个番茄样品进行检测,30个表现典型黄化曲叶病症状的样品均扩增得到约500 bp的特异条带,测定了其中7个样品的部分序列,经序列比对分析表明其为番茄黄化曲叶病毒（Tomato yellow leaf curl virus, TYLCV）。利用TYLCV特异引物TJ-F/TJ-R、TY-F/TY-R对样品BJDXXY、BJFS02、BJFS03、BJMY2231进行TYLCV基因组克隆和序列测定,经分析4个样品携带的TYLCV基因组长度均为2 781碱基,编码6个蛋白。基因组序列比较发现,这4个分离物与TYLCV-Israel株系同源性达到98%以上;通过建立系统发育树,发现BJDXXY、BJFS02、BJFS03与河北分离物（HBLF4）、山东分离物（SDSG）亲缘关系较近,BJMY2231与上海分离物（TYLCV-Israel）、江苏分离物（JSNJ1）亲缘关系较近。     

南疆温室番茄黄化曲叶病病毒种类的分子鉴定

为明确南疆温室番茄黄化曲叶病的病毒种类,利用双生病毒的兼并引物通过PCR扩增,对采集的20个番茄病株进行了分子检测.从20个病株中均扩增到约500 bp的目标片段,对其中4株进行克隆和测序,其相互间序列同源性为97.1％ ～99.3％,与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的同源性较高,为98.6％ ～ 99.5％.随机选取莎车分离物KS2-5进行全基因组的克隆和测序,KS2-5 DNA全长为2781 nt(序列号:JQ807735),具有典型的双生病毒基因组特征,与TYLCV其它分离物同源性达到98.9％～99.5％,而与其它粉虱传双生病毒的序列同源性较低,为68.3％ ～75.5％,表明危害南疆温室番茄的病毒种类为番茄黄化曲叶病毒TYLCV.     

Detection and typing of tomato yellow leaf curl viruses

The denomination Tomato yellow leaf curl virus (TYLCV) comprises several viruses that cause severe damage to tomato crops in warm and temperate regions worldwide. TYLCV viruses are widespread in the Mediterranean Basin, in which two species have been reported: Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV, previously TYLCV-Is). The availability of methods convenient for the diagnosis of these viruses is essential. We have investigated several alternatives for reliable detection and differentiation of TYLCSV and TYLCV. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) proved to be very useful for large-scale diagnosis in field situations, but lacked discriminating capacity and sensitivity in the stages of infection in which low virus titre is present. The DNA-based methods are suited to laboratory operations and plant disease clinics, where accuracy of detection and discrimination of viruses is required. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was the most reliable method to discriminate between TYLCSV and TYLCV, but is not suited to high sample turnover. For large-scale testing, tissue print hybridization assay provides a reliable and sensitive alternative to PCR.     

Pepper (Capsicum annuum) Is a Dead-End Host for Tomato yellow leaf curl virus

ABSTRACT Tomato yellow leaf curl (TYLC) is one of the most devastating pathogens affecting tomato (Lycopersicon esculentum) worldwide. The disease is caused by a complex of begomovirus species, two of which, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), are responsible for epidemics in Southern Spain. TYLCV also has been reported to cause severe damage to common bean (Phaseolus vulgaris) crops. Pepper (Capsicum annuum) plants collected from commercial crops were found to be infected by isolates of two TYLCV strains: TYLCV-Mld[ES01/99], an isolate of the mild strain similar to other TYLCVs isolated from tomato crops in Spain, and TYLCV-[Alm], an isolate of the more virulent TYLCV type strain, not previously reported in the Iberian Peninsula. In this work, pepper, Nicotiana benthamiana, common bean, and tomato were tested for susceptibility to TYLCV-Mld[ES01/99]and TYLCV-[Alm] by Agrobacterium tumefaciens infiltration, biolistic bombardment, or Bemisia tabaci inoculation. Results indicate that both strains are able to infect plants of these species, including pepper. This is the first time that infection of pepper plants with TYLCV clones has been shown. Implications of pepper infection for the epidemiology of TYLCV are discussed.     

湖南省番茄和牵牛花上双生病毒的分子鉴定

双生病毒是一类在全世界范围内广泛发生的单链环状DNA病毒。本文对从湖南采集到的6例(洋姜、番茄、萝卜、赛葵、甘薯、牵牛花)疑似双生病毒侵染的植物叶片进行了分子鉴定。利用滚环扩增技术(RCA)对样品DNA进行扩增,分别对其RCA产物进行酶切,并将酶切得到的片段测序后进行BLAST比对,结果显示番茄样品中的病毒分离物与番茄黄化曲叶病毒相似性最高(99%),牵牛花样品中的病毒分离物与甘薯卷叶病毒相似性最高(99%),证明这两个分离物是单组分DNA-A双生病毒。这是在湖南省首次发现并报道双生病毒的全核酸序列。     

Evidence of a new Tomato yellow leaf curl virus in Japan and its detection using PCR

A new isolate of Tomato yellow leaf curl virus (TYLCV) has been identified from tomato plants in Kochi Prefecture in Japan and designated TYLCV-[Tosa]. The complete nucleotide sequence of the isolate was determined and found to consist of 2781 nt. In phylogenetic analyses of entire nucleotide sequences, TYLCV-[Tosa] was delineated as a single branch and was more closely related to TYLCV-[Almeria] than TYLCV isolates Ng, Sz, or Ai reported in Japan, which had spread since 1996. Isolate TYLCV-[Tosa] is suggested to be a newly introduced, novel isolate of TYLCV that dispersed into Kochi Prefecture. In addition, a rapid method using the polymerase chain reaction to separate TYLCV isolates into four genetic groups was established. This method would be useful for reliable diagnosis based on genetic differences among isolates of TYLCV.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB192965 and AB192966     

番茄黄化曲叶病毒与番茄褪绿病毒复合侵染对番茄黄化曲叶病毒传播的影响

番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)是一种由烟粉虱传播的单链环状DNA病毒, 在田间可与多种病毒发生复合侵染, 如番茄褪绿病毒(tomato chlorosis virus, ToCV)等?本文对比了TYLCV单独侵染和TYLCV与ToCV复合侵染对烟粉虱获取和传播TYLCV的影响?结果表明, 与取食TYLCV单独侵染的番茄相比, 取食复合侵染番茄的烟粉虱对TYLCV的传毒率显著提高, 且番茄植株和烟粉虱体内TYLCV的病毒积累量也显著提高?试验结果说明复合侵染会提高烟粉虱的传毒率, 促进TYLCV的发生与流行?     

广东省番茄黄化曲叶病毒的分子鉴定及序列分析

番茄黄化曲叶病毒病是世界番茄生产上一种毁灭性病害,番茄黄化曲叶病毒Tomato yellow leaf curl virus(TYLCV)是引起该病害的主要病原病毒之一。本文采用滚环扩增及基因克隆方法,获得了侵染广东佛山和肇庆番茄的TYLCV 4个分离物全基因组;它们均为2 781 nt,编码6个ORF,其中病毒链上编码AV1和AV2,互补链上编码AC1、AC2、AC3和AC4。同源性比较结果表明,4个广东分离物基因组序列两两间同源性为99%以上;与已报道的TYLCV各分离物同源性在90%以上,而与来自中国不同地区的TYLCV分离物的同源率均在98%以上。系统进化分析显示,广东分离物与来自中国不同地区的TYLCV分离物亲缘关系较近,并聚类在一个分支。因此,侵染引起广东佛山和肇庆番茄黄化曲叶病的病毒应来自国内其他地区。本研究是对TYLCV广东分离物分子特征的首次报道。     

侵染广西番茄的番茄黄化曲叶病毒的分子鉴定

番茄黄化曲叶病毒病是番茄生产上的毁灭性病害,严重影响番茄产量及品质。2019年从广西百色市采集疑似番茄黄化曲叶病叶片样品,采用滚环扩增(RCA)及基因克隆等方法,获得了4个烟粉虱传双生病毒的全基因序列,4个分离物的全长序列分别为2 776、2 781、2 752、2 781 bp,均编码6个开放阅读框;核苷酸相似性比较发现,4个分离物彼此间的相似性均在90%以上,与已报道的番茄黄化曲叶病毒Tomato yellow leaf curl virus (TYLCV)各分离物间的相似性也在91%以上;系统进化分析表明,TYLCV广西分离物BS-2和BS-4与TYLCV-Hunan、BS-1和BS-3与TYLCV-YN6553等分离物处于独立的小分支,说明TYLCV广西分离物与TYLCV-Hunan、TYLCV-YN6553具有较近的亲缘关系。本研究首次报道TYLCV在广西发生。     

2009年河北省番茄黄化曲叶病毒病发生危害和分布

2009年,河北省主要番茄种植区发生一种疑似番茄黄化曲叶病毒病的新病害。本文在田间调查、取样的基础上,采用酶联免疫吸附测定(ID-ELISA)方法对病原进行鉴定,明确了番茄黄化曲叶病在河北省的分布与危害:至2009年9月,番茄黄化曲叶病毒病已在河北省南部的邯郸、邢台和石家庄,中部的保定和衡水等地严重发生,河北省中北部廊坊、沧州南部靠近衡水的部分地市亦零星发生此病害,应用TYLCV检测试剂盒检测的29个品种58个标样A405值达到2.1～7.5。唐山、承德地区尚未发现番茄黄化曲叶病的危害,4个县市9个番茄品种检测结果均呈阴性。     

Mercurialis ambigua and Solanum luteum: Two Newly Discovered Natural Hosts of Tomato Yellow Leaf Curl Geminiviruses

The yellow leaf curl disease of tomato is caused by a complex of virus species, two of which, tomato yellow leaf curl virus (TYLCV)-Sar and TYLCV-Is, are involved in epidemics of southern Spain. Plants of Mercurialis ambigua and Solanum luteum showing abnormal upward leaf curling and leaf distortion collected in the vicinity of tomato crops were found to be naturally infected with TYLCV-Is and TYLCV-Sar, respectively. These weed species, as well as Datura stramonium and S. nigrum, which had also been found to be naturally infected by TYLCVs in the same region in previous studies, were tested for susceptibility to TYLCV-Sar or TYLCV-Is by Agrobacterium tumefaciens-mediated and by Bemisia tabaci inoculation. Results indicated that both TYLCV-Sar and TYLCV-Is were able to infect D. stramonium and M. ambigua, whereas only TYLCV-Sar infected S. nigrum and S. luteum. Implications for the epidemiology of TYLCV are discussed. This is the first report of M.ambigua and S. luteum as hosts of TYLCV.     

不同番茄品种对5种植物双生病毒的抗病性鉴定

 由粉虱传双生病毒引起的番茄曲叶病［1］在我国最初仅分布在海南、云南、广东和广西,自2006年上海市和浙江省先后在番茄上发现番茄黄化曲叶病毒（Tomato yellow leaf curl virus,TYLCV）以来,该病害蔓延迅速,在多个省份的番茄上暴发成灾［2］。引起番茄曲叶病害的病原较复杂,在我国其主要病原为TYLCV、中国番木瓜曲叶病毒（Papaya leaf curl China virus, PaLCuCNV）、中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus, TYLCCNV)、泰国番茄黄化曲叶病毒（Tomato yellow leaf curl Thailand virus, TYLCTHV）和台湾番茄曲叶病毒（Tomato leaf curl Taiwan virus, ToLCTWV）［2~5］,而浙江省的主要病原为TYLCV和ToLCTWV。选育抗病品种是防治番茄黄化曲叶病最有效的手段。了解番茄品种对不同双生病毒的抗性,对因地制宜布局抗病品种具有重要意义。浙杂502、浙粉701、浙粉702是浙江省大规模种植的番茄品种,为了解这些品种对上述5种病毒的抗性,本研究利用5种病毒的侵染性克隆,在人工接种条件下,综合评定分析这3个番茄品种的抗病指标。     

Note:Bemisia tabaci biotype B associated with tomato yellow leaf curl disease epidemics on rhodes Island, Greece

In 2006 an outbreak of tomato yellow leaf curl disease occurred in tomato crops on Rhodes Island, Greece. Diseased plants were found to be infested with the B biotype of theBemisia tabaci (Gennadius) complex and greenhouse and open-field-grown tomato crops were infected withTomato yellow leaf curl virus (TYLCV) introduced from the Middle East. This is the first report of TYLCV and the B biotype ofB. tabaci on Rhodes Island in Greece. http://www.phytoparasitica.org posting Dec. 11, 2007.     

Identification of whitefly transmitted tomato yellow leaf curl geminivirus from Iran and a survey of its distribution with molecular probes

A virus causing a disease of tomato, prevalent in the southern provinces of Iran, with symptoms of leaf-curling, stunting, reduction of leaf size, leaf corrugation, shortening of internodes and severe reduction in fruit yield, was shown to be transmissible to healthy tomato plants by grafting and by whiteflies ( Bemisia tabaci ), but not by sap inoculation. Geminivirus DNA was detected in extracts of diseased tomato plants by dot-blot hybridization assays using as probes full-length cloned DNA of Australian, Italian (Sardinian) or Jordanian strains of tomato yellow leaf curl virus (TYLCV). Geminivirus coat protein was detected in whitefly inoculated plants by dot immunobinding assay using polyclonal antibody raised against Jordanian TYLCV. A limited survey using the dot-blot hybridization assay for virus detection indicated the presence of the virus in tomato-growing provinces of southern but not northern Iran. Whitefly transmission experiments to tomato under controlled greenhouse conditions showed that some isolates of TYLCV-like geminiviruses from different parts of Iran differ in symptomatology.     

京郊番茄主要病毒病毒原鉴定

近年来, 番茄病毒病, 特别是番茄黄化曲叶病毒（TYLCV）病在北京地区空前暴发, 给番茄生产造成严重威胁, 使番茄的产量和品质显著降低。2012-2013年在北京周边7个区县, 采集疑似感染病毒的番茄植株样品325份, 分别针对TYLCV、烟草花叶病毒（TMV）、黄瓜花叶病毒（CMV）3种病毒进行了PCR或ELISA 检测。检测结果表明, 目前在北京地区大棚或温室内发生的番茄病毒病以TYLCV为主; 露地番茄病毒复合侵染现象比较普遍, 病毒检出率100%, 其中TYLCV检出率达到75%以上。在TYLCV侵染的样品中, TYLCV和CMV复合侵染占20%左右, TYLCV和TMV复合侵染占15%左右。部分样品检测到TYLCV、CMV 和TMV 3种病毒复合侵染的现象。     

截形叶螨抗哒螨灵种群抗性遗传方式的研究

通过对室内选育出的截形叶螨敏感品系和抗哒螨灵品系的杂交和回交试验表明:所测得的截形叶螨正交F1SR(SS♀×RR♂)和反交F1RS(RR♀×SS♂)代的显性度DSR和DRS分别为0.40和0.60,表明抗性由不完全显性基因控制;两个D值(DRS和DSR)95%置信限有重叠,并且经t检验,两个D值不存在显著差异(P0.05),证明截形叶螨对哒螨灵的抗性遗传为常染色体控制;回交F2代(BC1RS和BC1SR)的实际死亡率和期望值经χ2检验,无显著性差异(χ2=9.72,df=9,P0.05),表明抗性遗传由单基因控制。     

A new approach to evaluate relative resistance and tolerance of tomato cultivars to begomoviruses causing the tomato yellow leaf curl disease in Spain

A simple procedure to evaluate relative resistance and tolerance of tomato cultivars to the begomoviruses causing tomato yellow leaf curl (TYLC) disease in Spain was developed. To estimate the resistance and tolerance levels of a cultivar, several formulae were developed based on the ratio of infected plants, virus titre (estimated by tissue–print hybridization) and symptom intensity. The formulae were applied to five commercial tomato cultivars (Amoretto, Birloque, Royesta, Tovigreen and Ulises) naturally infected by TYLC viruses. The analyses showed that Ulises, Birloque and Tovigreen exhibited a moderate resistance, and Ulises was also highly tolerant. There was a positive correlation between symptom intensity and virus titre in infected plants, suggesting that the hybridization technique could also be used as an early estimator of tolerance. Finally, molecular hybridization and nucleotide sequence analyses of the begomovirus intergenic region showed that the local TYLC virus population consisted of a single species, Tomato yellow leaf curl virus (TYLCV, formerly TYLCV-Israel), with low genetic variation (nucleotide identity between isolates higher than 97%).     

Detection of Tomato yellow leaf curl Sardinia virus in Tunisia

Surveys were undertaken for tomato yellow leaf curl viruses in the main Tunisian tomato-growing areas in fields and plastic houses. Symptoms included yellowing, leaf curling and stunting. Collected samples were submitted to molecular analysis using two approaches: (1) hybridization tests with two DNA probes corresponding to an intergenic region derived from a cloned dimer of an Egyptian full-length TYLCV and to the coat region of a Tunisian TYLCV isolate; (2) PCR amplification coupled to RFLP allowing both identification and clustering of Tunisian isolates. Only Tomato yellow leaf curl Sardinia virus was detected in Tunisia.     

Solanum nigrum: an indigenous weed reservoir for a tomato yellow leaf curl geminivirus in southern Spain

A tomato yellow leaf curl geminivirus (TYLCV-AL), was first identified in tomato plants in Almeria, southern Spain in 1992. This virus is transmitted by the tobacco whitefly, Bemisia tabaci (Gennadius), and is presently infecting tomato crops throughout the south eastern region of Spain. Solanum nigrum, collected from a field in south east Spain and exhibiting leaf curl symptoms, was squash blotted onto nylon membrane and gave a positive signal when hybridised to a TYLCV-Is DNA probe. Laboratory tests showed B. tabaci to transmit TYLCV-AL from infected tomato plants to S. nigrum seedlings. The virus could then be acquired by B. tabaci and transmitted back from infected S. nigrum plants to tomato, inducing typical TYLCV symptoms. These results indicate the importance of S. nigrum as a weed host/reservoir for a TYLCV and its possible role in the spread of this virus within Europe.