Serological and molecular detection of Tomato chlorosis virus and Tomato infectious chlorosis virus in tomato

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are two criniviruses inducing similar yellowing symptoms in tomato. An approximately 4 kb central region of the genomic RNA2 of French ToCV and TICV isolates was sequenced. TICV, for which no other sequences were available, appeared as a distant species in the genus, being close only to LIYV ( Lettuce infectious yellows virus ) for some, but not all, proteins. ToCV has more than 98% nucleotide identity with isolates from the US and Spain, and sequencing the CP gene of several isolates collected in different regions in southern France during 2 years suggested a unique origin. Polyclonal antisera were produced using capsid proteins of both viruses expressed in Escherichia coli . DAS-ELISA assays were developed for routine diagnosis and conditions for preparing samples for an optimized detection were determined. No cross-reactions were observed. However, some false-negative results, corresponding to samples giving ELISA readings close to the detection limit were regularly detected, particularly for ToCV (approximately 5% of the samples). A triplex RT-PCR assay was thus developed, which allowed detection of both viruses in a one-step protocol. An internal PCR control was included, which in addition showed that it could be used as a control for the entire RT-PCR procedure. Finally, combining DAS-ELISA in a first round, and triplex RT-PCR for doubtful samples, appeared the best way to achieve a reliable diagnosis of these viruses.     

Occurrence of Tomato spotted wilt virus (TSWV) in Jordan

Tomato spotted wilt virus was recorded for the first time in Jordan on tomato plants. Severe disease symptoms were observed in different tomato farms in the Jordan Valley. Using a specific primer pair a fragment of the capsid protein gene of the virus has been amplified by RT-PCR and IC-RT-PCR. The amplified PCR product was cloned and sequenced. Sequence analysis revealed that the Jordanian isolate of TSWV shared high nucleotide similarities with other isolates from different countries. The sequence of the capsid protein gene was deposited in GenBank under the accession number AY646682 . The response of different tomato breeding lines and hybrids, previously developed for resistance against Tomato yellow leaf curl virus (TYLCV) were tested for their reaction to TSWV infection. All tested lines and hybrids were susceptible to TSWV infection. This has been confirmed at the molecular level by using the SCAR 421 marker linked to the TSWV resistance gene Sw-5 .     

Occurrence and Diagnosis of Tomato chlorosis virus in Portugal

Tomato chlorosis virus (ToCV), a new whitefly-transmitted and phloem-limited Crinivirus infecting tomatoes in Europe, is reported for the first time in Portugal. Tomato plants with symptoms of interveinal chlorosis, collected during autumn 1998 and summer and autumn 1999 in Algarve, southern Portugal, were positive in RT-PCR assays using ToCV-specific primers. The amplified 439bp fragment was sequenced and showed 99% homology with the ToCV sequence in the GenBank database. A digoxigenin–DNA probe was produced and tested in dot-blot with total RNAs extracted from tomato samples. Both the RT-PCR and dot-blot hybridisation procedures enabled rapid and reliable detection of ToCV from field samples.     

Comparative whitefly transmission of Tomato chlorosis virus and Tomato infectious chlorosis virus from single or mixed infections

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are two criniviruses that are emerging worldwide, and induce similar yellowing diseases in tomato crops. While TICV is transmitted only by Trialeurodes vaporariorum , ToCV is transmitted by three whitefly species in two genera Trialeurodes vaporariorum , T. abutilonea and Bemisia tabaci . The efficiency of transmission by T. vaporariorum from plants infected by one virus or by both was compared, and the probability of virus transmission by a single whitefly was derived from group testing experiments. The estimated transmission probabilities ranged from 0·01 to 0·13, and were not significantly different between ToCV and TICV, or between single and mixed infections. Experiments using B. tabaci as a vector and source plants infected by TICV and ToCV did not reveal any functional trans-complementation for transmission of TICV by ToCV, suggesting that if this phenomenon occurs in nature, it is at a very low frequency. Possible reasons why TICV did not establish in southern France while ToCV is now endemic are discussed.     

Tomato infectious chlorosis virus — a new clostero-like virus transmitted byTrialeurodes vaporariorum

A previously undescribed virus disease of tomato, other crops and weed hosts was found in California. Affected tomato plants exhibited interveinal yellowing, necrosis and severe yield losses. Leaf dips and purified preparations contained closterovirus-like long flexuous, filamentous particles approximately 12×850–900 nm. The virus, designated as tomato infectious chlorosis virus (TICV), is transmitted in a semipersistent manner by the greenhouse whitefly,Trialeurodes vaporariorum. The host range of the virus is moderate (26 species in 8 plant families) but includes some important crops and ornamental species including tomato, (Lycopersicon esculentum), tomatillo (Physalis ixocarpa), potato (Solanum tuberosum), artichoke (Cynara scolymus), lettuce (Lactuca sativa) and petunia (Petunia hybrida). The virus has been found in a number of different locations in California and has a number of potential vehicles of movement including greenhouse grown ornamentals, tomato transplants, artichoke cuttings and potato seed. The virus has the potential to spread to other growing regions with resident populations of the greenhouse whitefly. The host range, particle size, insect transmission, and serology clearly distinguish TICV from previously described viruses.     

Tomato infectious chlorosis virus causes leaf yellowing and reddening of tomato in Italy

Since autumn 2000, severe and widespread chlorosis, sometimes associated with redness, has been observed in greenhouse tomatoes in different regions of Italy. A total of 104 samples were analyzed for tomato infectious chlorosis virus (TICV), by a one-step RT-PCR procedure. In some areas of central Italy and Sardinia, the symptom was consistently correlated with the presence of TICV. The RT-PCR procedure enabled rapid and reliable detection of TICV from field samples. Sequence analysis of the amplified 501-bp fragment, part of the HSP70 coding region, revealed an identity of 99% with the TICV sequence in the GenBank database. A digoxigenin-labeled DNA probe was also produced and successfully tested in dot blot assays. This is the first report of TICV causing epidemics in Europe.     

番茄褪绿病毒与番茄黄化曲叶病毒复合侵染对番茄褪绿病毒传播的影响

番茄褪绿病毒Tomato chlorosis virus(ToCV)是一种由烟粉虱Bemisia tabaci传播的正义单链RNA病毒,在田间常与番茄黄化曲叶病毒Tomato yellow leaf curl virus(TYLCV)复合侵染而造成番茄生产上重大的经济损失。为了明确ToCV与TYLCV的复合侵染对烟粉虱传播ToCV所造成的影响,本文采用RT-PCR以及qRT-PCR检测了复合侵染的番茄对烟粉虱获取和传播ToCV的影响。研究表明,烟粉虱取食复合侵染的番茄后对ToCV的传播效率显著提高,仅25头烟粉虱的传毒率即可达到100%,ToCV在烟粉虱以及番茄体内的累积量均显著提高。说明这种复合侵染促进了烟粉虱对ToCV的传播,在田间应当及时防控烟粉虱,警惕病毒与烟粉虱的蔓延。     

番茄褪绿病毒侵染黄瓜的首次报道

番茄褪绿病毒Tomato chlorosis virus(ToCV)是严重危害世界经济作物的一种病毒,寄主范围广泛。田间调查发现黄瓜Cucumis sativus表现出叶片黄化、脉间褪绿的疑似番茄褪绿病毒感病症状,同时叶片背面聚集了大量烟粉虱。采用RT-PCR方法对样品叶片和烟粉虱进行检测,ToCV感染率为65%,且发病叶片上烟粉虱携带ToCV。为进一步确定黄瓜是否为番茄褪绿病毒的新寄主,室内利用农杆菌侵染性克隆接种健康黄瓜,结果显示:接种30 d的黄瓜新生叶片出现褪绿症状。采用ToCV HSP70基因的引物对田间黄瓜叶片、烟粉虱和室内黄瓜新生叶片进行RT-PCR,扩增出约450 bp的条带,在NCBI上BLAST显示与KC887999.1的同源性最高,为99%。这些数据表明黄瓜是番茄褪绿病毒的寄主。这是ToCV感染黄瓜的首次报道。     

Synergistic Interaction Between Tomato chlorosis virus and Tomato spotted wilt virus Results in Breakdown of Resistance in Tomato

ABSTRACT Multiple viral infections frequently are found in single plants of cultivated and wild hosts in nature, with unpredictable pathological consequences. Synergistic reactions were observed in mixed infections in tomato plants doubly infected with the positive-sense and phloem-limited single-stranded RNA (ssRNA) crinivirus Tomato chlorosis virus (ToCV) and the negative-sense ssRNA tospovirus Tomato spotted wilt virus (TSWV). Synergism in a tomato cultivar susceptible to both viruses resulted in a rapid death of plants. A pronounced enhancement of ToCV accumulation mediated by TSWV co-infection was observed with no evident egress of ToCV from phloem tissues. No consistent alteration of TSWV accumulation was detected. More remarkable was the synergism observed in tomato cultivars which carry the Sw-5 resistance gene, which are resistant to TSWV. Pre-infection with ToCV resulted in susceptibility to TSWV, whereas co-inoculations did not. This suggested that a threshold level or a time lapse is needed for ToCV to interfere or downregulate the defense response in the TSWV-resistant plants.     

陕西杨凌番茄褪绿病毒的分子检测

近年来在陕西番茄生产区出现了一种新病害,病株表现为叶片褪绿,叶脉颜色变深及叶片增厚,果实变小发白,严重影响番茄产量及经济效益,2016年发病极为严重,部分产区甚至绝收。该病疑似由番茄褪绿病毒Tomato chlorosis virus(ToCV)引起。利用ToCV特异引物对感病番茄样品进行RT-PCR检测,结果从所采集的4份病样中均检测到ToCV预期大小的特异片段。对ToCV外壳蛋白CP基因和类热激蛋白HSP70h基因进行克隆与序列分析,ToCV-YL1 CP基因774个核苷酸序列与已报道的ToCVCP基因有较高的一致性,与山东青岛和山西晋中分离物同源性为100%。ToCV-YL1 HSP70h基因1 665个核苷酸序列与已报道的ToCV HSP70h基因有较高的同源性,与中国、韩国、日本、美国、希腊分离物同源性达到99%以上。研究表明ToCV已传播至陕西地区。     

警惕番茄褪绿病毒在我国的传播和危害

番茄褪绿病毒( Tomato chlorosis virus, ToCV )侵染番茄引起番茄褪绿病毒病。发病植株下部叶片黄化、脉间褪绿、边缘轻微卷曲, 叶片光合作用显著降低, 果实变小, 产量和品质明显下降。该病毒最早于1998年在美国佛罗里达州发现, 随后在世界多地陆续报道。我国首先于2004年在台湾报道, 2013年又在北京和山东发现并鉴定了该病毒, 同时在辣椒上检测到该病毒。ToCV属于长线形病毒科( Closteroviridae )毛形病毒属( Crinivirus )成员, 基因组为二分体正义单链RNA, 由粉虱传播。经初步调查和检测, ToCV在我国北京、山东、河北、天津等省市相继发生, 给当地番茄生产造成了严重危害。ToCV传播迅速, 成为我国番茄生产中又一重要病毒, 防控形势严峻。基于以上原因, 建议有关部门立即采取相应预防和防治措施, 组织开展相关研究和攻关, 控制该病毒在我国的传播和危害。     

侵染广东番茄的番茄褪绿病毒分子鉴定

在广东番茄上发现一种新病害,病株表现为叶片褪绿,叶脉颜色变深及叶片增厚等症状。利用番茄褪绿病毒Tomato chlorosis virus(ToCV)HSP70基因的两对特异引物对番茄病样进行RT-PCR检测,结果表明,从所采集的6份病样中均扩增到预期大小的DNA特异片段。对其中1份样品的扩增片段进行克隆与序列分析,结果表明,扩增片段包括1个长度为1 665个核苷酸的完整病毒基因,其核苷酸序列与已报道的ToCV HSP70基因有较高的同源性,表明广东番茄受到了ToCV的侵染。但ToCV广东番茄分离物的HSP70序列与国内外已报道的各分离物的同源性均低于82%,存在较大差异,其中与塞浦路斯tomato、约旦JU_20分离物的同源性最高,为81.8%。这是ToCV在广东发生的首次报道,也是该病毒HSP70基因序列存在显著差异分离物的首次发现。     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

Host Range Studies for Tomato chlorosis virus, and Cucumber vein yellowing virus Transmitted by Bemisia tabaci (Gennadius)

The Bemisia tabaci (Gennadius) biotype B transmitted host range of Tomato chlorosis virus (ToCV), genus Crinivirus, Family Closteroviridae, and Cucumber vein yellowing virus (CVYV), genus Ipomovirus, Family Potyviridae, was studied. New experimental hosts were identified for each of these viruses. Seventeen species in eight plant families were assessed as potential hosts for ToCV. Infection in asymptomatic Anthriscus cereifolium (chervil) test plants by ToCV was confirmed by using a Real-Time PCR assay designed for ToCV. The presence of readily transmissible, infectious ToCV virions in A. cereifolium was confirmed by re-isolation of the virus via whitefly-transmission from A. cereifolium to Lycopersicon esculentum and A. cereifolium. This is the first report of the experimental transmission of ToCV by B. tabaci to a species within the Umbelliferae. All other hosts assessed for the presence of ToCV were found to be uninfected. Ten species in five families were assessed as potential hosts for CVYV. The CVYV host range identified included some important crops and common weeds, such as L. esculentum, Nicotiana tabacum, A. cereifolium, Datura stramonium, Nicotiana benthamiana, Nicotiana clevlandii and Cucumis sativus. Symptoms were present on D. stramonium, N. benthamiana and C. sativus control plants. The presence of infectious whitefly transmitted CVYV virions was confirmed solely for D. stramonium and N. tabacum, following re-isolation of the virus via B. tabaci transmission from all infected species to C. sativus. This is the␣first report of experimental CVYV transmission by B. tabaci to non-cucurbitaceous crop and weed hosts belonging to the Solanaceae or Umbelliferae.