Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus Invade South-east Coast of China

An epidemic outbreak of severe yellow leaf curl disease was reported in field grown tomato within Zhejiang Province of China in the autumn–winter cropping season of 2006. A molecular diagnostic survey was carried out based on comparisons of partial and complete viral DNA sequences. Comparison of partial DNA‐A sequences amplified with degenerate primers specific for begomoviruses confirmed the presence of two types of begomoviruses. The complete DNA sequences of five isolates, corresponding to the two types, were determined. Sequence comparisons and phylogenetic analysis revealed that they correspond to two previously identified begomoviruses, Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus. The satellite DNAβ molecule was not detected in these samples by either PCR or Southern blot hybridization analysis. There has been no previous report of geminivirus disease incidence in Zhejiang Province, indicating that the introduction of these two tomato infecting geminiviruses into the agro‐ecological zone of South‐eastern China is a fairly recent event. The implications for disease control are discussed.     

Begomoviruses Associated with Yellow Leaf Curl Disease of Tomato in Iran*

The occurrence of Tomato yellow leaf curl virus (TYLCV; genus Begomovirus, family Geminiviridae) in the major tomato‐growing areas of Iran was determined using TAS‐ELISA and PCR. The nucleotide sequences of the coat protein (CP) gene and intergenic region (IR) of eight Iranian isolates were determined. CP nucleotide identities among the Iranian isolates were 96–98%, and showed 94–96% identity with TYLCV‐IR [IR:Ira:98] and TYLCV‐IL [IL:Reo:86]. However, they showed low identity (68–69%) with ToLCIRV‐[IR:Ira]. Sequence analyses of IR indicated that seven Iranian isolates had sequence identity of 93–100% with each other, and 76% identity with the Jiroft isolate; identities of 75–79% with TYLCV‐IR[IR:Ira:98] were observed in every case, and 59–62% identity with ToLCIRV‐[IR:Ira]. The IR nucleotide sequences of Iranian isolates showed 92–93% identity with TYLCV‐IL[IL:Reo:86], except the Jiroft isolate (75%). The CP and IR sequence analyses suggested that eight Iranian TYLCV isolates probably differ from ToLCIRV‐[IR:Ira]. Based on IR sequence comparisons and phylogenetic analyses, the Iranian isolates were divided into two groups. The first major group (A), consists of seven virus isolates, was most closely related to TYLCV‐IL[IL:Reo:86], and relatively divergent from TYLCV‐IR [IR:Ira:98] and ToLCIRV‐[IR:Ira]. However, the Jiroft isolate from group B did not show high similarity with TYLCV‐IR[IR:Ira:98], ToLCIRV‐[IR:Ira], and TYLCV‐IL[IL:Reo:86], suggesting that the isolate may be a divergent variant. The differences are in a range that suggests different strains or species from TYLCV‐IR[IR:Ira:98] and ToLCIRV‐[IR:Ira] are probably associated with tomato yellow leaf curl disease in Iran.     

Begomoviruses Associated with Leaf Curl Disease of Tomato in Java,Indonesia*

We report that several begomoviruses are associated with tomato leaf curl disease in Java, Indonesia. Tomato plants with leaf curl symptoms were collected from Bandung (west Java), Purwokerto (central Java), Magelang (central Java) and Malang (east Java) of Indonesia, the major tomato‐growing areas of the country. Viruses were detected using the polymerase chain reaction (PCR), with universal primers for the genus Begomovirus. PCR‐amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, the viruses were divided into three groups. With respect to amino acid (aa) identities of the N‐terminal halves of the coat proteins compared in this study, group I was most closely related to Ageratum yellow vein virus (AYVV) (97%), Ageratum yellow vein China virus‐[Hn2] (AYVCNV‐[Hn2]) (96%) and Ageratum yellow vein virus‐[Taiwan] (AYVV‐[Tai]) (95%), and ageratum‐infecting begomovirus from Java (99%). Group II had high sequence identity with a tentative species of tomato leaf curl Java virus (ToLCJAV) (96% aa) for the CP. Group III was most closely related to a proposed species of Pepper yellow leaf curl Indonesia virus (PepYLCIDV) (90% aa identity) by its partial CP sequence.     

Molecular Characterization of a Strain of Squash Leaf Curl China Virus from the Philippines

The complete nucleotide sequence of infectious cloned DNA components (A and B) of the causal agent of squash leaf curl disease in the Philippines was determined. DNA‐A and DNA‐B comprise 2739 and 2705 nucleotides, respectively; the common region is 174 bases in length. Five ORFs were found in DNA‐A and two in DNA‐B. Partial dimeric clones containing DNA‐A and DNA‐B, constructed in a binary vector and transformed into Agrobacterium tumefaciens, induced systemic infection in agro‐inoculated pumpkin plants (Cucurbita moschata). The total DNA‐A sequence was most closely related to that of Squash leaf curl China virus (SLCCNV) (88% identity), although the existence of B component of SLCCNV has not been reported. The deduced coat protein was like that of SLCCNV (98% amino acid sequence identity) and the Philippines virus has low sequence identity to Squash leaf curl virus (SLCV) and Squash mild leaf curl virus (SMLCV) (63 and 64% total nucleotide sequence identities, respectively). From these results, we propose that the Philippines virus be designated Squash leaf curl China virus‐[Philippines] (SLCCNV‐[PH]).     

云南番茄曲叶病是由烟草曲茎病毒引起的

从云南省德宏田间表现曲叶症状的番茄植株上分离到病毒分离物Y41,采集的带病植株在实验室可经烟粉虱(Bemisia tabaci)传播到健康的番茄.用针对非洲木薯花叶病毒(ACMV)、印度木薯花叶病毒(ICMV)及秋葵曲叶病毒(OLCV)的15种单抗对病样进行TAS-ELISA检测,结果表明,番茄曲叶病是由菜豆金色花叶病毒属(Begomovirus)病毒引起的,但其抗原表位型与我国广西报道的中国番茄黄化曲叶病毒(TYLCCV)不同.对Y41进行DNA-A全序列测定和分析表明,Y41 DNA-A全长2743个核苷酸,共编码6个ORF,其中病毒链编码AV1和AV2两个ORF,互补链编码AC1、AC2、AC3和AC4 4个ORF.对Y41及其它双生病毒CP进行同源性比较及系统进化关系分析表明,Y41属于"旧世界"的粉虱传双生病毒,与我国报道的烟草曲茎病毒(TCSV)及印度报道的番茄曲叶Karnataka病毒(ToLCKV)同源性最高,达到98.8%.进一步比较基因组发现,Y41与TCSV AV1、AV2、AC1、AC2、AC3、AC4各ORF同源性分别为98.8%、96.6%、86.4%、93.3%、89.6%和89.7%,基因间隔区(IR)、DNA-A同源性分别为92.1%和93.4%,且在基因间隔区内含有相似的重复子序列及排列方式.这些结果表明:Y41是TCSV在自然条件下侵染番茄的一个分离物.     

Production of monoclonal antibodies to Tomato leaf curl Bangalore virus

Purified Tomato leaf curl Bangalore virus (ToLCBV) was injected into mice and the splenocytes were used for establishing hybridoma lines. Initial screening of culture supernatants showed that 13 lines produced antibody, and after further screening four produced functional monoclonal antibodies. Upon characterisation, these were found to be of low affinity, probably due to host protein contamination and poor yield of native virus in the original preparations. In order to circumvent these problems, the coat protein of ToLCBV was over-expressed in Escherichia coli. Fusion experiments using recombinant coat protein as antigen yielded two primary hybridoma clones G₁₁ and E4 that exhibited good affinity of binding to the antigen. Sub-cloning yielded four monoclonal antibodies G₁₁E₇E₇, G₁₁E₇G₁₂, E₄E₂ and E₄G₆. G₁₁E₇E₇ and G₁₁E₇G₁₂ successfully detected ToLCBV in infected leaf extracts of tomato and Nicotiana benthamiana, viruliferous whiteflies and weed samples. These monoclonal antibodies could also detect other type III geminiviruses such as Pumpkin yellow vein mosaic virus and Bhendi yellow vein mosaic virus. Thus these monoclonal antibodies can be used for testing field-collected samples.     

广东番茄曲叶病毒G2分离物基因组DNA-A的分子特征

从采集于广东的番茄曲叶病病株上分离到病毒分离物G2 ,序列分析结果表明 ,其DNA_A为单链环状 ,全长2 74 4nt,共有 6个ORF ,其中病毒链上编码AV1(CP)、AV2 ,互补链上编码AC1、AC2、AC3和AC4。BLAST结果显示 ,与G2基因组有同源关系的病毒均属双生病毒科菜豆金色花叶病毒属。序列比较结果显示 ,G2与菜豆金色花叶病毒属病毒的DNA_A序列同源率均不超过 83% ,其中同源率最高的是PaLCuCNV_[G10 ](82 8% )。进一步比较发现 ,它们的基因间隔区 (IR)变异最大 (同源率为 30 9%～ 81 8% ) ;CP氨基酸序列的同源率较高 (77 6 %～ 99 2 % ) ,AC4蛋白氨基酸序列的同源率较低 (4 3 5 %～ 78 8% )。系统进化关系分析结果也显示 ,G2与已报道的菜豆金色花叶病毒属病毒的亲缘关系均较远。因此 ,G2可能是双生病毒科菜豆金色花叶病毒属中一个未报道的新种 ,命名为广东番茄曲叶病毒 (TomatoleafcurlGuangdongVirus ,ToLCGDV)     

Tomato Yellow Leaf Curl Virus Infection in a Monocotyledonous Weed (Eleusine indica)

Tomato yellow leaf curl virus (TYLCV) is one of the most important plant viruses belonging to the genus Begomovirus of the family Geminiviridae. To identify natural weed hosts that could act as reservoirs of TYLCV, 100 samples were collected at a TYLCV-affected tomato farm in Iksan from 2013 to 2014. The sample weeds were identified as belonging to 40 species from 18 families. TYLCV was detected in 57 samples belonging to 28 species through polymerase chain reaction using root samples including five species (Eleusine indica, Digitaria ciliaris, Echinochloa crus-galli, Panicum dichotomiflorum, and Setaria faberi) from the family Poaceae. Whitefly Bemisia tabaci-mediated TYLCV transmission from TYLCV-infected E. indica plants to healthy tomatoes was confirmed, and inoculated tomatoes showed typical symptoms, such as leaf curling and yellowing. In addition, TYLCV was detected in leaf and root samples of E. indica plants inoculated by both whitefly-mediated transmission using TYLCV-viruliferous whitefly and agro-inoculation using a TYLCV infectious clone. The majority of mastreviruses infect monocotyledonous plants, but there have also been reports of mastreviruses that can infect dicotyledonous plants, such as the chickpea chlorotic dwarf virus. No exception was reported among begomoviruses known as infecting dicots only. This is the first report of TYLCV as a member of the genus Begomovirus infecting monocotyledonous plants.     

Association of a Distinct Begomovirus and a Betasatellite with Leaf Curl Symptoms in Pedilanthus tithymaloides

Pedilanthus tithymaloides (Redbird flower) is an ornamental shrub that occasionally exhibits leaf curl and enation symptoms in Pakistan. Symptoms were shown to be associated with a monopartite begomovirus and a betasatellite. The complete nucleotide sequence of the begomovirus was found to be 2764 nucleotides in length and have the highest nucleotide sequence identity to a begomovirus previously isolated from tomato (90.3% nucleotide sequence identity), followed by Radish leaf curl virus (86.3%). The complete betasatellite sequence was determined to be 1358 nucleotides in length and has the highest sequence identity (97%) with Tobacco leaf curl betasatellite . The analysis shows the begomovirus associated with leaf curl disease of Pedilanthus to be a distinct and previously unreported begomovirus for which the name Pedilanthus leaf curl virus (PedLCV) is proposed. This virus is one of an increasing number of monopartite begomoviruses shown to be associated with a betasatellite.     

Expression of the Full‐length Coat Protein Gene of Tomato leaf curl Taiwan virus is Not Necessary for Recovery Phenotype in Transgenic Tomato

Transgenic tomato plants expressing full‐length (CPV1) and truncated coat protein (CP) gene (CPV2) of Tomato leaf curl Taiwan virus (ToLCTWV) were generated by Agrobacterium‐mediated transformation. Transgene integration and expression was confirmed by PCR and Southern blotting and Northern analysis, respectively. Resistance was evaluated both in plants of T0 and T1 progenies using viruliferous whiteflies under two different inoculum pressures (10–15 and 40–50 whiteflies/plant). Upon inoculation with ToLCTWV using viruliferous whiteflies, various levels of phenotypic reaction were observed. No complete resistance was observed in any of the plants tested. The reaction of the transgenic tomato lines carrying full‐length and truncated CP gene to ToLCTWV phenotype was (i) susceptible as non‐transgenic control, (ii) delayed symptom expression, (iii) complete susceptible (from delayed symptom expression phenotype) and (iv) recovered phenotype (either plants from symptom expression as non‐transgenic plants or delayed symptom expression phenotype). Dot blot quantification of the ToLCTWV using the replicase gene as a probe revealed that the recovered phenotypes accumulated a low level of ToLCTWV, and virus concentration was gradually reduced from 10 to 14 weeks postinoculation. The possible mechanisms of CP‐mediated resistance are discussed.     

Identification of Mungbean yellow mosaic Indian virus Associated with Tomato Leaf Curl Betasatellite Infecting Phaseolus vulgaris in Oman

Kidney bean (Phaseolus vulgaris) plants exhibiting foliar yellow mosaic symptoms and some leaf crumpling were identified in the Al Batinah region of Oman. Rolling circle amplification and polymerase chain reaction identified a bipartite begomovirus (family Geminiviridae) and a betasatellite in association with the symptomatic plants. Analysis of full‐length sequences showed the virus to be Mungbean yellow mosaic Indian virus (MYMIV) and the betasatellite Tomato leaf curl betasatellite (ToLCB). This is the first identification of a legume‐adapted begomovirus in Oman and the first identification of MYMIV in association with the betasatellite ToLCB. The isolate of MYMIV from Oman shows the greatest levels of sequence identity to isolates occurring in South Asia and South‐East Asia, suggesting that the virus has only recently been introduced. The significance of these findings is discussed.     

First Report of Tomato Yellow Leaf Curl Virus-Israel Species Infecting Tomato, Pepper and Bean in Tunisia

Tomato yellow leaf curl virus disease (TYLCVD) has been observed in Tunisia for more than 20 years. Until year 2004, only the Tomato yellow leaf curl Sardinia virus‐Sicily (TYLCSV‐[Sic]) was detected in tomato, pepper and bean crops. In the Sahel region, some tomato samples showing severe TYLCVD symptoms were collected from greenhouses in 2004 and 2005. Typing of these isolates revealed for the first time the presence of the TYLCV Israel in Tunisia. This result was confirmed by using several sets of specific primers and by sequencing. This species has also been detected on pepper and bean collected from fields in the same region. The sequencing of a tomato and a bean isolate showed that they both share more than 97% of sequence identity with the TYLCV from Dominican Republic ( AF024715 ). The TYLCV has been found in single and mixed infection with the TYLCSV‐[Sic].     

Tomato yellow leaf curl virus infection of tomato does not affect the performance of the Q and ZHJ2 biotypes of the viral vector Bemisia tabaci

Abstract To better understand the etiology of begomovirus epidemics in regions under invasion we need to know how indigenous and invasive whitefly vectors respond to virus infection. We investigated both direct and indirect effects of infection with Tomato yellow leaf curl virus (TYLCV) on the performance of the invasive Q biotype and the indigenous Asian ZHJ2 biotype of whitefly Bemisia tabaci. The Q biotype performed better than the ZHJ2 biotype on either uninfected or virus‐infected tomato plants. However, virus‐infection of host plants did not, or only marginally affected, the performance of either biotype of whiteflies in terms of fecundity, longevity, survival, development and population increase. Likewise, association of the vectors with TYLCV did not affect fecundity and longevity of the Q or ZHJ2 biotypes on cotton, a non‐host of TYLCV. These results indicate that the alien Q biotype whitefly, but not the indigenous ZHJ2 biotype, is likely to become the major vector of TYLCV in the field and facilitate virus epidemics.     

Virus–vector Relationships,Host Range,Detection and Sequence Comparison of Chilli leaf curl virus Associated with an Epidemic of Leaf Curl Disease of Chilli in Jodhpur,India

An epidemic of chilli leaf curl disease was recorded in 2004 in Jodhpur, a major chilli‐growing area in Rajasthan, India. Several isolates were efficiently transmitted by the whitefly (Bemisia tabaci), all of which induced severe leaf curl symptoms in chilli. A single whitefly was capable of transmitting the virus, and eight or more whiteflies per plant resulted in 100% transmission. The minimum acquisition access period (AAP) and inoculation access period (IAP) were 180 and 60 min, respectively. The virus persisted in whiteflies for up to 5 days postacquisition. Of 25 species tested, the virus infected only five (Capsicum annuum, Carica papaya, Solanum lycopersicum, Nicotiana tabacum and N. benthamiana). The virus was identified as Chilli leaf curl virus (ChiLCV), which shared the closest sequence identity (96.1%) with an isolate of ChiLCV from potato in Pakistan and showed sequence diversity up to 12.3% among the ChiLCV isolates reported from India and Pakistan. A betasatellite was identified, which resembled most closely (97.3%) that of Tomato leaf curl Bangladesh betasatellite previously reported from chilli and tomato leaf curl in India. The betasatellite was very different from that reported from chilli leaf curl in Pakistan, indicating that different betasatellites are associated with chilli leaf curl in India and Pakistan. We describe here for the first time the virus–vector relationships and host range of ChiLCV.     

番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F:5′-ACGCATGCCTCTAATCCAGTGTA-3′,下游引物TYLCV-R:5′-CCAATAAGGCGTAAGCGTGTAGAC-3′),依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒,这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种,从而为蔬菜安全可持续生产提供科技支撑。     

番茄黄化曲叶病毒的快速分子检测

番茄黄化曲叶病毒是当前世界范围内危害番茄生产的毁灭性病害。文章针对番茄黄化曲叶病毒全基因组序列的特异区段自主设计了1对特异性PCR引物(上游引物TYLCV-F：5′-ACGCATGCCTCTAATCCAGTGTA-3′, 下游引物TYLCV-R：5′-CCAATAAGGCGTAAGCGTGTAGAC-3′), 依据PCR扩增特异片段543 bp的有无可以快速、准确、高效、特异地检测出是否感染了TYLCV病毒, 这项技术可以方便地应用到工厂化育苗的带毒性检测、蔬菜大规模生产中植株发病情况的快速检测以及抗病毒育种, 从而为蔬菜安全可持续生产提供科技支撑。     

烟粉虱携带番茄黄化曲叶病毒检测及其传入山东省的考证

【背景】番茄黄化曲叶病毒（TYLCV）是由媒介昆虫烟粉虱传播的一种双生病毒,对蔬菜及烟草等经济作物造成严重危害。前人资料表明,该病毒于2006年传人我国南方地区,2007年传人山东省,2008年后在山东各地逐渐蔓延扩散。【方法】为了考证TYLcV传人山东省的时间,本研究利用mtCOI基因对于2005和2006年7—8月份在山东省不同地区作物上共采集的15份烟粉虱样品进行了生物型鉴定,并进一步检测了烟粉虱携带TYLCV情况,同时对PCR扩增产物进行了测序分析。【结果】2005年的4份样品烟粉虱生物型均为B型,均不携带TYLCV。2006年的11份烟粉虱样品为B型与Q型混合样品,其中,2份烟粉虱样品检测到TYLCV,进一步证实该病毒为TYLCV。【结论与意义】本研究首次证实了TYLCV早在2006年就已经传入山东省。研究结果不仅对于防控该病毒具有重要指导意义,而且对于其入侵生物学研究也具有重要参考价值。