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。     

Pyramiding Ty‐2 and Ty‐3 genes for resistance to monopartite and bipartite tomato leaf curl viruses of India

Breeding resistance to whitefly‐transmitted begomoviruses is an important goal of tomato breeding programmes worldwide. So far, resistance to begomoviruses in tomato has been achieved using wild species, and at least five resistance genes (Ty genes) have been studied. The present study was undertaken to combine Ty‐2 and Ty‐3 and to determine the effect of pyramiding on infection of tomato by three diverse begomovirus species. The diagnostic ability of the markers linked to Ty genes was assessed and marker‐assisted selection was used to develop pyramided tomato lines from the crosses between Ty stocks. Five stable pyramided tomato lines that differ in fruit morphology and yield potential were developed. The horticultural performance of pyramided lines in field trials showed that the yield and horticultural traits are well maintained in the plants. The response of these lines was assessed using agroinoculation and field tests in a disease hotspot. The pyramided lines and Ty‐3‐carrying lines exhibited a high level of resistance to the monopartite and two bipartite begomoviruses tested. The pyramided tomato lines developed in this study could be important genetic resources for sustainable tomato production in areas affected by tomato leaf curl virus disease. The combined results of disease resistance tests also showed that Ty‐3 is critical for achieving broad‐spectrum resistance. The limitations of relying on a single gene and the importance of pyramiding are discussed in the light of available evidence on frequent recombination in begomoviruses.     

The Natural Occurrence of Two Distinct Begomoviruses Associated with DNAbeta and a Recombinant DNA in a Tomato Plant from Indonesia

ABSTRACT Two begomoviruses (Java virus-1 and Java virus-2), two satellite DNAs (DNAbeta01 and DNAbeta02), and a recombinant DNA (recDNA) were cloned from a single tomato plant from Indonesia with leaf curl symptoms, and the role of these satellite DNAs in the etiology of begomovirus disease was investigated. The genome organizations of the two viruses were similar to those of other Old World monopartite begomoviruses. Comparison of the sequences with other begomoviruses revealed that Java virus-1 was a newly described virus for which the name Tomato leaf curl Java virus (ToLCJAV) is proposed. Java virus-2 was a strain of Ageratum yellow vein virus (AYVV) (AYVV-[Java]). ToLCJAV or AYVV-[Java] alone did not induce leaf curl symptoms in tomato plants. However, in the presence of DNAbeta02, both ToLCJAV and AYVV-[Java] induced leaf curl symptoms in tomato plants. In the presence of DNAbeta01, these viruses induced mild leaf curl symptoms in tomato plants. The recDNA had a chimeric sequence, which arose from recombination among ToLCJAV, AYVV-[Java], DNAbeta01, and DNAbeta02; it was replicated only in the presence of AYVV-[Java] in tomato plants.     

Sequence analysis and classification of apparent recombinant begomoviruses infecting tomato in the nile and mediterranean basins

ABSTRACT Numerous whitefly-transmitted viral diseases of tomato have emerged in countries around the Nile and Mediterranean Basins the last 20 years. These diseases are caused by monopartite geminiviruses (family Gemini viridae) belonging to the genus Begomovirus that probably resulted from numerous recombination events. The molecular biodiversity of these viruses was investigated to better appreciate the role and importance of recombination and to better clarify the phylogenetic relationships and classification of these viruses. The analysis partitioned the tomato-infecting begomoviruses from this region into two major clades, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus. Phylogenetic and pairwise analyses together with an evaluation for gene conversion were performed from which taxonomic classification and virus biodiversity conclusions were drawn. Six recombination hotspots and three homogeneous zones within the genome were identified among the tomatoinfecting isolates and species examined here, suggesting that the recombination events identified were not random occurrences.     

Tomato leaf curl Karnataka virus from Bangalore, India, Appears to be a Recombinant Begomovirus

ABSTRACT The genome of Tomato leaf curl virus (ToLCV) from Bangalore, India, a whitefly-transmitted geminivirus, was cloned (pIND9) and sequenced. The circular DNA of 2,759 nucleotides (U38239) is organized similarly to that of other begomoviruses with monopartite genomes. Comparison of the nucleotide sequence of pIND9 with other tomato-associated begomoviruses from India (Tomato leaf curl Bangalore virus [ToLCBV, Z48182]) and Tomato leaf curl New Delhi virus-Severe (ToLCNdV-Svr, U15015) showed moderate DNA sequence identities (82 to 87%) between capsid protein (CP) genes but low identities (66 to 67%) for the intergenic regions and the replication-associated protein (Rep) genes (75 to 81% identity). Phylogenetic trees generated with nucleotide sequences of the Rep and CP genes of 26 begomoviruses indicated that this ToLCV is distinct from other begomoviruses and that it may be a recombinant virus derived from at least three different viral lineages. Tomatoes (Lycopersicon esculentum) inoculated with the cloned DNA monomer of ToLCV (pIND9) via particle bombardment developed leaf curling and yellowing symptoms. The virus was transmitted by Bemisia tabaci biotype B from tomatoes infected via particle bombardment to healthy tomatoes and by sap inoculation from infected tomatoes to tomato, Nicotiana benthamiana and N. tabacum. This ToLCV is a distinct member of the genus Begomovirus from India that differs from the previously characterized Tomato leaf curl Sadasivanagar virus isolate Bangalore 1 (L12739), ToLCBV (Z48182), ToLCBV isolate Bangalore 4 (AF165098), and the bipartite ToLCNdV (U15015, U15016). Thus, this ToLCV is named Tomato leaf curl Karnataka virus (ToLCKV).     

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.     

Resistance to Monopartite Begomoviruses Associated with the Bean Leaf Crumple Disease in Phaseolus vulgaris Controlled by a Single Dominant Gene

ABSTRACT Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Málaga virus are monopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect common bean (Phaseolus vulgaris), causing bean leaf crumple disease (BLCD). This disease was found to be widespread in southern Spain and causes stunted growth, flower abortion, and leaf and pod deformation in common bean plants. Commercial yield losses of up to 100% occur. In the present study, we have identified and characterized a resistance trait to BLCD-associated viruses in the common bean breeding line GG12. This resistance resulted in a complete absence of BLCD symptoms under field conditions or after experimental inoculation. Our analysis showed that virus replication was not inhibited. However, a severe restriction to systemic virus accumulation occurred in resistant plants, suggesting that cell-to-cell or long-distance movement were impaired. In addition, recovery from virus infection was observed in resistant plants. The reaction of P. vulgaris lines GG12 (resistant) and GG14 (susceptible), and of F(1), F(2), and backcross populations derived from them, to TYLCV inoculation suggested that a single dominant gene conferred the BLCD resistance described here.     

侵染假酸浆的泰国番茄黄化曲叶病毒全基因组结构特征

为明确假酸浆Nicandra physalodes叶片黄化、皱缩症状是否由菜豆金色花叶病毒属病毒侵染引起,本研究利用分子检测方法和生物信息学技术鉴定了假酸浆样品中的病毒种类。从采集的病样中克隆并获得了2条菜豆金色花叶病毒属病毒DNA-A全序列和1条beta卫星全序列,经全序列分析发现,该双生病毒的两条DNA-A全序列与泰国番茄黄化曲叶病毒(tomato yellow leaf curl Thailand virus, TYLCTHV)云南分离物TYLCTHV-YN1732一致性最高,达99.3%,亲缘关系较近;beta卫星的全序列与云南番茄曲叶beta卫星(tomato leaf curl Yunnan betasatellite, TLCYnB)的分离物YN5230一致性最高,达99.3%,亲缘关系较近。重组分析显示,假酸浆上分离的TYLCTHV-YN5735-12是一个重组病毒,有两个重组事件,一个主要发生在AV1的编码区,由中国番茄黄化曲叶病毒(tomato yellow leaf curl China virus, TYLCCNV)和广西大戟曲叶病毒(euphorbia lea...     

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%).     

A Recessive Allele (tgr-1) Conditioning Tomato Resistance to Geminivirus Infection Is Associated with Impaired Viral Movement

ABSTRACT Begomoviruses (the family Geminiviridae) are transmitted by the whitefly Bemisia tabaci and contain monopartite or bipartite circular single-stranded (ss)DNA genomes. They have emerged as severe problems in the production of agricultural and horticultural crops worldwide. Here, we report the identification of a tomato breeding line, FLA653, that confers a high level of resistance to Tomato leaf curl virus (TLCV, monopartite). Genetic analysis indicated that the resistance is controlled by a single recessive allele named tgr-1, which is in contrast to previous reports that multiple genetic factors are involved in tomato resistance to begomoviruses. Particle bombardment of an infectious TLCV DNA construct into the detached leaves of FLA653 resulted in the viral replication, but the viral ssDNA accumulated at a much lower level than that in susceptible controls. In situ localization of TLCV in the bombarded leaves suggests that tgr-1 impaired TLCV movement, raising the possibility that it may specify a host factor essential for viral systematic infection. This makes tgr-1 a strong candidate for developing resistance in major crops carrying the gene homologue.     

侵染新疆加工番茄的中国番茄黄化曲叶病毒 DNA-A的基因组特征

 从新疆加工番茄上分离到病毒分离物XJ26-4,对其基因组DNA-A 全序列测定表明,XJ26-4 DNA-A 全长2 737 个核苷酸(GenBank 登录号:FN985163),具有典型的双生病毒基因组特征。进一步序列比较发现,XJ26-4 DNA-A 与中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus, TYLCCNV)各分离物的同源性最高,达到91. 2% ~ 99. 5% ,而与其他双生病毒的序列相似性均在79. 5% 以下,表明XJ26-4 是TYLCCNV 的一个分离物。这是首次明确新疆加工番茄受到粉虱传双生病毒的侵染。     

Genetic diversity and mixed infections of begomoviruses infecting tomato, pepper and cucurbit crops in Nicaragua

Begomoviruses were detected in Nicaraguan fields of tomato ( Lycopersicon esculentum ) and adjacently growing plants of pepper ( Capsicum annuum ), chilli pepper ( C . baccatum ), cushaw ( Cucurbita argyrosperma ) and Mexican fireplant ( Euphorbia heterophylla ) using polymerase chain reaction (PCR) and universal begomovirus primers. All tomato and Mexican fireplant plants showing symptoms were infected with begomoviruses, while only 30–46% of the pepper, chilli pepper and cushaw plants showing symptoms tested virus-positive. No begomoviruses were found in potato. The virus species were provisionally identified by sequencing 533 bp of the viral coat protein gene ( AV1 ). Tomato severe leaf curl virus (ToSLCV), Tomato leaf curl Sinaloa virus (ToLCSinV) and Pepper golden mosaic virus (PepGMV) were found to infect both tomato and pepper. A new provisional species designated Tomato leaf curl Las Playitas virus (ToLCLPV) was detected in a tomato plant. Squash yellow mottle virus (SYMoV) and PepGMV were found in cucurbits, the latter for the first time in this host. Euphorbia mosaic virus (EuMV) was detected in Mexican fireplant. Sequencing of a larger number of PCR-amplified clones from selected plants revealed intraspecific viral sequence variability, and also multiple begomovirus infections which could represent up to three species in a single tomato or cushaw plant. Phylogenetic grouping of virus sequences did not correlate with the host of origin.     

Tomato dwarf leaf curl virus, a new bipartite geminivirus associated with tomatoes and peppers in Jamaica and mixed infection with tomato yellow leaf curl virus

Genomic characterization using nonradioactive probes, polymerase chain reaction with degenerate primers for whitefly transmitted geminiviruses and nucleotide sequencing were used to describe a new bipartite geminivirus, associated with dwarfing and leaf curling of tomatoes and peppers in Jamaica. Partial DNA-A and DNA-B clones were obtained. DNA sequence analysis showed that tomato and pepper samples have a similar geminivirus associated with them. Nucleotide sequence identity > 92% between the common regions of DNA-A and DNA-B confirmed the bipartite nature of the Jamaican geminivirus isolates. Nucleotide sequence comparisons of DNA-A and DNA-B with those of geminiviruses representing the major phylogenetic groups of Western Hemisphere geminiviruses showed the greatest similarity to potato yellow mosaic virus and members of the Abutilon mosaic virus cluster of geminiviruses. This new virus is given the name tomato dwarf leaf curl virus (TDLCV) because of the dwarfing and leaf curling symptoms associated with infected tomato plants. Polymerase chain reaction and Southern hybridization showed mixed infections of TDLCV with tomato yellow leaf curl virus from Israel in 16% of the field samples of tomatoes and peppers.     

复合侵染水茄的两种菜豆金色花叶病毒属病毒基因组结构特征分析

为明确水茄Solanum torvum植株叶片邹缩、褪绿是否由菜豆金色花叶病毒属病毒侵染引起,从云南省西双版纳傣族自治州田间采集具有疑似感染症状的水茄植株叶片样品,应用菜豆金色花叶病毒属病毒简并引物和特异性引物进行PCR扩增、克隆和测序,通过生物信息软件分析比较其核苷酸序列特征,并对其进行系统发育分析。结果显示,从采集的疑似病叶中共克隆获得了5条菜豆金色花叶病毒属病毒DNA-A全序列和3条DNA-B全序列,经全序列分析发现,侵染水茄的2种菜豆金色花叶病毒属病毒分离物分别属于中国南瓜曲叶病毒(squash leaf curl China virus,SLCCNV)和野茼蒿黄脉病毒(Crassocephalum yellow vein virus,CraYVV)。SLCCNV水茄分离物的基因组具有典型的菜豆金色花叶病毒属病毒双组分结构特征,与来自泰国的SLCCNV分离物(AB330078)亲缘关系最近,相似性最高达到99.0%;CraYVV水茄分离物的基因组具有典型的菜豆金色花叶病毒属病毒单组分结构特征,与来自云南省景洪市的CraYVV分离物(EF165536)亲缘关系最近,相似性最高达到97.6%。表明水茄是这2种菜豆金色花叶病毒属病毒的新寄主,并首次发现双组分和单组分菜豆金色花叶病毒属病毒可复合侵染水茄。     

Detection and molecular characterization of viruses associated with tomato yellow leaf curl disease in cucurbit crops in Jordan

In this study, Tomato yellow leaf curl Sardinia virus (TYLCSV) and the strains Israel and Mild of Tomato yellow leaf curl virus (TYLCV-IL, TYLCV-Mld) were detected for the first time in four cucurbit crops in Jordan by nested polymerase chain reaction (nPCR). These viruses cause the tomato yellow leaf curl disease (TYLCD) in tomato. Cucumber, squash, melon and watermelon plants inoculated with TYLCV-IL[JO:Cuc], TYLCV-Mld, TYLCSV-IT[IT:Sar:88] and the Jordanian isolate of TYLCV (TYLCV-JV) did not show disease symptoms. However, virus-specific fragments were detected in uppermost leaves of symptomless plants by nPCR. A whitefly transmission test showed that Bemisia tabaci could transmit TYLCV-Mld from cucumber into tomato and jimsonweed plants. However, all infected tomato plants remained symptomless. In addition, results of semi-quantitative PCR (sqPCR) analysis showed that the relative amount of TYLCV-Mld DNA acquired by B. tabaci from cucumber plants was less than that acquired from tomato plants.     

Diversity,distribution and prevalence of vegetable-infecting geminiviruses in Burkina Faso

In West and Central Africa, as in many regions of the world, vegetables are severely affected by geminivirus diseases. In Burkina Faso, observation of various virus-like symptoms, especially on tomato, suggests the involvement of several geminiviruses and underlines the pressing need for additional information on their diversity, distribution, prevalence and host plant reservoirs. Large-scale surveys conducted in Burkina Faso confirmed the presence of tomato (yellow) leaf curl diseases (ToLCD-TYLCD) and geminiviruses in all localities with mean prevalences of 25% and 45%, respectively. Five geminiviruses including four begomoviruses (pepper yellow vein Mali virus (PepYVMLV), tomato leaf curl Burkina Faso virus, tomato leaf curl Mali virus and tomato leaf curl Ghana virus) and a dicot-infecting mastrevirus (chickpea chlorotic dwarf virus) were characterized on tomato. In addition, PepYVMLV and cotton leaf curl Gezira virus (CLCuGeV) were characterized on pepper and okra, respectively, in combination or not with alphasatellites and betasatellites for CLCuGeV. The most severe, prevalent and widely distributed virus on vegetables was PepYVMLV, which was characterized for the first time in combination with a genetically divergent DNA-B component that may constitute a key factor of PepYVMLV pathogenicity. Of the eight weeds identified as potential reservoir hosts of begomoviruses, four host PepYVMLV. The results confirm the importance of geminivirus diseases on vegetable crops in Burkina Faso and highlight the complex association of geminiviruses and satellites. The detection of begomoviruses in weeds growing close to crops points to the increasing necessity to consider reservoir plants and virus communities in the control of virus diseases.     

Occurrence of a new recombinant begomovirus species infecting tomato in the Al‐Batinah region of Oman

Whitefly‐transmitted begomoviruses are the most important limiting factor for tomato cultivation in Oman, particularly in the Al‐Batinah region, the major agricultural area of the country. Commercial farms in the Al‐Batinah region were surveyed during January–March 2013. Samples of tomato showing leaf curl disease symptoms typical of begomoviruses were collected and analysed. Full‐length sequences of five clones were shown to have relatively low percentage identity values to known begomoviruses, with the highest (88·6%) to isolates of Tomato leaf curl Oman virus (ToLCOMV), a begomovirus previously reported in Oman, indicating that these represent a newly identified species, for which the name Tomato leaf curl Barka virus (ToLCBrV) is proposed. Four isolates of ToLCBrV were found associated with Tomato leaf curl betasatellite (ToLCB). The five isolates of ToLCBrV characterized in this study were shown to be recombinants, with ToLCOMV as the major parent, and a fragment of Croton yellow vein virus (CrYVV) spanning the 3′ half of the replication‐associated protein. The significance of these findings is 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。     

河北省主推番茄品种抗黄化曲叶病鉴定与筛选初报

采用田间自然鉴定方法对河北省42个番茄生产主推品种进行抗番茄黄化曲叶病鉴定。结果表明,‘迪芬尼’、‘74 589’、‘佳西娜’、‘格利’、‘74 587’、‘荷兰6号’、‘Ty204’等7个品种田间表现明显抗番茄黄化曲叶病,发病率分别为0、0、0、1.35%、1.32%、0、1.76%。其余35个品种田间均表现明显感病,发病率最高的品种为‘鲁寿巨粉’,发病率达到99.22%,各品种发病率差异显著。     

陕西省早春茬番茄定植时间与番茄黄化曲叶病毒病发生的关系

为了明确早春茬番茄定植时间与番茄黄化曲叶病毒病发生的关系,采取小区对比试验对不同定植时间下番茄上烟粉虱种群消长动态、番茄黄化曲叶病毒病发病株率、发病程度及番茄产量和经济效益等进行了系统调查。结果表明:番茄田烟粉虱种群消长动态在年度间基本相同,4月上旬始见,5月中旬达到高峰,烟粉虱发生后15 d左右出现番茄黄化曲叶病毒病症状。4月上旬以前定植番茄黄化曲叶病毒病发生较轻,单位面积产量和经济效益较高,4月上旬以后定植发病较重,产量和经济效益较低。定植时间(X_1)与番茄黄化曲叶病毒发病株率(Y_1)及病情指数(Y_2)均呈显著正相关,其相关关系为Y_1=1.470 4X_1+7.947 2,Y_2=0.876 7X_1-6.441 7。生产实践中采取双膜或三膜覆盖栽培等措施适当提早定植,避免番茄感病期与烟粉虱发生高峰期相遇,在不使用任何防治措施下,可有效预防番茄黄化曲叶病毒病暴发成灾。