中国番茄黄化曲叶病毒——双生病毒的一个新种

用 2 0个单抗对中国番茄黄化曲叶病毒 (TYLCV CHI)和其他双生病毒进行了测定 ,在血清学水平上证实中国番茄黄化曲叶病毒与中国烟草曲叶病毒有较大的亲缘关系 ;同时报道了TYLCV CHI部分共同区、外壳蛋白N端基因和AV1基因的PCR及其克隆和序列分析 ,从分子水平上证实TYLCV CHI与世界各地的其他双生病毒不同 ,是一种新的粉虱传双生病毒     

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。     

番茄黄化曲叶病毒对番茄叶片光合特性和叶绿体超微结构的影响

以番茄为试验材料,研究番茄黄化曲叶病毒(TYLCV)侵染对植株叶片叶绿素含量、净光合速率、气孔导度、胞间CO2浓度和叶绿体超微结构的影响.结果表明:TYLCV侵染番茄后,叶片叶绿素a、b以及总叶绿素含量分别下降50.2%、24.19%和43.84%,叶片净光合速率和气孔导度分别下降43.28%、27.07%,胞间CO2浓度增加13.04%.与健康叶片相比,叶绿体变形,叶绿体基质片层大部分消解,基粒结构消失,叶绿体外膜和内膜剥离,质壁分离和细胞膜内陷,细胞器消解.研究表明,TYLCV侵染破坏了番茄叶片的叶绿体结构,严重影响番茄叶片的光合作用.     

上海地区番茄黄化曲叶病毒病的鉴定及嫁接接种法研究

番茄黄化曲叶病毒(tomato yellow leafcurl virus,TYLCV)是一种由烟粉虱(Bemisia tabaci)和嫁接传播的双生病毒,在热带、亚热带地区给番茄生产造成严重威胁.根据番茄黄化曲叶病毒的保守序列设计一对引物,运用PCR技术从上海地区的感病番茄中扩增出一条575bp的特异带,而健康植株无此带.测序表明该序列与番茄黄化曲叶病毒具有极高的同源性(97%～99%).将健康接穗嫁接到感染番茄黄化曲叶病毒的番茄砧木上,间隔15 d和30 d,分别提取接穗的DNA,并用PCR法检测病毒,发现嫁接15 d后在部分接穗中检测到TYLCV病毒,嫁接30 d后在所有的接穗中均检测到病毒,因此,嫁接法可以作为番茄黄化曲叶病毒病的接种鉴定方法.     

高抗番茄黄化曲叶病毒病番茄新品种浙粉702的选育及抗病性鉴定

高抗黄化曲叶病毒病番茄新品种浙粉702是以自育株系材料T7969F2-19-1-1-3为母本,T4078F2-3-3-3为父本,结合分子标记辅助技术选育的杂交一代粉红大果型番茄品种。母本‘T7969F2-19-1-1-3’系从以色列引进的耐贮运番茄品种‘NEMO-TAMMI’（F1）与抗叶霉病粉红株系材料‘T9179’杂交分离后代中经连续9代单株选择而成。父本‘T4078F2-3-3-3’系从荷兰引进的抗TYLCVD番茄品种‘奇诺亚’（F1）与粉红株系材料‘T9178’杂交分离后代中经连续8代单株选择而成。该品种2011年通过浙江省非主要农作物认定委员会认定。通过对浙粉702园艺性状、产量性状、品质性状和抗病性等进行研究,结果表明：浙粉702 品质优良,早熟,丰产,高抗番茄黄化曲叶病毒病,枯萎病,抗叶霉病和番茄花叶病毒病,适合我国喜食粉果地区种植,平均可达73.83 t.hm-2。     

高抗番茄黄化曲叶病毒病番茄新品种浙粉702的选育

高抗黄化曲叶病毒病番茄新品种浙粉702是以自育株系材料T7969F2-19-1-1-3为母本、T4078F2-3-3-3为父本,结合分子标记辅助技术选育的杂交一代粉红大果型番茄品种。母本T7969F2-19-1-1-3系从以色列引进的耐贮运番茄品种NEMOTAMMI(F1)与抗叶霉病粉红株系材料T9179杂交分离后代中,经连续9代单株选择而成。父本T4078F2-3-3-3系从荷兰引进的抗TYLCVD番茄品种奇诺亚(F1)与粉红株系材料T9178杂交分离后代中,经连续8代单株选择而成。该品种2011年通过浙江省非主要农作物认定委员会认定。通过对浙粉702的农艺学、产量、品质和抗病等性状鉴定,结果表明:浙粉702品质优良,早熟,丰产,高抗番茄黄化曲叶病毒病和枯萎病,抗叶霉病和番茄花叶病毒病,适合我国喜食粉果地区种植,平均产量可达73.83 t/hm2。     

侵染广西烟草的中国番茄黄化曲叶病毒及其伴随的卫星DNA分子的基因组特征

从中国广西靖西的烟草病株上分离到病毒分离物G102和G103,用双生病毒特异性引物均扩增出约500bp的片段,两者序列同源性达99%。对G102基因组DNA-A全序列测定表明,其全长为2728个核苷酸,与中国番茄黄化曲叶病毒(TYLCCNV)同源性最高,达96.5%。进一步研究发现,G102和G103都伴随有长为1342个核苷酸的卫星DNA分子(DNAβ),这两个DNAβ分子的全序列与TYLCCNV的DNAβ同源性最高,分别为92.9%和93.4%。这是首次明确广西分离的TYLCCNV也伴随有卫星分子。     

侵染稀硷的中国番茄黄化曲叶病毒及其卫星DNA全基因组结构特征

从云南红河稀硷上分离到病毒分离物Y64,全序列测定表明,Y64 DNA_A全长2730个核苷酸。基因组比较发现,Y64 DNA_A与中国番茄黄化曲叶病毒Y38分离物（TYLCCNV_［Y38］）同源性最高（99％）,与中国番茄黄化曲叶病毒广西分离物（TYLCCNV）的同源性次之（96％）,而与亚洲地区的其它双生病毒的同源性均在83％以下, 表明稀硷上的分离物Y64是TYLCCNV的1个分离物。利用DNAβ的特异性引物beta01和beta02,在病毒分离物Y64中扩增到卫星DNA分子（Y64β）。序列分析表明,Y64β全长1340个核苷酸,至少在其互补链上编码1个有功能的ORF（C1）。Y64β的全序列与TYLCCNV的各个分离物的卫星分子（Y38β、Y36β和Y8β）的同源性最高,分别为99.5％、99.5％和99.3％;与其它已报道的卫星DNAβ的同源性均低于66.4％。系统关系树研究表明,卫星DNAβ分子与其辅助病毒是共同进化的。     

Within-Host Dynamics of the Emergence of Tomato Yellow Leaf Curl Virus Recombinants

Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV) has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi), and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci. We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection–a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our results anticipate the outcomes of natural encounters between TYLCV and ToLCKMV.     

Recruitment of the Host Plant Heat Shock Protein 70 by Tomato Yellow Leaf Curl Virus Coat Protein Is Required for Virus Infection

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.     

Reservoir Weed Hosts of Tomato Yellow Leaf Curl Begomovirus from Tanzania

A survey was initiated to detect tomato yellow leaf curl virus (TYLCV) and identify its reservoir weed hosts in six regions (Arusha, Morogoro, Dodoma, Iringa, Kilimanjaro and Dar es Salaam) in Tanzania. Three farms were randomly selected in each region. Assessment of TYLCV incidence was done by relating the number of infected tomato plants to the total number of plants assessed along a diagonal in five quadrants measuring 4m ‐ 4m in size (one at each corner of the farm and one at the centre). Disease severity was scored on a scale of 0 to 4 (where 0 = no symptoms and 4 = very severe symptoms). Within and outside each farm, weeds showing TYLCV-like symptoms were collected and either squash-blotted, dot-blotted or both on nylon membranes. The membranes were hybridized with DIG-labelled probe synthesized for the detection of TYLCV from Sardinia (TYLCV-Sar) following standard protocols. Selected plant species were experimentally inoculated with screenhouse cultures of TYLCV representative isolates from the six regions using Bemisia tabaci to determine their host status. Results indicated that TYLCV incidence and severity were significantly higher (P = 0.05) in Dodoma region than the rest of the regions. In Iringa region, the incidence and severity of TYLCV were the lowest of all regions. TYLCV was detected in 12 of the 17 dot-blotted samples and in all the 21 squashed samples using the non-radioactively labelled riboprobes. Similarly, five plant species (Capsicum annuum, Datura stramonium, Lycopersicon esculentum, Nicotiana glutionsa and N. tabacum) tested in the screenhouse were infected by the six TYLCV isolates used. It is recommended that weeds within and outside tomato farms be removed to eliminate or reduce sources of virus inoculum. The dot and squash blot techniques are convenient for field detection of the virus, and are especially useful for the detection of early and latent infections so that management strategies can be initiated and implemented.