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.     

接种番茄斑萎病毒番茄植株对西花蓟马生物学特性的影响

西花蓟马Frankliniella occidentalis(Pergande)是我国的一种重要入侵害虫。本文研究了西花蓟马在番茄3种处理(健康CK、机械接种番茄斑萎病毒MI、机械损伤MD)叶片上的生长发育、存活及种群增长。结果表明:健康、机械接种番茄斑萎病毒、机械损伤叶片上的发育历期依次为12.68、12.99和11.79d。雌雄成虫寿命和雌虫繁殖能力在各处理叶片上差异不显著(P>0.05)。健康、机械接种番茄斑萎病毒、机械损伤叶片上的内禀增长率依次为0.1362、0.1526和0.1292d-1。本研究表明,接种番茄斑萎病毒的番茄叶片未缩短西花蓟马发育历期,也不能延长寿命及提高产卵量,不能明显加速种群数量增长。这意味着番茄斑萎病毒对西花蓟马在番茄叶片上的生物学特性未能产生明显的有利作用。     

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     

蚕豆萎蔫病毒—新疆番茄分离株的鉴定

在新疆番茄斑驳病株上分离出一种球状病毒,回接到番茄上产生斑驳症状。病毒粒体为２０面体,平均直径２５ｎｍ。经汁液摩擦接种可感染昆诺阿藜、苋色藜、蚕豆、番茄等１８种植物,不感染菜豆、豇豆、豌豆、六叶茄、黄瓜等。可由桃蚜传毒。在琼脂双扩散试验中,能与蚕豆萎蔫病毒（ＢＢＷＶ）抗血清产生明显的沉淀线,与豇豆花叶病毒（ＣｐＭＶ）、黄瓜花叶病毒（ＣＭＶ）抗血清均不发生反应。纯化病毒紫外扫描呈典型的核蛋白吸收叫线,病毒衣壳蛋白由两种蛋白亚基构成,其分子量分别为４５９００和２０４００道尔顿,由１８种氨基酸约２５５个氨基酸残基组成。病毒核酸是双组份的,它们的分子量为１３２００００和２３４００００道尔顿。根据上述结果认为该病毒是蚕豆萎蔫病毒侵染番茄的一个株系。     

银川番茄斑萎病毒的分子鉴定

为明确银川番茄(Lycopersicon esculentum)是否遭受了番茄斑萎病毒(TSWV)的危害, 采用国家标准TSWV RT- PCR检测技术对银川番茄上采集的14份疑似感染TSWV病叶样本进行分子鉴定, 对克隆得到的核衣壳蛋白基因N (Nucleocapsid)序列进行多序列比对和系统进化树分析, 随后对PCR阳性样本进行蛋白检测。结果表明, 14份病叶样本中有8份扩增出长度为394 bp的TSWV N基因序列, 且8条序列完全一致; 获得的银川番茄TSWV分离物与云南番茄、中国莴苣(Lactuca sativa)、中国鸢尾(Iris tectorum)和重庆辣椒(Capsicum annuum) TSWV分离物相对近缘, 与山东、黑龙江和北京等地及国外TSWV分离物相对远缘; 利用TSWV的抗体通过Western blot对8个PCR阳性样本进一步检测, 结果也证实8个阳性样本中存在TSWV感染。该研究首次通过分子鉴定及蛋白检测证明银川番茄上存在TSWV感染, 需要加快抗TSWV番茄品种的选育工作。     

用ELISA和Dot-ELISA方法检测植物病原的比较研究

比较了ELISA和Dot-ELISA方法检测番茄巨芽类菌原体(Tomato Big Bud Mycoplatma-Like Organism,TBB-MLO)、番茄斑萎病毒(Tomato Spotted Wilt Virus,TSWV)和青枯假单胞菌(Pseudomonas solanacearum)的差异,结果表明,两种方法对番茄斑萎病毒无明显差异,对番茄巨芽类菌原体,用Dot-ELISA比用ELISA的检测灵敏度高50—100倍,对青桔假单胞菌,用Dot-ELISA比用ELISA检泓抗原     

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。     

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.     

口蹄疫病毒结构蛋白P1-2A及蛋白酶3C基因的转基因番茄对豚鼠免疫反应的初步研究

构建了O型口蹄疫病毒China99株结构蛋白P1-2A、非结构蛋白3C以及部分2B基因(P1-2X3C)的植物双元表达载体pBin438/P1-2X3C,通过农杆菌介导法转化番茄子叶,经卡那霉素抗性筛选,获得40余株抗性植株,对得到的抗性植株进行分子生物学检测,65%的再生植株PCR检测阳性;RT-PCR结果证实P1-2X3C基因在转基因番茄中能够有效转录;ELISA和Western blot检测表明转基因植株中表达的目的蛋白具有免疫反应性。转基因番茄叶片蛋白粗提液经肌肉途径免疫豚鼠,于第3次免疫后28d用100ID50/0.2mL的同源强毒攻击,结果表明口蹄疫病毒P1-2X3C基因的转基因番茄表达产物具有良好的免疫原性,豚鼠3免后血清效价可达1:64~1:128,攻毒后两组免疫豚鼠保护率分别达3/5和5/5。     

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。     

侵染番茄的番茄花叶病毒的研究

从种传番茄苗中获得一病毒分离物Ｔｏ－Ｓｌ,人工摩擦接种７科２４种植物,Ｔｏ～Ｓｌ能侵染４科１５种植物,在番茄上产生花叶,在白肋烟上为局部枯斑。Ｔｏ－Ｓｌ的钝化温度为８５～９０℃,稀释限点为１０￣（－６）～１０￣（－７）．体外保毒期在一个月以上。病毒粒体杆状,长度主要分布于２８１～３００ｎｍ之间,平均长度２８８ｎｍ。病毒衣壳蛋白亚基只有一条多肽链,分子量为２１ｋＤａ。ｄｓＲＮＡ分析测得其基因组长度为６．４ｋｂ。琼胎糖双扩散和胶内交叉吸附试验证明,Ｔｏ－Ｓｌ与ＴＭＶ有血清关系,但有一定的差异,病毒粒体电泳分析也表明Ｔｏ－Ｓｌ与ＴＭＶ粒体有差异。在交叉保护试验中,ＴＭＶ和Ｔｏ－Ｓｌ之间均无保护作用。根据以上试验结果Ｔｏ－Ｓｌ被鉴定为番茄花叶病毒。这是我国首次系统报道番茄上番茄花叶病毒的侵染。     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

我国南方花生发生一种由蕃茄斑萎病毒引起的新病害

我们于1984和1985年6月上、中旬,在广州市郊、县,湛江市郊以及广西南宁市郊、县,北海市郊和合蒲县等花生产区,调查花生病毒病时,除了发现花生轻斑驳病毒病外,还发现一种新的病毒病害。其症状特征是:病株顶端叶片上出现很多褪绿黄斑或环斑,有的环斑变     

侵染番茄的黄瓜花叶病毒（CMV）株系特性的比较研究

从山东省主要番茄种植区病毒样本中分离到111个黄瓜花叶病毒(CMV)分离物,根据病毒的寄主范围和症状,初步鉴定为三个株系:番茄蕨叶株系(CMV-ToF)、番茄花叶株系(CMV-ToM)和番茄轻花叶株系(CMV-ToL)。三株系除在生物学特性存在明显差异外,其病毒粒体形态大小,电泳迁移率,病毒外壳蛋白亚基分子量、核酸组分以及病毒粒体血清学特性亦存在差别。     

Влнянне характера эаселення ноля капустноii совкоii(Barathra brassicae L.) эффективноеть трихограммы (Trichogramma evanescens Westw.) (Lepidoptera,Noctuidae; Hymenoptera,Trichogrammatidae)

TSWV belongs to the genus Tospovirus which was established in the family Bunyaviridae, a family of animal viruses. Besides TSWV, Impatiens necrotic spot virus (INSV) and ground nut bud necrosis virus (GBNV) were established as different Tospovirus species. Tospoviruses have quasispherical particles of 85 nm diametre which are surrounded by a membrane and contain 3 RNA species and 4 structural proteins. In Tospovirus infected plant cells virions were detected in cavaties of the endoplasmatic reticulum and additionally amorphous electron dense material accumulates in infected cells. Defective forms of TSWV lack the ability to form complete virus particles. TSWV is the only plant pathogenic virus that is transmitted by thrips which transmit the virus with different efficiency. The virus has an extensive plant host range of more than 360 different species. The developing symptoms depend on the Tospovirus species, the virulence of the virus strains and the environmental conditions.

Based on the reaction of TSWV isolates with N‐specific polyclonal antisera, 3 serogroups were established. The most frequently used technique for serologically based diagnosis of Tospoviruses is DAS ELISA with N‐specific or preadsorbed antisera against complete virus. For TSWV epidemiology distinct weeds and cultural host plants play an important role for the survival of virus and vector. Breeding for resistance is the most important preventive measure of control.     

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.     

Monoclonal Antibodies against the Recombinant Nucleocapsid Protein of Tomato spotted wilt virus and its Application in Virus Detection

Tomato spotted wilt virus (TSWV) is the type member of the tospovirus genus and causes significant losses in a wide range of economically important ornamental and vegetable crops worldwide. The nucleocapsid gene, located on the ambisense S RNA segment of TSWV was expressed in Escherichia coli using pET-32a as vector and correct expression of recombinant protein was confirmed by Western blot using an anti-TSWV monoclonal antibody (MAb). The recombinant protein was purified using Ni-NTA agarose and the purified protein was used for the production of MAbs. Three murine MAbs against the recombinant nucleocapsid protein were produced. Triple antibody sandwich enzyme-linked immunosorbent assay and immunocapture RT-PCR methods were then established for reliable and efficient detection of TSWV using the produced MAbs.     

Incidences and progression of tomato chlorosis virus disease and tomato yellow leaf curl virus disease in tomato under different greenhouse covers in southeast Spain

Epidemics of whitefly‐transmitted Tomato chlorosis virus, Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus have been present in the south east of Spain since the 1990s. A survey was performed in 40 greenhouses and nethouses during 2003 to establish the relationship between the disease incidence and the quality of greenhouse or nethouse coverings, providing a physical protection of crops against whiteflies. For tomato chlorosis virus disease (ToCD), the incidence correlated with the type of greenhouse cover and was most reduced under higher quality covers. Control of tomato yellow leaf curl disease (TYLCD) was achieved only for crops grown in the highest quality greenhouses. TYLCD incidence in tolerant tomatoes remained below 100% within the 5 months of sampling, despite the disease progress rate at the initial stage of the cultivation being higher than that of ToCD, which did reach 100% incidence in many greenhouses. Linear regression analysis showed that the development of ToCD and TYLCD in most of the greenhouses was best described by the monomolecular model and the Gompertz model, respectively. Tomato infectious chlorosis virus was not detected in parallel surveys carried out during this study, although it has been described previously in the area studied.     

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.     

Variety,mulch and stage of inoculation effects on incidence of tomato spotted wilt virus disease in cucumber (Cucumis sativus L.)

Abstract

Tomato spotted wilt virus (TSWV) vectored by thrips is one of the major diseases affecting cucumber yield. Control of thrips is an underlying factor in its management. A study was conducted to determine the effect of time of inoculation, variety and mulch on disease incidence. Four varieties were inoculated with TSWV at cotyledon, 3 – 4 leaf and flower bud stages in a RCBD experiment replicated four times in a greenhouse. In the field, a 2×8 factorial design where two cucumber varieties were raised on seven types of mulches (red, yellow, silver, clear, black, white, and straw) with unmulched plots as controls was used. Variety Marketer was more tolerant to the disease compared to other varieties. Most varieties were generally tolerant to TSWV at cotyledonous but susceptible at 3 – 4 leaf and flower bud stages. Silver and clear mulches significantly suppressed thrip populations, yield and quality under field conditions.