重庆烟草地区ELISA检测

为调查重庆主要烟草地区的病毒病种类,采用没脸免疫吸附法(ELISA)对采自重庆涪陵、彭水、万州、黔江、武隆、酉阳的258份的烟草样品进行了病毒病种类的检测.检测结果表明,其中274份样品呈阳性。主要受到7种病毒病的侵染,主要包括:烟草花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯X病毒(Potato virus X,PVX)、芜菁花叶病毒(Turnip mosaic virus,TuMV)、蚕豆萎蔫病毒2号Broad bean wilt virus 2,BBWV-2)、烟草蚀纹病毒(Tobacco etch virus,TEV)。其中TMV的检出率最高,达50%,已成为重庆地区烟草优势病原。     

延边地区烟草病毒种类及病毒病的防治

对田间烟草进行病毒病种类的调查和鉴定结果表明,延边地区感染烟草的病毒主要是烟草花叶病毒(TMV)和黄瓜花叶病毒(cMV),而以两种病毒的混合侵染为主,达80-1 00％。弱毒疫苗N14．和CMV卫星RNA生防制剂S5,混合使用,或CMV卫星RNA生防制剂S512单独使用防治烟草病毒病,与不使用的对照比较,防治效果明显,达70.2—77.1％,上等烟比率达22.3—30.9％,而对照只有9．2-1 9.2％,亩收入比对照增加21．7—8 0.8％。     

马铃薯Y病毒N株HC-pro基因的序列分析与表达

马铃薯Ｙ病毒(Potato virus Y,PVY)是马铃薯Ｙ病毒属(Potyvirus)的典型成员,是烟草、马铃薯和辣椒等茄科作物病毒病的主要病原之一,分布于世界各地.近年烟草上的 PVY在全国范围内特别是陕西、黄淮和东北烟区流行呈上升趋势,且以PVY坏死株系（PVY-N)为主,重病田发生率达40%以上,并常与烟草花叶病毒、黄瓜花叶病毒混合侵染,严重影响烟草品质造成巨大的经济损失.     

酶联免疫吸附法（ELISA）检测四川“榨菜”病毒种类

1990至1991年在“榨菜”病毒病发生的始期和盛期,从四川九个县(市)近郊、远郊采集样品492份,用TuMV、CMV、TMV、PVX、PVY和CaMV六种抗血清,经酶联免疫吸附间接法进行检测,属于TuMV和TuMV与其它五种病毒之一或之二复合感染的有422份,占总样品数的85.77％,其中由TuMV单一感染的305份,占61.99％。此外,还有PVX、PVY、CaMV单一感染病样27份,占5.49％,而情况不明的43份,占8.74％,总检出率91.26％。检测结果表明,发病始期或盛期,近郊或远郊均以TuMV和TuMV与CMV复合感染的病毒为主,是四川“榨菜”生产上主要的病毒种类。     

四种广普性植物病毒高效mPCR检测方法的建立

本研究建立了能同时检测出烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、马铃薯X病毒(PVX)和马铃薯Y病毒(PVY)的多重RT-PCR体系。TMV、CMV、PVX、PVY是四种广普性植物病毒,寄主范围广泛,并且常常发生复合侵染。本研究以上述四种病毒的CP基因部分序列设计引物,以反转录的cDNA为模板,建立多重RT-PCR反应体系,分别扩增出211～417bp的不同长度的基因片断,并通过序列测定来确认扩增序列的特异性。将反转录合成的cDNA进行浓度稀释,来对多重RT-PCR与单重RT-PCR的灵敏度进行比较,结果证明,多重RT-PCR体系能够同时快速检测这四种病毒,并且有很高的灵敏度。     

从烟草上分离的黄瓜花叶病毒

黄瓜花叶病毒(CMV)危害烟草已成为烟区严重病害之一,如山东烟区CMV 引起的烟草病毒病发病率达50%以上。GMV 在福建烟区的发病率也达15～30%。上述调查系根据烟草田间症状表现以及温室内不同鉴别寄主的反应和抗性测定得到。为了进一步研究与肯定CMV 引起的烟草病毒病,有必要由病株分离病毒和进行电子显微镜观察鉴定。     

表达黄瓜花叶病毒卫星RNA的转基因烟草耐烟草花叶病毒

表达黄瓜花叶病毒(CMV)的卫星RNA的转基因烟草可以抗CMV的侵染.为了决定这些植物对其它病毒,如烟草花叶病毒(CMV)的安全性,我们对这些植物接种CMV.结果发现 卫星RNA可减弱TMV引起的症状,井降低病情指数,然而却对TMV在植物中的积累没有明显影响.这一发现有助于对卫星RNA抗病机制的理解,有助于含卫星RNA的生防制剂及表达病毒卫星RNA的转基因植物的应用.本文在国际上首次报道卫星RNA可减弱非相关病毒引起的症状.1 材料和方法1.1 病毒及植物烟草花叶病毒(TMV),烟草G140种子及枯班三生烟种子均为中国科学院微生物研究所病毒室保存;含黄瓜花叶病毒卫星RNA-R1基因的烟草G140(烟Sat-G140)为中国科学院微生物研究所病毒室培育,所用材料为第三代种子.     

表达黄瓜花叶病毒卫星RNA的转基因烟草耐烟草花叶病毒

表达黄瓜花叶病毒(CMV)的卫星RNA的转基因烟草可以抗CMV的侵染.为了决定这些植物对其它病毒,如烟草花叶病毒(CMV)的安全性,我们对这些植物接种CMV.结果发现 卫星RNA可减弱TMV引起的症状,井降低病情指数,然而却对TMV在植物中的积累没有明显影响.这一发现有助于对卫星RNA抗病机制的理解,有助于含卫星RNA的生防制剂及表达病毒卫星RNA的转基因植物的应用.本文在国际上首次报道卫星RNA可减弱非相关病毒引起的症状.1 材料和方法1.1 病毒及植物烟草花叶病毒(TMV),烟草G140种子及枯班三生烟种子均为中国科学院微生物研究所病毒室保存;含黄瓜花叶病毒卫星RNA-R1基因的烟草G140(烟Sat-G140)为中国科学院微生物研究所病毒室培育,所用材料为第三代种子.     

应用RNAi技术培育抗2种病毒病的转基因烟草

分别提取烟草普通花叶病(TMV)和烟草黄瓜花叶病（CMV）的病毒RNA。经反转录和外壳蛋白阅读框PCR扩增,获得TMV和CMV外壳蛋白基因cDNA, 分别进行两种病毒已知株系cDNA序列比对获得各自的保守序列,设计干涉序列,将干涉片段扩增产物连接到pMD18-T的相邻酶切位点,制备融合序列,并将其正向和反向序列插入pUCCRNAi载体,再转化到pCAMBIA2300-35S-OCS表达载体中。利用农杆菌LBA4404侵染烟草K326,获得3份含有TMV和CMV外壳蛋白基因干涉序列的转化材料,经分子鉴定证实干涉序列已导入烟草,并采用荧光定量PCR技术对其mRNA表达差异进行分析。抗病性调查表明转化烟株对TMV和CMV抗性都显著增强。     

辽宁省番茄花叶病主要病毒和株系的研究

从辽宁南部及沈阳地区采集番茄花叶病样本111个(1985-1987),经鉴定得知主要毒原为番茄的烟草花叶病毒(TMV)占总调查的59.46％。黄爪花叶病毒(CMV)亦有发生仅占总调查的9.00％。还有一些系混合侵染(TMV和CMV)占21.62％。尚未认清毒原种的约占2.70％,接种无症的占7.20％。通过试验认为我省番茄上主要毒原为烟草花叶病毒,并进行了常规鉴定、提纯、电镜观察、光谱测定和血清测定等。对番茄上烟草花叶病毒16个分离物做了株系分化研究,按照Pelbam(1970,1972)〔14、15、16、17〕的抗性分类法应用番茄GCR一套品种接种观察,得知辽南及沈阳地区分布很广的番茄TMV分离物,均属TMV-O株系。故在番茄抗TMV育种中尚应考虑到抗1及2株系的能力。     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

黑龙江省及长春市烟草病毒病的种类鉴定

１９９１－１９９３年在黑龙江省主要烟区的１１个县及长春市采样,得１２９个毒株。经鉴别寄主测定,抗血清反应（板式酶联法或斑点酶联法）及电镜或免疫电镜观察,有ＴＭＶ（４３．４％）,ＰＶＹ（１７．８％）,ＣＭＶ（３．９％）,ＴＲＶ（０．８％）,ＴＳＷＶ等病毒,ＴＭＶ与ＰＶＹ混合侵染的占１０．１％,ＰＶＹ与其它病毒混合侵染的占１１．６％,另有５个标样为马铃薯Ｙ病毒组成员,１０个为未知。     

Disease intensity of some tomato viroses in Serbia

In this paper we present the data on the disease intensity of the tomato plants grown in glass and plastic-houses, and in the open field. The infection was caused by the following viruses: Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato black ring virus (TBRV), Tomato ringspot virus (ToRSV), Tomato aspermy virus (TAV), and Cucumber mosaic virus (CMV). These viruses represented most frequent tomato pathogens in Serbia. According to the obtained results, it could be concluded that 92.94% of the tested tomato plants grown in glass and plastic-houses, and 89.82% grown in the open field were infected by one of the above viruses. Most of the plant samples were infected by two or more viruses. The most frequent viruses — tomato pathogens in Serbia were ToMV, PVY and TMV.     

广东省烟草花叶病病原病毒的鉴定

烟草花叶病是广东省产烟区的主要病害之一。我们在南雄等八个县进行调查,1983年至1984年的一般发病率为2～20％。根据血清学反应、病毒粒体形态、鉴别寄主反应及寄主范围、媒介昆虫种类、物理性质、交互保护反应等各项检验结果,鉴定广东省烟草花叶病病原是:三个黄瓜花叶病毒可能株系(普通株系CMV-C,烟草坏死株系CMV-TN,烟草黄色坏死株系(CMV-TYN),烟草花叶病毒(TMV),马铃薯病毒Y(PVY),烟草脉带花叶病毒(TVBMV)和烟草褪绿斑驳病毒(TCMV)(暂定)。     

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Construction of Plant Expression Vectors and Identification of Transgenic Potato Plants

Potato virus X (PVX), potato virus Y (PVY) and potato leaf roll virus (PLRV) infection in potato may result in the loss of centrification of seed potatoes and affect the quality and yield of potatoes in agricultural production. The authors cloned coat protein (cp) genes of PVX, PVY and PLRV and constructed two kinds of plant expression vector which contain PVX and PVY or PVY and PLRV cp genes. Three major commercial cultivars of potato and one cultivar of tobacco were transformed via Agrobacterium tumefaciens mediated procedure. Transgenic plants were confirmed by PCR analysis. Transgenic tobacco plants containing both PVX and PVY cp genes were significantly resistant to PVX and PVY infection via mechanical inoculation.     

双价外壳蛋白基因植物表达载体的构建及马铃薯转基因植株的鉴定

在克隆了马铃薯Ｘ病毒（ＰＶＸ）、马铃薯Ｙ 病毒（ＰＶＹ）和马铃薯卷叶病毒（ＰＬＲＶ）的外壳蛋白基因的基础上,构建同时包含ＰＶＸ和ＰＶＹ 与ＰＶＹ 和ＰＬＲＶ 两个外壳蛋白基因植物表达框架的表达载体,通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ）介导转化烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ ）和生产上常用的几个马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ ）优良品种：“Ｆａｖｏｒｉｔａ”、“虎头”、“克４”。经ＰＣＲ检测证明外源基因已整合到植物的染色体上,得到批量转基因植株。在转ＰＶＸ＋ＰＶＹ 外壳蛋白基因的烟草上接种ＰＶＸ （５ μｇ／ｍ Ｌ）、ＰＶＹ（２０ μｇ／ｍ Ｌ）病毒,得到有一定抗性的植株     

Changes in Glucose-6-Phosphate Dehydrogenase, Ribonucleases, Esterases and Contents of Viruses in Potato Virus Y Infected Tobacco Superinfected with Tobacco Mosaic Virus

Effects of the superinfection with tobacco mosaic virus (TMV) on susceptible tobacco plants infected with potato virus Y (PVY) were determined. Dynamic changes in the TMV and/or PVY contents, the ribonucleases (RNases), the phosphomonoesterase (PME), the phosphodiesterase (PDE) and the glucose-6-phosphate dehydrogenase (G6P DH) activities were studied. The PVY infection caused a substantial reduction in the multiplication of TMV. The content of TMV in the PVY inoculated leaves amounts to 6 and 9 % in the PVY systemically infected leaves when compared with single TMV. Surprisingly, the challenging virus (TMV) enhanced the content of inducing virus (PVY) in the locally inoculated leaves up to 130 – 141 %. In contrast, the reduction of PVY content down to 35 – 40 % by TMV was seen in the PVY systemically infected leaves. The activities of the RNase, the PME, the PDE and the G6P DH were increased (when compared with the healthy plants) during the acute phase of single virus multiplication (PVY or TMV). The increase in the activities of the enzymes in the leaves with mixed infection was at least as high as the sum of the increases of single infections. Moreover, a higher increase than the sum was seen for G6P DH and PDE (by about 20 – 35 %). This revised version was published online in July 2006 with corrections to the Cover Date.