Development of a stable attenuated double-mutant of tobacco mosaic virus for cross-protection

Tobacco(Nicotiana tabacum)and tomato(Solanum lycopersicum)are two major economic crops in China.Tobacco mosaic virus(TMV;genus Tobamovirus)is the most prevalent virus infecting both crops.Currently,some widely cultivated tobacco and tomato cultivars are susceptible to TMV and there is no effective strategy to control this virus.Cross-protection can be a safe and environmentally friendly strategy to prevent viral diseases.However,stable attenuated TMV mutants are scarce.In this study,we found that the substitutions in the replicase p126,arginine at position 196(R^(196))with aspartic acid(D),glutamic acid at position 614(E^(614))with glycine(G),serine at position 643(S^(643))with phenylalanine(F),or D at position 730(D^(730))with S,significantly reduced the virulence and replication of TMV.However,only the mutation of S^(643) to F reduced the RNA silencing suppression activity of TMV p126.A double-mutant TMV-E614G-S643F induced no visible symptom and was genetically stable through six successive passages in tobacco plants.Furthermore,our results showed that TMV-E614G-S643F double-mutant could provide effective protection against the wild-type TMV infection in tobacco and tomato plants.This study reports a promising mild mutant for cross-protection to control TMV in tobacco and tomato plants.

Control Effects of Combination of Plant Induced Resistant Agents against Tobacco Mosaic Virus (TMV)

[ Objective] The paper was explore the control effects of combination of plant induced resistant agents against tobacco mosaic vires （TMV）. [ Method ] The control effects of 6 different combinations of plant induced resistant agents against TMV of flue-cured tobacco cultivar HangDa were studied under the environ- ment of simulated disease nursery. [ Result] The combination of 2 induced agents polypeptide-agent and 3-acetonyl-3-hydroxyoxindole （AHO） had good control effect against TMV, which could obviously delay the incidence time of TMV in infected tobacco plants. With water and Duxiao as control, their average control effects against TMV of tobacco plants during field period reached 69.64% and 43.25% after transplanting for 70 d. They also showed significant superiority accord- ing to Duncan＇s test （p = 0.05 ） in the aspects of plant height and leaf number, and the growth and development condition of leaves was good. Tobacco seedlings carrying TMV virus had no direct correlation with whether the symptoms performed, the seedlings carrying virus would perform symptom only when the incidence condition was suitable. The peak period for the incidence of TMV in seedlings carrying virus was during 19 d after transplanting. Spraying effective agents during nursery stage and field period, as well as promoting quick growth at the initial stage of tobacco seedlings after transplanting were the key measures to control its inci- dence. [ Conclusion] The study provided theoretical basis for preparing the control measures against TMV.

Effect of chitosan on tobacco mosaic virus(TMV) accumulation, hydrolase activity, and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus(TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV(2 μg/mL) and chitosan(1 mg/mL) were lower in the early period of infection(3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases(proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid(PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles(18 nm in diameter, 300 nm long), these suspensions contained abnormal(swollen, "thin" and "short") virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that "thin" virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.

Occurrence Regularity and Control of Tobacco Mosaic Virus (TMV) in Wuxi Tobacco Area of China in 2011

On the basis of general situation and new characteristics of tobacco mosaic virus （TMV） in Wuxi tobacco area in 2011, the paper expounded the objec-tive reasons of TMV via systemic investigation, field experiment and date sorting. Meanwhile, combined with mcteorological conditions and results of systemic inves-tigation, the study especially analyzed meteorological conditions, outbreak and prevalence regularity of TMV and control efficacy of chemical reagents against TMV. The results showed that climatic conditions were the main conditions affecting TMV. There were three occurrence peaks of TMV in 2011, as a result of meteorologi-cal conditions of the months from April to June. The peaks were concerned with a wide range of rainfall about half a month before, low temperature, high humidity and scant sunshine and temperature jump after rain. The results of control effects showed that the chemical reagents could obviously prevent TMV, but once tobacco plants were infected, spraying chemical reagents would not have effective control effect against TMV.

Analysis of Spatial Distribution Pattern of Tobacco Mosaic Virus (TMV) Based on Geostatistics

Using geostatistical method, the semi-variation function models of tobacco mosaic virus （TMV） in east-west and north-south directions were established, and the distribution pattern of TMV in large scale space was studied. The results showed that the distribution pattern of the disease in east-west and north-south directions belonged to linear model with abutment, and the spatial distribution pattern within the investigated areas was aggregated model. The spatial correlation distances in east-west and north-south directions were 29.953 4 and 47.813 8 km, and the spatial variabilities were 95.71% and 80.05%, respectively. This indicated that they had strong spatial correlation. Isoline map accessed by Kringing interpolation method could clearly reflect the spatial aggregated model.

Agroinoculation as a Simple Way to Deliver a Tobacco Mosaic Virus- Based Expression Vector

烟草花叶病毒(TMV)表达载体30B是一个目前广泛应用的植物病毒表达载体,但用其生产外源蛋白时,必须先将它体外转录成RNA,才能被用来接种宿主植物。由于RNA体外转录费用昂贵、操作复杂,因此限制了30B表达载体的进一步应用。针对这一不足,我们用农杆菌接种法(agroinoculation)接种该病毒载体,即将30B cDNA置于花椰菜花叶病毒(CaMV)的35S启动子和终止子之间,再将整个表达框架插入到农杆菌T-DNA的左边界和右边界之内,构建成质粒p35S-30B,将转入该质粒的农杆菌注射到植物的叶片中,30B cDNA随T-DNA进入植物细胞后,被转录成可自我复制的RNA形式,进而发生系统侵染。为了检测此接种方式的可行性,绿色荧光蛋白(GFP)报告基因被克隆到p35S-30B中,构建成p35S-30B∶∶GFP,用含有该质粒的农杆菌进行注射操作。证实该病毒载体可通过简便的农杆菌接种法侵染Nicotiana benthamiana,在被接种植物的系统叶中,GFP的表达量可占植物总可溶蛋白的5.2％。

Construction of RNAi Vector Which Resist Cucumber Mosaic Virus and Transformation of Tobacco

[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.

紫苏精油中紫苏醛的抗TMV活性及作用方式

紫苏精油(PEO)是从紫苏叶中提取的一种具有挥发性芳香气味的淡黄色油性物质,具有多种生物活性,但关于其抗烟草花叶病毒(TMV)活性尚未见报道。本研究测定了紫苏精油及其主要成分的抗TMV活性,明确了其主要活性成分,并在此基础上进行了作用方式研究。结果表明:PEO在800μg/mL剂量下对TMV的活性高达65.58%,其主要活性成分紫苏醛在该剂量下的保护、治疗和钝化活性分别为80.41%、73.42%和34.93%,均显著高于对照药剂商品化药物壳寡糖,而其保护和治疗活性优于宁南霉素。作用方式研究结果表明:紫苏醛诱导了烟草的过敏反应(HR),透射电镜(TEM)观察显示紫苏醛对TMV粒子没有直接作用。紫苏醛在800μg/mL剂量下处理心叶烟,具有显著的诱导抗病活性,为58.46%。紫苏醛处理诱导了烟草病程相关基因非表达子1(NPR1)、病程相关蛋白1基因(PR1)和病程相关蛋白5基因(PR5)3个致病相关基因(PR基因)表达上调,也诱导了苯丙氨酸解氨酶基因(PAL)、呼吸爆发氧化酶B基因(RBOHB)和原叶绿素酸酯氧化还原酶基因1(POR1)过表达。此外,紫苏醛提高了烟草叶片中水杨酸(SA)和H2O2含量,增强了超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)4种防御酶的活性。紫苏醛在800μg/mL剂量下处理心叶烟24 h,结果表明:烟草叶片中的SA和H2O2含量最高,分别为1032.08 pmol/L和23.40μmol/g FW,防御酶活性也达到最大值,CAT、PAL、POD和SOD活性分别比对照提高了1.76、1.95、2.17和3.78倍。该研究结果表明,紫苏醛可能通过诱导系统性获得抗性(SAR)来增强植物对病原体感染的抵抗力,这可能是通过SA信号转导途径介导的。因此,紫苏醛作为一种新型抗病毒药物和免疫诱导剂在农业上具有广阔的应用前景。

基于CP基因的中国云南省烟草TMV群体遗传多样性分析

为探明云南烟草上烟草花叶病毒(tobacco mosaic virus,TMV)的群体遗传多样性特征,本研究使用TMV外壳蛋白(coat protein,CP)基因的特异性引物对采自云南各烟区的疑似烟草花叶病样品进行检测,通过克隆测序获得138个TMV CP基因序列,对其进行了一致性比对,及系统发育、错配分布、遗传变异、群体多样性等分析。变异分析显示,138条CP基因序列共有变异位点数122个,占总位点数的25.42%,变异类型只有核苷酸替换,核酸多样性为0.018,单倍型多样性为0.970;一致性比对结果显示,138条序列的核苷酸和氨基酸序列一致性均为96%~100%;重组分析未检测到重组信号;对来自云南烟草,NCBI上其他地区烟草及云南地区其他寄主的TMV分离物的CP基因进行系统发育分析,结果显示,所有TMV分离物在进化上可分为4个组,本试验获得的分离物分布在1、2、3组,其中只有来源昭通地区的分离物倾向于聚成簇,表明云南烟草TMV分离物的适应性进化受地理适应性所驱动,与寄主适应性基本无关;错配分布分析显示,云南烟草TMV群体呈多峰分布,表明该群体近期未经历群体扩张事件;群体间遗传分化分析结果表明,除昭通和曲靖烟草TMV群体与其他TMV群体之间很容易发生遗传漂变促使群体发生遗传分化外,其余大部分TMV群体之间的基因流都可以抵制遗传漂变。本研究首次报道了云南各烟区TMV分离物群体遗传变异情况,研究结果可为TMV的抗病育种和制定更为有效的防治策略提供重要参考。

High-density SNP genetic linkage map construction and quantitative trait locus mapping for resistance to cucumber mosaic virus in tobacco(Nicotiana tabacum L.)

Genetic linkage maps are essential for studies of genetics, genomic structure, and genomic evolution, and for mapping quantitative trait loci (QTL). Identification of molecular markers and construction of genetic linkage maps in tobacco (Nicotiana tabacum L.), a classical model plant and important economic crop, have remained limited. In the present study we identified a large number of single nucleotide polymorphism (SNP) markers and constructed a high-density SNP genetic map for tobacco using restriction site-associated DNA sequencing. In 1216.30 Gb of clean sequence obtained using the Illumina HiSeq 2000 sequencing platform, 99,647,735 SNPs were identified that differed between 203 sequenced plant genomes and the tobacco reference genome. Finally, 13,273 SNP markers were mapped on 24 high-density tobacco genetic linkage groups. The entire linkage map spanned 3421.80 cM, with a mean distance of 0.26 cM between adjacent markers. Compared with genetic linkage maps published previously, this version represents a considerable improvement in the number and density of markers. Seven QTL for resistance to cucumber mosaic virus (CMV) in tobacco were mapped to groups 5 and 8. This high-density genetic map is a promising tool for elucidation of the genetic bases of QTL and for molecular breeding in tobacco.

Comparative transcriptome analysis reveals differential gene expression in resistant and susceptible tobacco cultivars in response to infection by cucumber mosaic virus

Cucumber mosaic virus(CMV) is one of the most severe viral diseases transmitted by aphids infecting Solanum crops in China, causing great losses of crop yields and income in rural communities.The tobacco cultivars NC82 and Taiyan 8 are closely related but differ in resistance to CMV.NC82 is susceptible to infection and Taiyan 8 is resistant, but the mechanisms underlying this difference in resistance are not clear.In this study, we conducted RNA sequencing to analyze changes in gene expression induced in the leaves of Taiyan 8 and NC82 upon systemic infection with CMV, compared with gene expression in the leaves of mock-inoculated plants.Leaves were sampled at one, three, eight, and 15 days after infection.In total, 3443 and 747 differentially expressed genes were identified in Taiyan 8 and NC82, respectively.Gene ontology and pathway enrichment analyses revealed that the different responses to CMV infection between cultivars were based on microtubulebased processes, pentose and glucuronate interconversions, plant–pathogen interaction,and hormone signal transduction pathways.Genes encoding pathogenesis-related proteins, disease-resistance proteins, lipoxygenase, cellulose synthase, an auxin response factor, and an ethylene receptor showed different expression patterns.The differences in gene expression following CMV infection likely contributed to the different resistance levels of these two tobacco cultivars.The comprehensive transcriptome dataset described here,which includes candidate response genes, will serve as a resource for further studies of the molecular mechanisms associated with tobacco defense responses against CMV.

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus(TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000(PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 102 viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.

Effects of Early Infection of Tobacco Mosaic Virus on Photosynthetic Proteins and Its Control

[Objectives] This study was conducted to explore the optimal period of TMV control and the physiological sites that interfere with TMV infection. [Methods] Proteome analysis was performed on the host tissues(tobacco plants) at different time periods of viral infection, to verify the changes in the expression of differential protein genes and N and PR1-a in the photosynthetic pathway and porphyrin metabolism and chlorophyll metabolism pathways in proteome; and the tobacco plants were treated with different preparations. [Results] The expression levels of N and PR1-a in the tobacco leaves treated with preparation B reached the highest level, and the effects on the expression levels of the differential protein genes were also the most significant. The control effects of the preparations were tested by the half-leaf method, and the results showed that preparation B had a significant control effect against the early infection of the virus.[Conclusions] This study lays a foundation for the prevention and control of tobacco mosaic virus on crops.

A TOM1 homologue is required for multiplication of Tobacco mosaic virus in Nicotiana benthamiana

The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus(TMV) in A.thaliana.In this study,we cloned an AtTOM1-like gene from Nicotiana benthamiana named as NbTOM1.Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues of N.tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities,respectively.Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N.benthamiana.The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.

Control Effects of Different Agents on Tobacco Mosaic Virus Disease

[Objectives] This study was conducted to screen out suitable agents for controlling tobacco mosaic virus disease and the best control period in Zhangzhou tobacco area, providing a theoretical basis for the control of virus diseases, thereby improving the quality of flue-cured tobacco and the income of tobacco farmers. [Methods] The effects on tobacco mosaic virus disease under the interaction between different agents and different application periods were investigated. The incidence of tobacco mosaic virus disease was investigated, and its control effect was analyzed. [Results] Different agents and different application periods had different control effects on tobacco mosaic virus disease. The incidence of tobacco mosaic virus disease: At 30 and 45 d after transplanting, the incidences of A2B1 treatment were the lowest, at 0.85%, 1.71%, respectively;and at 60 d after transplanting, the incidence of A3B1 treatment was the lowest, only 10.68%. The control effect: At 30 and 45 d after transplanting, A2B1 treatment had better control effects, reaching 79.39% and 73.06%, respectively. [Conclusions] 3% hypersensitive protein sprayed at 1 d before transplanting and 7 and 15 d after transplanting achieved the best effect, followed by 10% ningnanmycin sprayed at 1 d before transplanting and 7 and 15 d after transplanting. In tobacco production, it is recommended to apply 1 000 times dilution of 3% supersensitive protein microgranules for three times(at 1 d before transplanting and 7 and 15 d after transplanting), which can effectively prevent tobacco mosaic virus disease.

烟草花叶病毒(TMV)免疫诱抗剂的制备与验证

为了制备烟草花叶病毒(TMV)免疫诱抗剂,研究采用壳寡糖、高锰酸钾、内生真菌发酵液、紫外线、银杏叶提取物等试剂处理,筛选出TMV弱毒/无毒株系制备的最优方法,并在室内开展免疫激活效应。结果表明:紫外线、0.1%高锰酸钾溶液、内生真菌发酵液16倍浓缩液、100μg/mL壳寡糖溶液对TMV表现出良好的钝化效果,钝化液对TMV有较高的抑制率。钝化TMV能引起烟草多酚氧化酶(PPO)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)5种防御酶活性显著升高,从而提高烟株对TMV感染的抗性。烟苗和大田烟叶TMV防控试验也证明钝化TMV在烟叶生产中可以作为TMV免疫诱抗剂使用,提高烟株的TMV免疫力和抵抗力。

与辣椒抗TMV L^(3)基因连锁的分子标记的筛选及种质资源抗TMV鉴定

以含抗辣椒TMV的L^(3)基因的材料L15为基础,利用与L^(3)基因连锁的3个分子标记189D23MF、PMFR11和PMFR21对种质资源进行鉴定.对L15、3份感病材料H8/H9/H11×L15 F_(1)、46份辣椒种质资源进行室内人工TMV接种并对其进行表型抗性鉴定和RT-PCR检测.对筛选出的标记在F_(1)及BC_(1)P_(1)群体中进行验证.结果表明:分子标记189D23MF在L15和12份辣椒种质上均未扩增出条带;SCAR标记PMFR11在L15和46份辣椒种质上呈现共显性分离,SCAR标记PMFR21为显性标记.通过室内人工接种鉴定方式,2份材料L15、H11×L15表现为高抗.H9×L15表现为抗,H8×L15及5份材料A1、A35、A39、A55、L16表现为中抗,11份材料表现为感病,30份材料表现为高感.在F1及BC1P1群体中PCR扩增结果表明SCAR标记PMFR21可用于辣椒抗TMV分子标记辅助选择.

链霉菌F01菌株对TMV的抗性及其发酵条件优化

【目的】从源自秦岭的土壤微生物中筛选具有抗病毒活性的生防菌并进行分类鉴定,对其抗烟草花叶病毒(TMV)的作用方式进行研究并对发酵条件进行优化,为防治烟草花叶病毒病提供新资源。【方法】采用土壤分离法分离生防菌,采用形态学和分子生物学方法对其进行分类鉴定。采用无菌过滤法制备F01菌株发酵液,以心叶烟为材料,采用半叶枯斑法测定生防菌F01菌株抗TMV的作用方式,并采用单因素法优化F01菌株的发酵体系。【结果】从秦岭土壤中分离到1株生防放线菌株F01,经形态学和分子生物学鉴定为链霉菌。F01菌株发酵液对烟草花叶病毒具有良好的防治效果,其抗病作用方式包括体外病毒钝化和体内诱导抗病性,对TMV的抑制率为74.2%。优化后的发酵体系为:以高氏1号为培养基,以小米粉和酪蛋白胨为碳、氮源,在28℃、pH 8.0条件下培养5 d,发酵液对TMV有显著的抑制效果,抑制率为81.7%。【结论】分离得到的链霉菌F01菌株具有良好的抗TMV活性,优化后的发酵条件简单,发酵产物对TMV有显著抑制效果,具有进一步开发为抗TMV工程菌株的潜力。

The technics of breeding a new transgenic tobacco cultivar resistant to mosaic diseases and its extention

This paper reports a new transgenic tobacco line NC89 resistant to both tobaccomosaic virus(TMV)and cucumber mosaic virus(CMV).It deals with the details of field experi-ments of the whole process of segregation and selection of the progenies of transgenic plant popu-lation.By using the gene transfer technics,some transgenic plants resistant to mosaic disease froma flue-cured tobacco cultivar NC89 were obtained in green house in 1989.Thereafter,throughfield inoculation and selection from the 2nd and 3rd generations,a homozygous line resistant toboth viruses and with good qualities was bred out.The inheretant stability of the resistance of thenew line was confirmed through the 4th to 6th generations.In 1992,the transgenic resistant linewas planted about 7000 hectares in Henan province.The relative effect of controlling mosaic dis-eases of tobacco was 50—70%.The out-put value increased 30—50% per unit area.This is a newexample of growing a transgenic virus-resistant Plant in large scale in China.

青蒿中番茄斑萎病毒和烟草花叶病毒的分子鉴定及相关序列分析

番茄斑萎病毒(tomato spotted wilt virus, TSWV)和烟草花叶病毒(tobacco mosaic virus, TMV)是2种重要的植物病原病毒,对多种经济作物的产量和品质均造成严重影响。2021年-2022年,在云南省丽江市烟草种植区不同烟区采集叶片黄化、皱缩以及无症状的青蒿Artemisia caruifolia样品共计14份,利用免疫金标速测卡和RT-PCR对其病原病毒进行检测。利用免疫金标速测卡检测结果显示,在所检样品中有9份样品检测出TSWV,检出率为64.28%,有3份样品检测出TMV,检出率为21.43%,2种病毒复合侵染的检出率同样为21.43%;利用RT-PCR对复合侵染的3份样品进行分子检测,结果显示,在3份复合侵染青蒿样品中获得3条TSWV N基因序列、3条TMV cp基因序列和2条TMV RdRp部分序列。TSWV青蒿分离物与分离自云南的TSWV-2分离物相似性最高,为99.6%;TMV青蒿分离物与分离自辽宁的TMV-Shenyang分离物和分离自云南的TMV-Yongren-1相似性最高,均大于99.4%。这是首次发现TSWV和TMV 2种不同属病毒复合侵染青蒿。