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 objective reasons of TMV via systemic investigation, field experiment and date sorting. Meanwhile, combined with meteorological conditions and results of systemic investigation, 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 meteorological 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.

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.

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.

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.

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。

15种植物的单宁提取物对烟草花叶病毒(TMV)的抑制作用

测定了 15种植物的乙醇粗提物对TMV的体外抑制作用 ,其中 11种具有明显效果 ,这 11种植物粗提物经水 /氯仿萃取 ,水萃取物具有明显抑制TMV效果 ,而氯仿萃取物抑制效果差。水萃取物用明胶去除单宁后 ,基本失去抑制TMV的能力 ,表明单宁在其中起着重要作用。从杠板归与假槟榔种子中提取的总单宁对TMV具有明显的抑制作用 ;病毒粒体样品在分别加入这 2种植物总单宁后 ,电镜下表现为聚集结合 ;TMV分别与这 2种植物的单宁混合后 ,通过透析 ,可恢复大部分侵染力 ,表明单宁对病毒并无直接毒害作用 ;TMV外壳蛋白 (CP)与单宁混合后 ,在蛋白质的聚丙烯酰胺凝胶电泳中表现为结合现象 ;单宁对TMV体内抑制复制效果不明显 ;单宁在接种TMV前后施用并没有降低TMV侵染力 ;这些结果表明这 2种植物单宁对TMV抑制作用可能是与TMV CP结合而引起病毒侵染力下降或影响病毒CP对侵染位点的识别。

解淀粉芽孢杆菌Ba33对烟草的促生及抗TMV作用

从烟田土壤中分离得到解淀粉芽孢杆菌（Bacillus amyloliquefaciens）Ba33菌株。5叶期三生NN烟（Nicotiana tabacum var．samsun NN）喷施Ba33发酵液3次后再接种TMV（烟草花叶病毒），与喷清水对照相比，能显著降低枯斑数量，枯斑抑制率为67．8％。于4～6叶期普通烟草（Nicotiana tobacum L．）NC89烟苗接种TMV前后分别用Ba33发酵液、NB培养基或清水连续灌根3次，观察到Ba33发酵液灌根处理的烟苗鲜重、最大叶长和叶宽均高于清水对照处理，叶色浓绿，长势良好；接种TMV10d后进行实时荧光定量PCR检测．Ba33发酵液灌根处理的植株新叶中RNA含量低于清水对照和NB培养基灌根处理。认为Ba33具有拮抗TMV、促进烟草生长的作用．是具有潜在应用价值的生防菌株．

榆黄蘑中一种抗病毒蛋白的纯化及其抗TMV和HBV的活性

采用阴离子交换层析和凝胶层析方法从新鲜食用菌榆黄蘑 (Plearotuscitrinopileatus)中进行了抗病毒蛋白的纯化 ,结果获得了一个纯化蛋白YP4 6 4 6 ,经SDS PAGE可确定其分子量为 2 7.4kD。以半叶法在枯斑寄主心叶烟上检测该蛋白对烟草花叶病毒 (TMV)的抑制率 ,发现有较好的抗TMV活性 ,其抑制TMV的中浓度为 0 .2 4 μg/mL。同时以HepG2 .2 .2 .15细胞株为模型 ,对所获得的蛋白进行体外抗乙型肝炎病毒效果的评价。结果表明该蛋白对HBsAg的 5 0 %抑制时的浓度为 0 .0 8μg/mL 。

不同类型烟草种质资源对TMV的抗性鉴定

为揭示从美国新引进的不同类型烟草种质资源对烟草普通花叶病毒(TMV)的抗性,采用温室盆栽试验,通过苗期高压喷枪接种,对新引进的304份烟草种质资源进行了TMV的抗性鉴定。结果表明,供试材料中有94份免疫资源、15份抗病资源、56份中抗资源、122份中感资源和17份感病资源。免疫资源包括14份白肋烟、28份雪茄烟、2份晾烟、14份烤烟、1份马里兰烟、4份香料烟、31份黄花烟或黄花烟杂交材料。白肋烟、雪茄烟、晾烟、烤烟、马里兰烟、香料烟、黄花烟和杂交材料七大资源类型中,免疫资源在同类供试资源中所占比例存在明显差异,按比例排序依次为黄花烟和杂交材料>烤烟>白肋烟>雪茄烟>晾烟>香料烟>马里兰烟。

椴叶鼠尾草抗烟草花叶病毒活性成分研究

为寻找新型植物病毒抑制剂,以烟草花叶病毒(TMV)为供试病毒,研究发现入侵植物椴叶鼠尾草提取物具有抗TMV活性。通过半叶枯斑法不同施药方式筛选发现,在质量浓度为1 mg/mL时,石油醚萃取物的保护作用和钝化作用最强,抑制率为40.95%和52.85%;乙酸乙酯萃取物的治疗作用最强,抑制率为52.85%,与阳性对照药剂宁南霉素无显著差异。石油醚萃取物各馏分中,Fr7的保护作用和治疗作用最好,抑制率为40.51%和45.52%,Fr6的钝化作用最好,抑制率为74.63%。乙酸乙酯萃取物各馏分中,Fr1′的保护作用最强,抑制率为61.77%,Fr3′的治疗作用最强,抑制率为55.02%,Fr4′的钝化作用最强,抑制率为60.22%。运用现代色谱波谱技术分离鉴定了5个化合物分别为tilifodiolide、eupatoric acid、熊果酸、齐墩果酸和2-甲基-5,7-二羟基色原酮。

一株对TMV具有显著拮抗活性的恶臭假单胞菌的筛选与鉴定

由烟草普通花叶病毒(Tobacco mosaic virus,TMV)引起的烟草病毒病是我国烟草上主要的病害之一,是影响我国烟草产量和质量的主要制约因素.本研究从TMV重病区中未发病的K326烟株根际土壤中筛选出对TMV具有高度拮抗活性菌株A3,用半叶法接种,对TMV的抑制效果可达95%以上.利用革兰染色及其一系列生理生化试验,结合该菌株的16S rDNA序列测定,对该菌株进行了鉴定.试验结果表明,菌株A3的革兰染色为阴性,专性需氧型,能以柠檬酸盐作为碳素的营养来源,可以将硝酸盐还原成亚硝酸盐,具有过氧化氢酶活力,但不能分解明胶、淀粉等大分子物质;在平板培养基上形成的菌落约3 mm,边缘整齐,湿润光滑,扁平突起,黏稠,呈淡红色;其16S rD-NA序列与GenBank数据库中假单胞菌属的细菌菌株的16S rDNA序列有98%～99%的高度相似性.结合其生理生化特性鉴定以及16S rDNA序列分析鉴定的结果,判定菌株A3为恶臭假单胞菌Pseudomonas putida,并对其生产可用性进行了讨论.

非(弱)杀菌化学物质诱导烟草对病毒病(TMV)抗性的研究

研究了 12种非 (弱 )杀菌化学物质诱导烟草对烟草花叶病毒病 (TMV)抗性的效果 ,供试化学物质均能诱导烟草对 TMV的抗性。新复级差测验结果表明 ,不同非 (弱 )杀菌化学物质对烟草抗病毒病性具有不同的诱导作用 ,其中 ,水杨酸表现突出 ,平均防治效果为 5 0 .30 % ,灌根 +喷雾和灌根两种处理方法与喷雾处理差异极显著 ,但灌根 +喷雾处理和灌根处理之间差异未达到显著差异 ,低浓度处理诱导效果高于高浓度处理。