【作者】 刘勇；

【导师】 周雪平；

【作者基本信息】 浙江大学 ， 植物病理学， 2006， 博士

【摘要】 烟草病毒病是危害烟叶生产最重要的病害之一，危害烟草的病毒种类多达30多种，花叶型病毒病田间常见，曲叶型病毒病的危害呈上升趋势。为了弄清云南烟草病毒病的种类，对云南省烟草花叶型和曲叶型病毒病样品进行了血清学和分子检测，并对烟草病毒病控制进行了研究。采用抗原直接包被法和双抗体夹心法对采自云南烟草花叶病样本进行了病毒种类检测，并利用三抗体夹心法对黄瓜花叶病毒（Cucumber mosaic virus，CMV）的亚组类型进行了鉴定。在云南采集的520个病毒病样本中，烟草花叶病毒（Tobacco mosaic virus，TMV）、CMV和马铃薯Y病毒（Potato virus Y，PVY）总检出率分别为71.74％、55.01％和6.35％，云南、湖南和福建64个CMV阳性样品中，属亚组Ⅰ的样品为57个，占89.1％，属亚组Ⅱ样品为10个，占15.6％。采用烟草曲茎病毒（Tobacco curly shoot virus，TbCSV）、云南烟草曲叶病毒（Tobacco leaf curl Yunnan virus，TbLCYNV）、中国番茄黄化曲叶病毒（Tomato yellow leaf curl China virus，TYLCCNV）的特异引物，对云南烟草曲叶病样本的病原进行了PCR检测；采用粉虱传双生病毒PA和PB通用引物，对代表性样品进行了扩增、克隆测序和序列比对。检测表明云南烟草曲叶病是由TbCSV、TbLCYNV和TYLCCNV三种病毒引起的。109个烟草曲叶症状的样本中，TbCSV总检出率为41.3％、TbLCYNV总检出率为16.5％、TYLCCNV总检出率为53.2％。其中两种或三种病毒复合侵染的检出率为8.2％。500 bp序列比较表明，这些病毒分离物之间的相似性为77％～100％。依据500 bp序列构建的同源关系树上，所有TYLCCNV的分离物因地理隔离聚为两大类，一类为保山分离物，相互间核苷酸序列相似性为93％，另一类为文山和红河分离物，相互间核苷酸序列相似性为95％，两类之间核苷酸序列相似性为89％。对其中一个分离物Y264 DNA-A的全序列测定表明，Y264 DNA-A全长2741个核苷酸（nts）。具有典型的begomoviruses结构，共编码6个开放读码框（ORFs）。DNA-A全长与中国番茄黄化曲叶病毒分离物Y10的相似性最高，为91.3％，各ORFs编码的氨基酸序列与TYLCCNV的相似性最高。因此，Y264为TYLCCNV的一个分离物。采用三抗体夹心法对云南烟草漂浮苗样本进行了病毒种类检测，在云南采集的1400多个烟草漂浮苗样本中，TMV的检出率占总阳性样本数的95％以上；CMV和马铃薯Y属病毒（包括PVY和TEV）的检出率占总阳性样本数的5％以下。在漂浮苗移栽后至团棵期，在发病率高（20％以上）的田块中采集的480个花叶病样本中，不同田块TMV检出率48.1％～100％，而CMV和马铃薯Y属病毒（包括PVY和TEV）检出率0％～12.2％。表明危害云南烟草漂浮苗的主要病毒种类为TMV。采用半叶法测定了9种药剂对烟草病叶汁液中TMV的钝化效果，采用叶面喷雾法测定了药剂对烟草的药害浓度与药害症状。室温下一定浓度的药剂与1：10000的TMV病叶汁液等体积混合3 min时，30％有效氯漂白粉10倍液、98％磷酸三钠·12H₂O 10倍液、7％有效氯次氯酸钠150倍液、美国消毒剂125倍液和75％酒精TMV的抑制率可达100％；10％二氧化氯200倍液和40％育宝150倍液与TMV混合30 min时对TMV的抑制率可达100％和96.30％；0.6％菌毒净125倍液、3.95％病毒必克800液和24％毒消800倍液与TMV混合30 min时对TMV的抑制率低于80％。除0.6％菌毒净较安全外，供试消毒剂在常用浓度下接触烟草叶片，产生较严重药害，症状因消毒剂种类不同，主要为褪绿斑点、坏死斑点和畸形。试验了在漂浮育苗池中使用消毒剂对TMV的钝化效果和烟苗出苗和生长的影响。枯斑寄主测定表明：与TMV病叶汁液2万倍稀释液接触12 hr，对TMV的抑制率高于80％的消毒剂及浓度为：30％有效氯漂白粉15 mg／L和150 mg／L；7％有效氯次氯酸钠2μl／L和20μl／L；美国消毒剂5μl／L和50μl／L。漂浮池水中加入低浓度消毒剂，出苗率和成苗率与对照差异不显著，但对烟苗后期长势的影响因消毒剂种类而有差异。对烟苗长势有促进作用的消毒剂有：磷酸三钠15mg／L～300mg／L、硫酸铜50mg／L～100mg／L、高锰酸钾10mg／L～40mg／L和24％毒消10μl／L。对烟苗长势低浓度无明显影响而高浓度有抑制作用的消毒剂有：40％育宝、30％有效氯漂白粉、7％次氯酸钠、美国消毒剂和0.6％菌毒净。为避免消毒剂对烟苗生长的影响，池水中消毒剂使用终浓度不能超过下列浓度：40％育宝100μl／L、30％有效氯漂白粉300mg／L、7％次氯酸钠20μl／L、美国消毒剂50μl／L、0.6％菌毒净100μl／L。更多还原

【Abstract】 Tobacco virus disease is one of most destructive diseases to tobacco leaf production, and more than 30 virus species could infect tobacco. Mosaic disease in field is very common, while the incidence of leaf curl disease is increasing. In order to elucidate virus types in tobacco plants in Yunnan, China, tobacco mosaic and leaf curl disease samples are collected in Yunnan and virus types were tested by serological and molecular methods, virus disease control measures are evaluated.Tobacco leaf samples showing mosaic symptoms collected from Yunnan province were tested for virus types by using antigen direct coated, double antibody sandwich or triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA), and TAS-ELISA was then used for subgrouping of Cucumber mosaic virus (CMV) samples. Among 520 tobacco samples collected in Yunnan province, 71.74%, 55.01% and 6.35% samples were infected by Tobacco mosaic virus (TMV), CMV and Potato virus Y (PVY), respectively. Among 64 CMV samples from Yunnan, Hunan and Fujian province, 89.1% samples were infected by subgroup I isolates, and 15.6% were infected by subgroup II isolates.The leaf curl samples were tested for virus types by PCR using three primer specific to Tobacco curly shoot virus (TbCSV), Tobacco leaf curl Yunnan virus (TbLCYNV) and Tomato yellow leaf curl China virus (TYLCCNV), respectively. The representative samples were amplified using begomoviruses universal primers PA and PB, and PCR products were cloned and sequenced. PCR detections demonstrate that tobacco leaf curl disease in Yunnan is associated with three begomoviruses including TbCSV, TbLCYNV and TYLCCNV. Among 109 leaf curl tobacco samples collected, 41.3%, 16.5% and 53.2% samples were infected by TbCSV, TbLCYNV and TYLCCNV, respectively, 8.2% samples were mix-infected by two or three virus. Alignment of the 500 bp sequences showed that nucleotide sequence identities among these samples ranged from 77%～100%. TYLCCNV samples could be further divided into two groups based on geographic distribution, samples collected from Baoshan form one group with 93% nucleotide sequence identity, while those from Wenshan and Honghe form another group with 95% nucleotide identity. However, nucleotide sequence identity between thetwo groups is only 89%.Based on the 500 bp sequence, isolate Y264 was chosen to be sequenced completely. Sequence of Y264 comprised 2741 nucleotides (nts), having a genomic organization typical of begomoviruses originating from the Old World. Y264 is most closely related to TYLCCNV-[Y10] with 91.3 % nucleotide sequence identity for DNA-A and 87.6%～95.7% amino acid sequence identities for the encoded proteins, therefore Y264 should be considered as a isolate of TYLCCNV.Tobacco float seedlings with or without mosaic symptom collected before transplant were tested for virus types by TAS-ELISA, among 1400 float seedling samples collected in Yunnan province, 95% positive samples were infected by TMV, while less than 5% positive samples were infected by CMV or PVY. Tobacco leaf samples showing mosaic symptoms were collected in field plants at rosette period when mosaic incidence was above 20% and tested for virus types, among 480 samples, 48%～100% samples were infected by TMV, and 0%～12.2% samples were infected by CMV, PVY or Tobacco etched virus, indicating TMV is the dominant virus infecting tobacco float seedling in Yunnan province.Inactivation effects to TMV of 9 chemicals were tested and their injury symptom and dosage to tobacco seedling leaf were evaluated. The local lesion assay indicated when the following chemical solutions were mixed (1/1,V/V) with TMV infected leaf extracts diluted in 1:10000 for 3 min, TMV was inactivated with 100% efficiency, these chemicals were 1:10 solution of 30% chloride of lime, 1:10 solution of 98% Trisodium phosphate, 1:150 solution of 7% sodium hypochloride, 1:125 solution of anonymity disinfectant used in USA and 75% alcohol solution. 1:200 solution of 10% chlorine dioxide and 1:150 solution of 40% Yubao could inactivate TMV with 100% and 96.3% efficiencies, respectively, when the solutions were mixed with diluted TMV leaf extracts for 30 min, and the following chemicals had only less than 80% efficiency in the same time for mixture: 1:125 solution of 0.6% Jundujin, 1:800 solution of 3.95% Bingdubike and 1:800 solution of 24% Duxiao. Except for 0.6% Jundujing, the other tested chemicals in normal dosage could induce seedling leaf injury with chlorotic spot, necrotic spot and disorder symptoms which varied among different chemicals.Tests were also carried out for evaluation the inactivation effect to TMV and influence to tobacco seed germinate and seedling growth of chemicals used in water pool for tobacco float seedling growth. The local lesion assay indicated when the following chemical solutions were mixed (1/1,V/V) with TMV infected leaf extracts diluted in 1:10000 for 12 h, they could inactivate TMV with more than 80% efficiency, these chemicals were 30% chloride of lime at 15 mg/L and 150 mg/L, 7% sodium hypochlorite at 2μl/L and 20μl/L, anonymity disinfectant used in USA at 5μl/L and 50μl/L. The seed germinate rate and seedling rate show no obvious difference between treatment and control, but seedlings grew slowly in later growth for some chemicals. The following chemicals could promote seedling growing with recommended concentrations, these chemicals were 15 mg/L～150 mg/L Trisodium phosphate, 50 mg/L～100 mg/L copper sulfate, 10 mg/L～40 mg/L potassium permanganate and 10μl/L Duxiao. The following chemicals show no influence to seedling growth at low concentration but inhibite seedling growth at high concentration, these chemicals were 40% Yubao, 30% chloride of lime, 7% sodium hypochlorite, anonymity disinfectant used in USA and 0.6% Jundujing. To avoid negative effects to seedling growing, the following concentrations were recommended: 40% Yubao at 100μl/L, 30% chloride of lime at 300mg/L, 7% sodium hypochlorite at 20μl/L, anonymity disinfectant used in USA at 50μl/L and 0.6% Jundujing at 100μl/L.更多还原