壳寡糖诱导对烟草体内TMV-CP基因表达的抑制作用

 采用实时荧光定量PCR方法, 研究了壳寡糖诱导对烟草体内TMV-CP基因表达量的影响。结果表明, 壳寡糖诱导后烟草表达了对TMV侵染的系统获得抗病性, 植株显症推迟4~7 d, 症状减轻, 平均严重度比对照降低了82.9%。同时烟草叶片中CP基因的表达量显著降低。壳寡糖诱导处理的植株在接种病毒时(0 h), CP基因的表达量(拷贝数/2μL)为0.918, 接种后168 h增加到167.730。而在不诱导对照植株内, CP基因的表达量在0 h为1.218, 接种后168 h猛增到648.623。换言之, 不处理对照的表达量在此期间增长了532.53倍, 而壳寡糖诱导处理植株中仅增长了182.71倍。试验证明壳寡糖诱导的系统获得抗病性强烈抑制了烟草体内TMV-CP基因的表达。     

壳寡糖对烟草花叶病毒的抑制作用及其对烟草酶活性的影响

以壳寡糖喷施处理感染烟草花叶病毒的心叶烟和普通烟NC89,结果表明壳寡糖对烟草具有一定的体外保护作用。利用浓度为40μg/ml壳寡糖对烟草进行处理,以接种烟草花叶病毒前10d喷施的处理效果最好,对心叶烟的枯斑抑制率为78.0%;对普通烟NC89的防治效果为32.4%,均与药剂对照好普相当。而在病毒侵染植株的前4d内喷洒,对植株几乎没有防护作用。以壳寡糖处理烟叶,可提高其叶片中苯丙氨酸解氨酶、多酚氧化酶、超氧化物歧化酶、过氧化物酶4种防御酶的活性,以及可溶性蛋白的含量。     

用超声波法使烟草花叶病毒侵染烟草细胞的研究

 本文首次采用超声波法把烟草花叶病毒(TMV)粒子导入带壁烟草细胞,建立病毒-细胞高效侵染体系。将烟草细胞置于含TMV粒子的缓冲液中,用声强为0.5 W/cm²的脉冲超声波处理5 min,培养48 h后使用荧光免疫法检测烟草细胞转染率为59.7%。经酶联免疫吸附测定(ELISA)检测,TMV粒子在烟草细胞体内增殖48 h后达到高峰。电镜观察细胞体内有大量的TMV粒子。用枯斑寄主法测定表明TMV子代有较高的致病力。     

牛蒡低聚果糖诱导烟草对烟草花叶病毒的抗性

在室内条件下,研究了牛蒡低聚果糖对烟草花叶病毒抗性的影响及其影响机制。结果表明,在牛蒡低聚果糖处理叶（第三叶）中,接种烟草花叶病毒24h时对照处理叶片中该病毒含量大约是牛蒡低聚果糖处理叶片中的2.6倍,相对防效达61%;在未处理叶（第四叶）中,接种烟草花叶病毒24h时对照处理叶片可检测到TMV-CP的表达,而牛蒡低聚果糖处理叶则检测不到TMV-CP的表达。通过对抗病相关基因的检测发现,牛蒡低聚果糖处理和接种烟草花叶病毒都可诱导抗病相关基因（包括PR-1a、PR-2、PR-3、PI-1、PI-2和PAL）在烟草局部叶和系统叶中大量表达。这些结果表明,牛蒡低聚果糖可诱导烟草对烟草花叶病毒产生抗性,其作用机制可能在于诱导了抗病相关基因在植株体内的表达,增强了植株的系统抗性。     

蚕豆萎蔫病毒2号分离物PV131和P158侵染寄主植物的细胞病理比较观察

 透射电镜观察比较了蚕豆萎蔫病毒2号(BBWV-2)分离物PV131和P158侵染昆诺藜(Chenopodium quinoa)和蚕豆(Vicia faba)的细胞超微结构变化。结果显示:受PV131分离物侵染的昆诺藜叶肉细胞质中膜结构大量增生,形成有膜状结构、大小泡囊及电子致密物质的特殊膜增生区;病毒粒子散布在细胞质中,形成大块结晶体和管状结构,该管状结构直径约75nm,横切面由9个病毒粒子组成;受PV131侵染的蚕豆叶肉细胞中也形成膜增生区和管状结构,未见病毒结晶体。受P158分离物侵染的昆诺藜和蚕豆叶肉细胞病变情况与PV131分离物相似,形成膜增生区和相同的管状结构,在蚕豆叶肉细胞中也未观察到病毒结晶体。上述结果表明:2个BBWV-2分离物虽然在不同寄主植物上引起的细胞病变程度有差异,但其细胞病理学特征是由病毒基因结构所决定,与寄主的种类无关。     

壳寡糖对烟草防御酶活性及同工酶酶0谱的影响

以黄苗榆烟草为试材、摩擦接种法接种烟草花叶病毒,比色法测定了几种防御酶活性,研究壳寡糖对烟草-烟草花叶病毒这一互作系统激发产生的一系列抗病生理生化反应的可能性.试验结果初步表明壳寡糖处理可导致烟草叶片PO、CAT、PPO、PAL和β-1.3葡聚糖酶活性不同程度地提高.PO同工酶酶谱分析得知喷药后接毒处理可看到7条酶带且谱带色浓,酶活性强.由此初步认为壳寡糖诱导抗病毒机制与提高PO活性及其增多同工酶酶谱的酶带有关;分析SOD、CAT和PPO三种酶似乎与壳寡糖诱导抗病毒机制相关不大或无相关性;单因素的喷药和接毒多可导致几种防御酶活性的提高,尤其是PAL和β-1.3葡聚糖酶活性提高明显,但喷药后接毒的酶活变化及升高幅度似乎与两单因素的加权效应无关.     

葡萄扇叶病毒引起的寄主细胞病变研究

 电镜观察了葡萄扇叶病毒杭州分离物(Grapevine fanleaf virus Hangzhou isolate,GFLV-H)侵染苋色藜(Chenopodium amaranticolor)和昆诺藜(C.quinoa)的细胞超微结构变化。病毒粒子存在于叶肉组织薄壁细胞的细胞质内,常成单纵列排列在小管结构中,小管形成聚集体。在系统感染的昆诺藜细胞中位于管状结构中的病毒粒子穿过胞间连丝。2种感病寄主细胞质内膜结构增生,在核周围形成含细纤维状物质的小囊泡。液泡边缘存在少许小泡,有多泡体和髓鞘样结构伸入液泡,叶绿体和线粒体等细胞器也发生不同程度的变化。     

水稻基腐病菌毒素对烟草活性氧代谢及细胞超微结构的影响

 用水稻基腐病菌毒素注射烟草叶片后,烟草细胞中O₂^-_·和NO大量增加,H₂O₂含量明显降低,SOD、CAT和POD等酶的活性发生变化。毒素引起烟草细胞死亡,但没有细胞程序性死亡(PCD)的DNA梯特征。病菌毒素处理烟草叶片后,电导率增大,细胞膜透性增加。电镜观察烟草细胞的超微结构表明,毒素处理8h后,叶绿体变形,叶绿体基质片层大部分消解,基粒结构消失,叶绿体外膜和内膜剥离,质壁分离和细胞膜内陷,细胞器消解;处理48h后,细胞内含物完全消解,细胞膜消失,细胞壁皱缩变形。本研究表明,毒素引起烟草细胞死亡与植物抗病反应HR中细胞坏死的生理生化机制不同,但可能存在相似的信号传导途径。     

海带多糖对烟草花叶病毒的抑制作用及其对烟草酶活性的影响

为明确海带多糖抗病毒剂对烟草抗烟草花叶病毒（Tobacco mosaic virus,TMV）病的诱导抗性及其对TMV感染后的保护作用,采用间接酶联免疫吸附法（ELISA）研究0.5 mg/mL海带多糖水剂、0.5 mg/mL香菇多糖水剂、6.25 mg/L氨基寡糖素水剂和0.1 mg/mL盐酸吗啉胍.铜可湿性粉剂对TMV侵染的预防保护作用和对TMV感染植株的治疗作用,并检测施用海带多糖后烟草体内过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）及超氧化物歧化酶（SOD）活性的变化和海带多糖对感染TMV后烟草叶绿素含量的影响。接种前喷施0.5 mg/mL海带多糖后,再接种TMV病毒,其抑制率可达42.42%,与喷施6.25 mg/L氨基寡糖素抑制率44.96%的效果相当,预防保护作用较好;但两种药剂处理感病植株的治疗效果较差,其抑制率仅分别为38.93%和40.13%。海带多糖能够系统地诱导烟草体内POD、PAL及SOD活性,从而控制了感染TMV后烟草叶绿素含量下降。说明海带多糖可诱导植物产生抗病性。     

苹果链格孢菌侵染对感病苹果品种叶细胞超微结构的影响

 将Alternaria mali分生孢子接种在苹果元帅品种叶片的下表皮,保湿培养3~24h,观察细胞超微结构,接种后3h,表皮及叶肉细胞的质膜发生内陷,出现囊泡,暗染色颗粒及管状结构,叶绿体膜结构解体,电子致密物质沉积,基粒排列混乱松散,外膜破裂等。接种后24h,叶片表面出现病斑,细胞质浓缩,细胞壁出现颗粒物,细胞变形,病健组织交界处,叶绿体基浓缩,出现脂质球,同时还观察到,距离病斑2mm处,许多叶肉细胞中出现密度均匀、形状不规则的物质,小叶脉木质部中出现电子致密物质沉积。     

Infection and necrosis of cowpea mesophyll cells by tobacco necrosis virus and two strains of tobacco mosaic virus

When cowpea mesophyll tissue with or without any epidermal layer was inoculated with tobacco necrosis virus (TNV), local necrotic lesions were produced. In epidermal strips isolated after inoculation of intact leaves local lesions were never observed. Homogenates of epidermal strips removed within 30 min after inoculation of the leaf with the cowpea strain of tobacco mosaic virus (Cp-TMV) or with TNV and incubated on agar for 2 or 4 days were not infectious. However, when clusters of mesophyll cells or vein pieces were still attached to the epidermal strips after stripping, the homogenates showed virus activity. When cowpea leaves were inoculated with Cp-TMV or a common strain of TMV (TMV-U) infective virus material was present in the mesophyll tissue as measured in the homogenates, at the moment of stripping, i.e. within 10 min after inculation.It may be concluded that cowpea mesophyll cells can act as primary sites of viral ingress into the leaf and that the epidermis is not required for necrosis production after virus inoculation.Samenvatting De mogelijkheid werd onderzocht om cowpea-mesofylcellen zonder de aanwezigheid van epidermiscellen met TNV te infecteren. Kleine lokale necrotische lesies werden 40–72 uur na inoculatie zichtbaar waaruit blijkt, dat bij cowpea de epidermis niet noodzakelijk is voor de vorming van TNV-lesies. Geïsoleerd epidermisweefsel vertoonde nooit lokale lesies. Homogenaten van met TNV geïnoculeerde en daarna geïsoleerde cowpea-epidermisstukjes werden getoetst op virusactivitiet. Als de stukjes volledig vrij waren van mesofylcellen of nerfweefsel, dan vond daarin geen virusvermeerdering plaats tijdens een incubatie van 2 of 4 dagen op agar. Als na het strippen nog enkele mesofylcellen of nerfstukjes aanwezig waren, kon wel enige virusactiviteit in de homogenaten worden aangetoond.In cowpeabladeren die geïnoculeerd werden met de cowpea-stam van TMV of de normale stam van TMV had infectieus virusmateriaal al binnen 10 min na inoculatie het mesofyl bereikt. Blijkbaar is in cowpeabladeren de epidermis niet noodzakelijk voor de binnenkomst van virus of voor de necroseproduktie na virusinoculatie.     

寡聚糖诱导黄瓜对白粉病抗病反应的超微结构研究

 中科6号(2%氨基寡糖素)处理黄瓜植株叶片,5 d后接种白粉菌Sphaerotheca fuliginea(Schlecht.) Poll.,可诱导黄瓜Cucumis sativus产生对白粉病的抗病性,寄主细胞对病原菌的侵入产生了防卫反应结构和物质以及过敏性坏死反应。表现为寄主细胞壁加厚,染色加深,寄主细胞壁下产生多层次结构的乳突,在寄主细胞壁与质膜之间有黑色物质沉积;吸器外质膜皱褶,染色加深,吸器外基质中出现染色加深的颗粒状电子沉积物;寄主细胞质紊乱,细胞器解体,整个寄主细胞解体、坏死。     

转CP基因线辣椒对CMV和CMV-RNA的抗病性比较

 本试验以转化CMV-CP和TMV-CP基因线辣椒纯合系作试材,比较了接种CMV粒体和CMV-RNA后的发病特点和叶片中的病毒含量。结果表明:转化线辣椒不仅能抵抗CMV粒体的侵染,而且还能抵抗CMV-RNA的侵染。不论接种CMV粒体或CMV-RNA,CP(+)线辣椒的系统症状都延迟出现,显症株率和病害严重度级别大幅度降低,病毒增殖和运转受到抑制,接种叶片与新生叶片中的病毒含量明显减低。这一结果证实CMV-RNA不能克服线辣椒由CP基因介导的抗病性。     

Encasement of Erysiphe graminis haustoria after treatment with bromuconazole

Preventive application of bromuconazole caused reduction in size and increased encasement rate of haustoria of Erysiphe graminis DC. For example, seven days after inoculation, 60 and 70% of haustoria had been encased in leaves treated with 8 mg litre⁻¹ and 16 mg litre⁻¹ respectively; the average length of the digitations was 8–10 μm in treated cells compared to 24 μm in untreated cells. The encasement process extended from the neck region to the whole haustorium. Haustorial bodies from treated plants had electron-dense cytoplasm and their organelles were more difficult to identify than in control plants. Extrahaustorial matrix was reduced to an unusually thin, osmiophilic pellicle, surrounded by abundant heterogeneous encasement material. Curative treatment induced similar changes, especially in the margin of the colonies. In the centre of the colony, haustoria were less affected by the fungicide; deposition of collar-like material, modification of extrahaustorial matrix and membrane and accumulation of plant cytoplasm around the digitations resulted in an intermediate, ‘swollen’ state of digitate haustoria. The possible pathway of encasement events is discussed.     

京郊大蒜病毒病的研究及其鳞茎中病毒的脱除

 京郊大蒜病毒病发生普遍,主要症状为条纹花叶、矮化和叶片扭曲畸形。病体细胞中含大量线状病毒粒体和风轮状内含物。线状病毒粒体长度范围为250-1875nm,以长度550-800nm粒体居多。其中长700-800nm的粒体被鉴定为大蒜花叶病毒(GMV),回接脱毒大蒜叶片产生条纹花叶症状。血清学鉴定表明GMV与洋葱黄矮病毒(OYDV)有近缘关系。长度500-600nm的粒体可能为大蒜潜隐病毒(GLV),回接脱毒大蒜不产生花叶症状。下述两种病毒为京郊大蒜的主要病毒。此外,个别标样含烟草花叶病毒,但不是大蒜的主要病毒。对800nm以下线状病毒粒体归属尚待研究。
应用营养茎尖、生殖茎尖和根尖分生组织培养技术,可以脱去上述主要病毒,获得脱毒大蒜。     

Viral inclusions in monocotyledons infected by maize streak and related geminiviruses

Isolates of maize streak virus (MSV) were examined by thin-section electron microscopy in plants, assessed for characteristic features of infection and compared with other related geminiviruses infecting monocotyledons from Africa, islands in the Indian Ocean, and the Pacific Island of Vanuatu. Arrays of virus particles, often crystalline, were most often seen in the nucleus. The morphology of the nuclear crystalline arrays was characteristic of certain isolates or groups of isolates (strains). Infected nuclei could be seen in cells from the phloem parenchyma, vascular bundle sheath and mesophyll tissue, and also in epidermal guard cells of plants infected with the maize strain of MSV. The particle arrays varied in morphology from regular rows of virions forming distinctive blocks, to randomly arranged aggregates in certain areas of the nucleus. We consistently failed to find viral crystalline arrays associated with infection of panicum streak virus (PSV) and sugar cane streak virus (SSV) isolates either in these hosts or in maize. Occasionally arrays of MSV particles were found outside the nuclear envelope in physiologically active cells. Accumulations or sheets of MSV particles were seen lining the walls of some phloem companion cells. Crystalline aggregates of particles were frequently observed in the cell vacuole, after lysis of the nuclear membrane of dead cells which made up the chlorotic lesions, the typical symptom of virus infection. Virus preparations from all hosts contained typical geminate particles regardless of the morphology of the virion arrays. The effect on chloroplasts appeared to vary between isolates and this is discussed in relation to lesion colour. The arrangement of virions in the nucleus as a taxonomic character is diagnostic for MSV. Inclusions with crystalline structure found in sieve elements of infected plants were not immunogold labelled when thin sections were probed using antiserum to the virus particles.     

一种细菌蛋白CZ控制烟草花叶病毒(TMV)的作用机理初探

以活性蛋白CZ处理NC89烟(Nicotianatabacumvar.NC89)12 h后,摩擦接种TMV,1、5、8、12、16 d分别取各处理叶片测过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的活性及丙二醛(MDA)和叶绿素的含量。结果表明,POD和PAL2种酶的活性均比对照明显提高;CZ处理使NC89烟叶片内MDA的含量降低,而叶绿素的含量增高。电镜下观察到活性蛋白能打破TMV粒体的规则排列,与TMV粒体不可逆的结合,从而降低侵染力。     

大豆凝集素基因lec-s转化烟草>增强对烟草花叶病毒的抗性

 将编码大豆凝集素的lec-s基因插入植物表达载体pBI121中,构建植物重组表达质粒pBI121:: lec-s。由根癌土壤杆菌EHA105介导的叶盘法转化烟草,获得了转基因烟草株系。PCR和RT-PCR检测证明lec-s基因已转入烟草植株中。接种烟草花叶病毒（Tobacco mosaic virus,TMV）进行抗病性试验结果表明,转基因烟草叶片上的病斑数显著减少,说明转基因烟草表现出对TMV的抗性。定量RT-PCR检测发现,接种TMV后,抗病防卫基因（PR-1a、GST1、Pal和hsr515）在转基因烟草叶片中显著上调表达。这些结果表明,大豆凝集素基因lec-s转化烟草可对TMV产生抗性,其作用机制可能在于lec-s基因参与了植物的防卫信号通路,诱导了抗病防卫基因在转基因植株体内的表达,增强了植株对TMV的系统抗性。