MDMV HC-Pro在玉米叶片中的积累及免疫定位

 本文以感病自交系Mo17、掖107和抗病自交系黄早四为材料,研究了玉米矮花叶病毒(MDMV)和MDMV辅助成份-蛋白酶(HC-Pro)在叶片中的积累动态和细胞内定位。结果表明,在接种后3 d,MDMV就在掖107和Mo17接种叶的上位叶积累到了相当高的程度,6 d后达到高峰;而MDMV在黄早四中的浓度一直低于在掖107和Mo17中的浓度。在接种掖107和Mo176 d后,MDMV HC-Pro在上位叶中的积累到达高峰,而在黄早四植株中MDMV HC-Pro直到第9 d才到最高,而且浓度一直低于感病自交系掖107和Mo17。麦二叉蚜从发病植株上获毒后的传毒效率变化与MDMV、MDMV HC-Pro在叶片中的积累动态一致。通过胶体金标记对MDMV HC-Pro进行免疫定位,发现在细胞质中、与质膜相联系的风轮状内含体、片层凝集状内含体、细胞质高密度物质和病毒粒子周围有金标记,说明在这些部位有MDMV HC-Pro的存在。     

人工饲料条件下大麦黄矮病毒GAV株系对麦长管蚜种群适合度的直接影响

植物病毒与介体蚜虫存在复杂的互作关系。前人关于植物病毒对蚜虫调控作用的研究主要集中在植物病毒通过寄主植物对蚜虫的间接影响上,未见植物病毒对介体蚜虫适合度直接调控的报道。鉴于此,我们以麦长管蚜Sitobion miscanthi (Takahashi)为试虫,以其传播的大麦黄矮病毒-GAV(Barley yellow dwarf virus GAV,BYDV-GAV)为测试病毒,以全纯人工饲料加入BYDV-GAV病毒提取液饲养麦长管蚜4 d,使之在不接触寄主植物条件下获毒,然后分别在全纯人工饲料和无毒小麦叶片上继续饲养,直至死亡。利用生命表技术分析麦长管蚜生长发育和繁殖参数。研究结果表明:在无毒小麦叶片饲养条件下,与未获毒对照麦长管蚜相比,获毒后麦长管蚜生活史参数成虫历期和产仔天数显著降低,繁殖力显著增加;种群参数内禀增长率、净繁殖率、周限增长率显著增加,平均世代周期显著降低。在全纯人工饲料条件下,与未获毒对照相比,获毒后麦长管蚜仅成虫历期和产仔天数显著下降,而其他生活史参数及种群参数均无显著差异。说明BYDV-GAV使得介体麦长管蚜在小麦叶片上的适合度显著提高,这是由麦长管蚜与寄主植物互作引起的,而病毒对介体麦长管蚜的适合度无直接调控作用。     

豇豆退绿斑驳病毒(CCMV)突变体的混合侵染研究

 本研究采用PCR点突变的方法,对豇豆退绿斑驳病毒(CCMV) RNA3的cDNA克隆进行突变,突变体TP7是在外壳蛋白基因之前突变形成一个独特的Sal I酶切点;TP8是在3a基因之前突变形成一个独特的Bam H I酶切点;AG 1是在外壳蛋白基因之后突变形成一个独特的Not I酶切点。TP7、TP8、AG1体外转录产物分别与CCMV RNA1、RNA2的c DNA克隆的体外转录产物混和后接种豇豆,结果表明,接种后第6 d TP7植株首先表现出症状,9d后TP8、AG1和野生型CCMV接种的植株表现出症状,TP7的症状发展最快。
采用TP7、TP8、AG1接种20 d的毒源植株,按TP7+TP8、TP7+AG1、TP8+AG1等量混合汁液接种豇豆,接种20 d,通过RT-PCR分析接种植物,在TP7+TP8和TP7+AG1混合侵染中,TP7在混合侵染中占优势,在TP8+AG1的混合侵染中,TP8和AG1的种群十分接近。     

叶霉菌非亲和小种对番茄系统抗性的诱导及植株内水杨酸动态

 番茄叶霉菌小种4是番茄Cf5品系的非亲和小种,接种Cf5植株第3叶后,经不同诱导间隔期以亲和小种5接种第3叶和第4叶,15 d后检查叶霉病发病情况。试验表明,在诱导间隔期为3 d和5 d时,小种4诱导接种的第3叶和未经诱导接种的上位第4叶发病面积比不接种或接种小种5的对照显著降低,以5 d间隔期处理效果最好。上述2个叶位的发病分别比对照降低90%和85%。小种4接种第3叶后该叶位和上部未接种第4叶内水杨酸含量迅速增加,以接种后3 d含量最高,分别达4.02 μg/g鲜重和3.21μg/g鲜重,比对照分别高2倍和1.8倍。接种后5 d内始终保持较高水平。接种8 d后逐渐下降,但仍高于对照。水杨酸含量的增加早于抗性表现,因而可能在该系统的抗性诱导中起作用。     

人工接种CGMMV的增殖及其对西瓜 光合作用的影响

为明确西瓜感染黄瓜绿斑驳花叶病毒CGMMV后病毒的增殖过程及其对光合作用的影响,本研究以辽宁省田间主栽西瓜品种‘京欣’为试材,测定了人工接种CGMMV后,西瓜植株体内病毒含量、光合色素、光合作用相关指标以及参与光合作用的关键酶活性的变化,结果显示:接种3d后,即可检测到病毒,24d植株内病毒含量达最高;叶绿素a、叶绿素b及类胡萝卜素的含量均有所降低,叶绿素a在9d后均低于健康对照,而叶绿素b和类胡萝卜素在15d后明显低于健康对照,第18天时,健康对照的叶绿素b含量达接种处理的1.33倍;ALA脱水酶活性除第15天与对照无显著差异外,整个测定期间均低于健康对照,21d时健康对照的ALA脱水酶的活性达29.9U/h,为接种处理的2.18倍;PEP羧化酶的活性显著低于健康对照,测定期间接种处理的PEP羧化酶活性变化不大;乙醇酸氧化酶活性明显升高,测定期间其活性均显著高于健康对照;西瓜叶片的光合速率、气孔导度和蒸腾速率均远小于健康对照,而细胞间隙CO_2浓度却比对照高,本研究为揭示西瓜感染CGMMV后的光合作用变化规律奠定了基础。     

基于Real-time PCR监测番茄黄化曲叶病毒在番茄植株中的动态变化

 本研究根据番茄黄化曲叶病毒(Tomato yellow leaf curl virus, TYLCV)分离物-SH2基因组序列,设计了一对引物,以番茄25S rRNA基因为内参,建立了番茄黄化曲叶病毒SYBR GreenⅠ实时荧光定量PCR检测方法。该方法可检测到浓度为4.64×10^-7 ng/μL的植物DNA中含有TYLCV,其灵敏度是常规PCR的1 000倍。利用该方法,研究了温室条件下以侵染性克隆接种TYLCV后的番茄植株中病毒DNA含量的变化情况。根、茎、叶中病毒DNA定量检测结果表明,TYLCV在3种植物器官中都呈现一个上升、稳定和下降的变化规律;病毒DNA在植株根部最早累积,累积的速度较慢,在叶部和茎部累积较快;叶部和茎部接种18 d后病毒DNA含量达到稳定期;在不同的器官中,病毒的含量不同,在茎部的含量最高,接种33 d后茎部病毒DNA的含量约为根部的100倍。本研究通过对TYLCV含量的动态监测,明确了病毒DNA在番茄植株中的累积和变化规律,为研究TYLCV侵染机制、病毒与寄主互作及病害防治提供了理论依据。     

TeMV与PWV侵染对西番莲代谢生理及组织微结构的影响

以西番莲品种‘台农1号’为实验材料,通过电镜扫描技术比较观测TeMV和PWV侵染与健康西番莲的叶片、幼嫩果实外果皮组织形态结构,分析测定其叶片生理生化变化,研究西番莲病毒侵染对西番莲叶片和幼嫩果实外果皮组织结构和生理生化的影响。结果表明：TeMV与PWV侵染后,西番莲植株叶片的海绵组织细胞间隙缩小,发生成团堆积状排列,栅栏组织细胞萎缩,呈不规则长条形;幼嫩果实外表皮组织中石细胞层发生严重断裂,排列松散,海绵层细胞变得松散,且发生较严重的木质化现象;叶片与幼嫩果实外表皮表面纹理粗糙、褶皱,气孔保卫细胞萎缩,气孔周围组织皱缩、粗燥;感病果实外果皮表面上的腺毛凸起无序。病毒侵染后,感病植株第1～3叶的蔗糖含量较第4～6叶的高23.11%;健康叶片则相对平衡,降低了淀粉积累、PG酶、纤维素酶与PPO酶活性,提高了感病植株第1～3叶的α-淀粉酶、GS酶与POD酶活性。西番莲受TeMV与PWV侵染后,植株叶片第1～6叶营养物质分配不平衡,相关物质的合成分解与抗氧化防御酶系统受伤害,叶片与果实组织形态结构受到不同程度的损坏,植株正常吸收功能遭到破坏。     

海南红麻曲叶病的病原检测与鉴定

 红麻曲叶病是2012年在海南省海口市发现的一种新病害,病株表现为叶片向上卷曲、叶脉肿大、叶脉变深绿色等症状。PCR检测结果显示,该病样中均存在菜豆金色花叶病毒属病毒。基因克隆及序列分析结果表明,该病毒分离物(HN08)基因组仅含A组分(DNA-A),其全长为2 738 nt,与木尔坦棉花曲叶病毒(CLCuMuV)各分离物的相似性均大于89.0 %,其中与中国各分离物的相似性均大于99.0 %。该病毒分离物也伴随有β卫星分子,其全长为1 346 nt,与CLCuMuV各分离物伴随的β卫星分子(CLCuMuB)序列相似性大于83.0 %,其中与分离物Fz1的序列相似性最高,为99.7 %。构建了HN08 DNA-A及其β卫星分子侵染性克隆,通过农杆菌注射接种红麻,接种后30 d,HN08 DNA-A及其β卫星分子混合接种的红麻植株新出叶片开始产生曲叶症状;接种后60 d,二者混合接种的植株大部分叶片表现为严重的曲叶症状,且与田间自然病株症状相同,而二者各自单独接种的红麻植株没有产生明显的症状。PCR及Southern blot检测进一步证实这些症状是由HN08 DNA-A及其β卫星分子的共同侵染引起的。因此,海南红麻曲叶病是由CLCuMuV及其伴随的β卫星分子(CLCuMuB)共同侵染引起的。本文首次报道了红麻是CLCuMuV自然新寄主。     

一种新的90 kD胞外蛋白激发子诱导烟草系统获得抗性研究

 就棉疫病菌(Phytophthora boehmeriae Saw.)培养滤液中提纯获得的90 kD胞外蛋白激发子诱发烟草系统获得抗性进行了研究。以10 nmol/L激发子溶液注射处理Samsun NN烟草叶片24 h后,在处理叶片及其上、下各2片叶片接种TMV,结果是处理叶及其上、下各2片叶片上的枯斑数显著少于对照,诱抗防效达40.9%~53.1%;接种TMV7d后处理叶片的枯斑平均直径为1.23mm,显著小于对照叶片上的枯斑直径2.97mm,但是处理叶片的上、下叶片上的枯斑平均直径与对照没有显著差别。以10 nmol/L激发子溶液注射处理Samsun NN烟草叶片分别立即接种或于1、2、4、7、15 d接种TMV,结果证明该激发子诱导烟草对TMV的抗性以处理后第1d至第4 d接种为较好,防效达30.2%~5 0.4%;处理后立即接种和第7d接种TMV诱抗防效仅4%左右,处理叶片上的枯斑数与对照没有显著差别,表明较强的诱导抗性可持续时间<7d。用不同浓度激发子处理烟叶,所测定的0.5~100 nmol/L各浓度均可显著地诱发烟草对TMV产生获得抗性,诱导抗性效果为34.9%~5 8.2%,诱导抗性效果随浓度的降低呈下降趋势。以10 nmol/L激发子溶液注射处理W38烟草叶片,2 d后分别注射接种烟草野火病菌(Pseudomonas syringae pv.tabaci)菌液或喷雾接种烟草赤星病菌(Alternaria alternata)分生孢子,结果是,注射接种烟草野火病菌5d后处理叶片及其上、下叶片上的野火病病斑均显著小于对照;处理叶片上的赤星病病斑数及病斑面积明显小于对照,表明该激发子可诱导烟草对野火病和赤星病产生抗性。上述结果表明,90kD蛋白激发子诱导烟草产生的获得抗性是一种典型的系统获得抗性,该系统获得抗性对病原菌具广谱抗性。     

放线菌代谢物质对油菜花叶病毒病的治疗及防护作用研究初报

在完整的油菜植株上,利用喷叶法测定放线菌代谢物质对病毒病的治疗效果,以处理组的发病率此对照组减少的百分率表示疗效。从1958年到1962年先后测定6,376号次放线菌,找到不吸水灰色放线菌类羣(Actinomyces ahygro-scopicus Yen)的1695、3494、4084和4313四个菌株的代谢物质,在接种油菜花叶病毒(总6号)24小时后喷叶,有50%左右的治疗效果,接种前24小时喷叶有100%的防护效果。接种24小时后喷叶而未发病的植株叶内未能测出有侵染性病毒的存在。从喷药一次的效果来看,显然胜于0.5%2-硫尿嘧啶在油菜上的作用。1695、3494和4084三株放线菌发酵液的有效成分经15磅30分钟的处理后,在植株上测定效果,差异并不大。但是,所有有效浓度对植株均能引起不同程度的黄化等生理反应,使生长遭受一定程度的抑制,类似2-硫尿嘧啶所引起的生理反应。病毒与1695放线菌发酵液混合(1分钟),立即被钝化。发酵液中的有效成分能被叶细胞吸收,通过剪叶法及不同部位局部喷药及接种等初步证明,代榭物质昀有效成分运转的距离似乎极短。1695的发酵液在植株上的治疗效果常因接种与喷药相隔时间的长短而异。接种后3小时内喷的效果为100%,6小时降至90%,1—3天为25—45%,5—7天为5—10%。同时,在接种后3天内喷而发病的植株体内其病毒含量比对照少,3天以后喷的则此对照反而有增多的趋势。1695的发酵液在胶州白菜上对孤丁1号的治疗效果(接种后隔24小时喷)为92%,比崐山土种油菜上对总6号病毒的疗效高出三分之一。此种差异可能由于寄主植物的差异引起的。     

Mature plant resistance of potato against some virus diseases. I. Concurrence of development of mature plant resistance against potato virus X,and decrease of ribosome and RNA content

Four of five weeks after planting a group of potato plants ‘Bintje’ was inoculated with potato virus X (PVX). Other groups were inoculated at intervals of 14 days. Tubers produced by plants inoculated 35 days after planting were all infected. The plants inoculated 49 days or later after planting produced few infected tubers. The latter had developed mature plant resistance against PVX infection. The ribosome and RNA contents of leaves were measured by application of adsorption chromatography. A rapid decrease in ribosome and RNA contents occurred in plants at the time of rapid increase in the rate of mature plant resistance. The decrease was most distinct in the fifteenth leaf and therefore the contents in this leaf seem to give a good indication of the rapid increase in resistance.     

香蕉束顶病毒(BBTV)侵染对寄主内源激素的影响

 本试验用ELISA方法测定了香蕉感染束顶病毒(BBTV)后植株体内的3种内源激素,赤霉素(GA₃)、玉米素类(iPAs)和脱落酸(ABA)的变化。结果表明,感株的GA₃水平在侵染过程中虽有微弱增加,但含量和增长速度显著低于健株对照;iPAs在接种BBTV第14天后明显下降,并维持较低水平;ABA在BBTV侵染后被大量诱导增加并不断积累,在接种后第35天测定含量最高,为对照的3.34倍。试验还同时检测了BBTV在侵染过程中的运转。用间接ELISA测定的接种叶和顶叶的BBTV含量显示:接种21天后BBTV在接种叶和顶叶中还大量增殖,呈系统性分布。但寄主的症状在接种35天后才逐渐在顶叶表现。以上结果表明:香蕉束顶病的症状表现似乎主要与病株中的内源激素的失调有关,而与BBTV在体内的运转并不直接相关     

Transgenic Nicotiana occidentalis Plants Expressing the 50-kDa Protein of Apple chlorotic leaf spot virus Display Increased Susceptibility to Homologous Virus, but Strong Resistance to Grapevine berry inner necrosis virus

ABSTRACT The 50-kDa protein (P50) encoded by the open reading frame 2 of Apple chlorotic leaf spot virus (ACLSV), a putative movement protein, was expressed in transgenic Nicotiana occidentalis plants. P50 in transgenic plants was mainly detected in a modified form in the cell wall fraction, similar to that in infected leaves. The P50-expressing plants (P50 plants) complemented the systemic spread of the P50-defective mutants of an infectious cDNA clone of ACLSV (pCLSF), indicating that P50 in transgenic plants was functional. Severity of symptoms was greatly enhanced and accumulation of virus in upper leaves was increased in P50 plants inoculated with pCLSF or ACLSV compared with that in nontransgenic control plants (NT plants). Conversely, transgenic plants expressing the coat protein of ACLSV (CP plants) showed a significant delay in symptom development and a reduction of virus accumulation. However, most P50 plants inoculated with Grapevine berry inner necrosis virus (GINV), another species of the genus Trichovirus, neither developed obvious symptoms nor supported virus accumulation in inoculated or upper leaves. In contrast, systemic symptoms developed and virus accumulated equally in NT and CP plants inoculated with GINV. After inoculation with Apple stem grooving virus or Apple stem pitting virus, there was no difference in symptom development and virus accumulation among P50, CP, and NT plants. Our results indicate that transgenic plants expressing a functional P50 were more susceptible to homologous virus and, on the contrary, showed strong resistance to the heterologous virus GINV.     

Interference of Long-Distance Movement of Grapevine berry inner necrosis virus in Transgenic Plants Expressing a Defective Movement Protein of Apple chlorotic leaf spot virus

ABSTRACT Transgenic Nicotiana occidentalis plants expressing a movement protein (P50) and partially functional deletion mutants (DeltaA and DeltaC) of the Apple chlorotic leaf spot virus (ACLSV) showed resistance to Grapevine berry inner necrosis virus (GINV). The resistance is highly effective and GINV was below the level of detection in both inoculated and uninoculated upper leaves. In contrast, GINV accumulated in inoculated and uninoculated leaves of nontransgenic (NT) plants and transgenic plants expressing a dysfunctional mutant (DeltaG). On the other hand, in some plants of a transgenic plant line expressing a deletion mutant (DeltaA', deletion of the C-terminal 42 amino acids), GINV could spread in inoculated leaves, but not move into uninoculated leaves. In a tissue blot hybridization analysis of DeltaA'-plants inoculated with GINV, virus could be detected in leaf blade, midribs, and petiole of inoculated leaves, but neither in stems immediately above inoculated leaves nor in any tissues of uninoculated leaves. Immunohistochemical analysis of GINV-inoculated leaves of DeltaA'-plants showed that GINV could invade into phloem parenchyma cells through bundle sheath of minor veins, suggesting that the long-distance transport of GINV might be inhibited between the phloem cells and sieve element (and/or within sieve element) rather than bundle sheath-phloem interfaces. Immunogold electron microscopy using an anti-P50 antiserum showed that P50 accumulated on the parietal layer of sieve elements and on sieve plates. The results suggested that resistance in P50-transgenic plants to GINV is due to the interference of both long-distance and cell-to-cell movement of the virus.     

Pathogenesis of Alfalfa mosaic virus in Soybean (Glycine max) and Expression of Chimeric Rabies Peptide in Virus-Infected Soybean Plants

ABSTRACT Infection of soybean (Glycine max) plants inoculated with particles of Alfalfa mosaic virus (AlMV) isolate 425 at 12 days after germination was monitored throughout the life cycle of the plant (vegetative growth, flowering, seed formation, and seed maturation) by western blot analysis of tissue samples. At 8 to 10 days after inoculation, the upper uninoculated leaves showed symptoms of virus infection and accumulation of viral coat protein (CP). Virus CP was detectable in leaves, stem, roots, seedpods, and seed coat up to 45 days postinoculation (dpi), but only in the seedpod and seed coat at 65 dpi. No virus accumulation was detected in embryos and cotyledons at any time during infection, and no seed transmission of virus was observed. Soybean plants inoculated with recombinant AlMV passaged from upper uninoculated leaves of infected plants showed accumulation of full-length chimeric AlMV CP containing rabies antigen in systemically infected leaves and seed coat. These results suggest the potential usefulness of plants and plant viruses as vehicles for producing proteins of biomedical importance in a safe and inexpensive manner. Moreover, even the soybean seed coat, treated as waste tissue during conventional processing for oil and other products, may be utilized for the expression of value-added proteins.     

Effects of rust and post-infection drought on photosynthesis, growth and water relations in groundsel

Exchanges of CO² and water vapour were examined in groundsel, Senecio vulgaris , grown and infected with rust, Puccinia lagenophorae , under laboratory conditions. The effects of drought were examined by withholding water from plants from 2 days until approximately 10 days after infection, after which further daily water losses were restored. Net photosynthesis was inhibited and dark respiration was stimulated in rust-infected leaves but, in young healthy leaves on the same plant, gross and net photosynthesis were stimulated and photorespiration was inhibited. The growth of plants was stunted 15 days after infection because the growth of leaf area was inhibited and, thereby, the photosynthetic capacity of the plant was reduced. Drought inhibited the growth of leaf area in both healthy and infected plants and, when combined with rust, had additive deleterious effects on net photosynthesis per plant and on growth. After sporulation, rusted plants transpired more rapidly than did healthy controls and leaf water potential fell to reach - 0.95 and-1.92 MPa in well-watered and drought-stressed plants respectively, compared with -0.18 and - 1.2 MPa in healthy controls, 20 days after infection. Rust impaired the normal increase in Water-Use Efficiency in response to drought.     

Analysis of the spatial distribution of identical and two distinct virus populations differently labeled with cyan and yellow fluorescent proteins in coinfected plants

ABSTRACT Apple latent spherical virus (ALSV) expressing yellow and cyan fluorescent proteins (ALSV-YFP and ALSV-CFP) was used to investigate the distribution of identical virus populations in coinfected plants. In Chenopodium quinoa plants inoculated with a mixture of ALSV-YFP and ALSV-CFP, fluorescence from YFP and CFP was always distributed separately in both inoculated and upper uninoculated leaves. Inoculation of each ALSV-YFP and ALSV-CFP to different leaves of a C. quinoa plant resulted in the separate distribution of each virus population among different upper leaves. When C. quinoa leaves were first inoculated with ALSV-CFP and then ALSV-YFP was reinoculated into the same leaves at various times after the first inoculation, ALSV-YFP infected only tissues where ALSV-CFP infection had not been established. The spatial separation was also found in Nicotiana benthamiana leaves coinoculated with Bean yellow mosaic virus (BYMV)-YFP and BYMV-CFP. In contrast, both YFP and CFP fluorescence signals were observed in the same tissues of N. benthamiana leaves mixed infected with ALSV-YFP and BYMV-CFP. YFP fluorescence from ALSV-YFP in mixed-infected leaves was brighter and longer than in leaves infected with ALSV-YFP singly.     

草甘膦异丙胺盐杀灭香蕉病毒病植株的研究

本研究比较了41%草甘膦异丙胺盐水剂喷雾、假茎注射和叶鞘点滴3种施用方法对香蕉病毒病病株的灭除效果。结果表明:(1)香蕉假茎注射3、5和10mL草甘膦异丙胺盐,随着注射剂量的增加,香蕉株高抑制率、叶片死亡率、植株枯死率增加,注射10mL,药后30d的植株死亡率为90.0%,显著高于其他2个处理。(2)草甘膦异丙胺盐稀释成200倍液喷雾处理香蕉,药后30d,植株枯死率达93.3%,显著高于300、400倍液处理。(3)3种使用方法比较,药后15d,喷雾法处理的叶片、植株枯死率显著高于假茎注射和叶鞘点滴法,而对株高抑制率差异不显著,药后30d,3种使用方法的灭除效果等于或高于90.0%,处理间差异不显著。考虑操作的方便,叶鞘点滴法和假茎注射法可作为防除感染病毒病香蕉植株的高效使用方法,注射和点滴剂量为10mL。