Amino Acid 129 of Cucumber mosaic virus Coat Protein Determines Local Symptom Expression and Systemic Movement in Tetragonia expansa, Momordica charantia and Physalis floridana

Local symptom expression and systemic movement of Cucumber mosaic virus (CMV) in Tetragonia expansa, Momordica charantia and Physalis floridana were mapped to the amino acid at position 129 of CMV coat protein (CP), using pseudorecombinants, chimeric RNAs, a site-directed mutant of RNA 3 and four strains of CMV : pepo-, SO-, MY17- and Y-CMV. Local and systemic symptoms caused by three strains, pepo-, SO- and MY17-CMV, and those by Y-CMV differed in the three host species. The three strains expressed local chlorotic spots at 24°C and systemic chlorotic spots and ringspots at 36°C, whereas Y-CMV developed local necrotic spots at 24°C but no systemic symptoms at 36°C in T. expansa. In M. charantia the three strains caused systemic chlorotic spots, whereas Y-CMV caused local necrotic spots. The three caused systemic mosaic and Y-CMV systemic necrosis in P. floridana. With pseudorecombinants combined with pepo- and Y-CMV RNAs, CMV RNA 3 was responsible for symptom expression and systemic infection. Inoculation with Y-CMV RNA 1, RNA 2 and chimeric RNA 3s exchanged CP gene fragments between pepo- and Y-CMV showed that NruI-XhoI fragment of CP was essential for symptom expression. Comparative analysis of the NruI-XhoI fragments revealed that only the amino acid at position 129 was common among the three strains but different from that of Y-CMV. Inoculation with a point mutant constructed by substituting one nucleotide resulting in an amino acid change from Ser to Pro at position 129 in Y-CMV CP verified the previous experiments. These results indicate that the amino acid at position 129 of CMV CP is the determinant for local symptom expression and systemic movement in the three host species. CMV CP containing Ser at position 129 may induce resistant responses in these plants. Received 29 June 2001/ Accepted in revised form 28 August 2001     

Synergistic Disease in Pepper Caused by the Mixed Infection of Cucumber mosaic virus and Pepper mottle virus

ABSTRACT The occurrence of more than one virus species in a single plant is not uncommon in cultivated and native plant species. A mixed virus infection may lead to greater disease severity than individual viral components and this is sometimes referred to as a synergistic disease. Although, in some cases, synergism has been demonstrated for various plant growth parameters such as plant height, weight, and yield, proof of synergy typically has not been demonstrated for symptom severity when the mixed virus infection was not lethal. We demonstrated synergy in bell pepper plants co-infected with Cucumber mosaic virus (CMV) and Pepper mottle virus (PepMoV) relative to each virus alone for stem height (two of three trials) and aboveground fresh weight (one of three trials) using factorial analysis and Abbott's equation for synergy. This approach allowed affirmation of the type of response (i.e., synergistic rather than antagonistic) and statistical proof of synergy. A detailed evaluation of symptom severity for each viral treatment revealed three phases associated with host plant developmental stages. A numerical symptom severity rating scale was developed and used in each of two equations to demonstrate statistical proof for synergy based on symptom severity for co-infected plants. Virus accumulation in noninoculated leaves was determined by enzyme-linked immunosorbent assay. In singly infected plants, CMV titers declined in mildly symptomatic leaves representing later stages of plant development, but titers increased in similar leaves of co-infected plants. In contrast, PepMoV titers did not differ in singly or co-infected plants.     

地高辛标记的cDNA探针检测烟草花叶病毒、黄瓜花叶病毒及马铃薯Y病毒

 根据已发表的烟草花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和马铃薯Y病毒(Potato virus Y,PVY)的外壳蛋白基因序列,设计特异引物,分别以提取的TMV、CMV和PVY侵染的病叶总RNA为模板,反转录PCR进行体外扩增,分别得到长度为0.44、0.77、0.80 kb的目的片段,并克隆到pGEM-T easy质粒载体上,以构建的重组质粒为模板,用PCR方法合成了相应的地高辛标记的双链DNA探针。以合成的探针通过斑点杂交技术检测烟草病叶总RNA和烟草病叶汁液。TMV、CMV和PVY的3种地高辛探针检测各自感染的烟草病叶总RNA的稀释低限分别为1:1000、1:10000、1:320,检测各自侵染烟草病汁液的最大稀释倍数分别为1:100、1:100、1:10,而每种探针与健康烟草和其它2种病毒的反应均为阴性。     

Effect of Figaren, an Antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants. Received 10 October 2000/ Accepted in revised form 6 February 2001     

黄瓜绿斑驳花叶病毒单克隆抗体的研制

将纯化的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus, CGMMV)制剂免疫BALB/c小鼠,最后一次免疫后第3天取其脾细胞与SP2/0细胞融合,经采用选择性培养基、有限稀释法克隆和间接ELISA方法进行筛选,成功获得了3株分泌CGMMV特异性单克隆抗体的杂交瘤细胞株并分别命名为3C9,3F7,2G10。用ELISA方法对所获得的3个杂交瘤细胞株进行亚型鉴定均为IgG2a,kappa链。 间接ELISA效价测定结果分别为3C9：1.024×107,3F7：2.56×106,2G10：1.28×106。此3株杂交瘤细胞所分泌的单克隆抗体均能与本研究室保存的其他3种不同的CGMMV分离物发生特异性反应,而不与其他3种同属成员病毒 烟草花叶病毒(Tobacco mosaic virus, TMV)、辣椒轻斑驳病毒(Pepper mild mottle virus, PMMoV)、齿瓣兰环斑病毒(Odontoglossum ringspot virus,ORSV) 发生反应。     

Transmissibility of Cucumber mosaic virus by Aphis gossypii Correlates with Viral Accumulation and Is Affected by the Presence of Its Satellite RNA

ABSTRACT Satellite RNAs (satRNAs) are associated with Cucumber mosaic virus (CMV) in tomato, most often causing severe epidemics of necrotic plants, and not associated with specific host symptoms. Laboratory studies on virus transmission by the aphid vector Aphis gossypii were performed to better understand the dynamics of field populations of CMV. The presence of satRNAs correlated with lower concentrations of virus in infected plants and with a decrease in the efficiency of transmission from satRNA-infected plants. Both the concentration of virus in CMV-infected tomato and the efficiency of transmission varied more extensively with nonnecrogenic satRNAs than with necrogenic satRNAs. A negative effect of satRNAs on virus accumulation can account, in part, for a decrease in the field transmission and recovery of CMV + satRNAs. Aphids behaved differently and probed less readily on plants infected with CMV + necrogenic satRNAs compared with plants containing non-necrogenic satRNAs. Aphid-mediated satRNA-free CMV infections were observed in test plants when aphids were fed on source plants containing CMV + nonnecrogenic satRNA; no comparable satRNA-free test plants occurred when aphids were fed on source plants containing necrogenic satRNAs. These results indicate that factors associated with transmission can be a determinant in the evolution of natural populations of CMV and its satRNA.     

烟草品种对烟草花叶病毒和黄瓜花叶病毒的抗性鉴定

 2010-2011年采用大田人工接种鉴定的方法,对生产中大面积推广使用的24份烟草品种进行了烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）的抗性鉴定。结果表明,供试品种对TMV和CMV的抗病性存在较大差异,对TMV表现抗病的有Coker86、吉烟5号、Coker176、CV87、辽烟8号、CV91、中烟90等7份材料;表现中抗的有秦烟98、双抗70、C151等3份材料;表现中感的有秦烟96、G80、金星6007、龙江981、K326、秦烟201、NC89等7份材料;表现感病的有G28、云烟97、净叶黄、红花大金元、RG11、云烟85、云烟87等7份材料。对CMV表现抗病的有Coker86、龙江981、C151、秦烟201、云烟87等5份材料;表现中抗的材料是金星6007;表现中感的有CV91、RG11、Coker176、中烟90、K326、红花大金元、净叶黄、G80、G28等9份材料;表现感病的有秦烟98、云烟85、秦烟96、NC89、双抗70、云烟97、CV87、辽烟8号、吉烟5号等9份材料。其中兼抗TMV和CMV两种病毒病的材料有2份,分别是Coker86和C151。同时研究还发现,抗病性不同的烟草品种在受到病毒危害以后,对烟叶的产量和品质的影响也不同。明确了中国24个烟草品种的抗病性水平,为抗病品种的利用与品种合理布局提供科学依据,同时为烟草抗病毒病育种的亲本选择提供抗源信息。     

葫芦种子传黄瓜绿斑驳花叶病毒的检测*

从感染黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus, CGMMV）的葫芦植株上收取种子,通过苗期症状观察法、双抗体夹心酶联免疫吸附法（DAS-ELISA）、免疫捕获反转录PCR（IC-RT-PCR）法测定葫芦种子的带毒情况,并用生物学接种方法测定葫芦种子携带病毒的侵染活性。苗期症状观察法结果表明,199株幼苗有2株表现花叶斑驳症状,种子传毒率为1.01%;而利用DAS-ELISA和IC-RT-PCR法随机检测30粒葫芦病株种子,CGMMV检出率为100%。种子各部位携带的CGMMV接种葫芦表现典型的花叶斑驳症状,表明葫芦种子携带的CGMMV具有侵染活性。DAS-ELISA检测葫芦种子CGMMV的灵敏度为1/5120种子研磨液。     

Evolution of Virulence in Natural Populations of the Satellite RNA of Cucumber mosaic virus

From 1986 to 1992, an epidemic of tomato necrosis caused by Cucumber mosaic virus (CMV) plus CMV satellite RNAs (satRNAs) occurred in eastern Spain. From 1989 onward, the frequency of tomato necrosis di-minshed, and it almost completely disappeared after 1991. Analyses of plants infected with CMV and with CMV satRNA and of the phenotype (necrogenic or nonnecrogenic for tomato) induced by some CMV satRNA variants, showed that the disappearance of tomato necrosis was due to changes in the genetic composition of the satRNA population (i.e., to its evolution toward decreased virulence). Analysis of components of the fitness of satRNA variants, necrogenic or nonnecrogenic for tomato, showed that necrogenic and nonnecrogenic variants did not differ in infectivity or in their accumulation level in tomato and that they represented the same fraction of encapsidated RNA. Other fitness components were positively correlated with the greater virulence of necrogenic variants, in that they were favored in mixed infections with nonnecrogenic variants and were more effectively passed into CMV progeny than were nonnecrogenic variants. On the other hand, necrogenic CMV satRNA variants caused a more pronounced depression in the accumulation of CMV than did nonnecro-genic variants, which could affect the efficiency of aphid transmission. Thus, the evolution of virulence in the CMV satRNA population can be explained by trade-offs between factors that determine virulence and factors that affect transmission, as predicted by theoretical models on the evolution of virulence in parasites.     

黄瓜绿斑驳花叶病毒北京和山东分离物的生物学测定及其基因组比较

为揭示黄瓜绿斑驳花叶病毒的分子流行学特征,通过RT-PCR和cDNA克隆技术获得并分析了从北京甜瓜和山东西瓜上分离的2个黄瓜绿斑驳花叶病毒(CGMMV)分离物CGMMV-Beijing和CGMMV-Shandong的全序列.结果表明CGMMV-Beijing和CGMMV-Shandong的基因组分别由6423和6427个核苷酸组成,包括5'及3'端非编码区和4个开放性的阅读框,分别编码129kDa和186kDa复制相关蛋白、29kDa的移动蛋白及17.4kDa的外壳蛋白.寄主范围测定显示在供试的6科11种植物中CGMMV-Beijing和CGMMV-Shandong均可侵染葫芦科的葫芦、南瓜和西瓜以及茄科的本氏烟和藜科的苋色藜,不侵染供试的其它植物.CGMMV-Beijing和CGMMV-Shandong基因组的核苷酸同源性为99.5%,186kDa蛋白的核酸和氨基酸同源性分别为99.7%和99.8%,移动蛋白的核酸和氨基酸同源性分别为99.3%和98.1%,外壳蛋白的核苷酸同源性为100%.多序列比对分析结果显示,CGMMV的各分离物之间的分子差异并不明显,核苷酸同源性均在98%以上.将CGMMV-Beijing和CGMMV-Shandong与侵染葫芦科植物的烟草花叶病毒属其它成员比较,结果显示其亲缘关系较远,基因组的核苷酸同源性介于56.7%～59.6%,外壳蛋白的氨基酸同源性在44.7%～54.0%之间.综合分析显示,CGMMV分离物之间的差异并不明显,可能具有共同的侵染来源.     

黄瓜绿斑驳花叶病毒低风险参照物质的研制

采用RT-PCR方法扩增黄瓜绿斑驳花叶病毒的外壳蛋白基因与3,非编码区,并将其构建到马铃薯X病毒(PVX)载体中.重组质粒经线性化及体外转录后接种烟草,获得含有病毒外壳蛋白基因的感病植株.感病组织可用于分子检测的质控,也可作为毒源来繁殖阳性参照物质.基于PVX载体制备的参照物质可降低检疫性病毒的生物安全风险,尤其对稳定性强、可造成严重经济损失的高风险病毒的检疫更具实用价值.     

Shoot meristem tissue of tobacco inoculated with Cucumber mosaic virus is infected with the virus and subsequently recovers from infection by RNA silencing

Distribution of Cucumber mosaic virus (CMV) in shoot meristem tissue of CMV-inoculated tobacco was successively analyzed with immunohistochemical microscopy and in situ hybridization. CMV signals were detected in the tissue at 7 days postinoculation (dpi), but then they decreased and disappeared after 14dpi. Detailed observation confirmed CMV invasion of shoot apical meristem at 6–8dpi. Short interfering RNA corresponding to CMV RNAs was first detected at 7dpi and was detected up to 24dpi. These results suggest that the shoot meristem tissue is infected with CMV but subsequently recovers from the infection by RNA silencing.     

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。     

两种类型黄瓜花叶病毒卫星RNA的序列、结构以及对辅助病毒的致病性调控分析

黄瓜花叶病毒(Cucumber mosaic virus,CMV)卫星RNA(satellite RNA,satRNA)通常影响CMV的致病性。本研究设计了检测satCMV的简并引物,通过RT-PCR从山东省泰安市3种自然发病寄主中检测到satCMV,发现了两类长度有明显差异的satCMV:白菜与萝卜中克隆的satCMV为339个核苷酸,二者的核苷酸一致率为99.41%;烟草中克隆的satCMV为383个核苷酸,与白菜和萝卜中satCMV的核苷酸序列一致率均为71.06%。系统进化分析表明这两类长度有明显差异的satCMV亲缘关系较远。两种类型的satCMV与CMV-Fny混合接种对CMV致病症状的调控存在明显差异。利用m Fold软件分析了两类不同长度的CMV卫星RNA基因组及其互补链的RNA结构特征,表明两类sat CMV与CMV互作的活跃度以及核心结构域存在差异。两类satCMV全基因组的克隆以及结构分析为研究不同类型satCMV与CMV的互作及其对CMV致病性的影响奠定了基础。     

黄瓜绿斑驳花叶病毒对西瓜产量、品质及种子带毒的影响

研究了西瓜不同时期接种黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)后对其生长、产量、品质和种子带毒的影响。西瓜植株接种CGMMV越早其生长受到抑制越明显,砧木期、嫁接后定植前、定植期、压蔓期、授粉期各处理的株高、叶片数、叶片长与对照相比差异极显著(p0.01),而结瓜以后接种CGMMV对西瓜生长状况无显著影响。压蔓期前3个生育期接种该病毒,西瓜产量损失分别为56.49%、55.48%和51.14%。而压蔓期以后接种该病毒,西瓜产量损失与对照相比仍分别降低17%、13.1%和14.35%。关于倒瓤情况,压蔓期前接种该病毒可导致西瓜果实100%倒瓤,压蔓期、授粉期接种处理西瓜倒瓤率分别为48%和34%,即使结瓜后再接种病毒西瓜倒瓤率仍高达18%。血清学检测表明,压蔓期前接种的西瓜其果肉和种子均携带CGM-MV,带毒率达100%;结瓜后再接种CGMMV西瓜种胚和果肉的带毒率仍然高达64%。检测种胚和果肉病毒含量表明,接种时期越早病毒含量越高,但结瓜后接种仍有较高的病毒含量,种胚和果肉的病毒含量分别达5.98 mg/mL和5.64 mg/mL。     

Amino acid 129 in the coat protein of Cucumber mosaic virus primarily determines invasion of the shoot apical meristem of tobacco plants

We previously reported that a strain of Cucumber mosaic virus (Pepo CMV) invaded the shoot apical meristem (SAM, tunica corpus) of tobacco plants at 6–8 days postinoculation (dpi), contrary to earlier observations. To identify a viral factor determining the ability to invade the SAM, we inoculated plants with two other CMV strains, MY17 and Y, and tested the three strains in this study. Immunohistochemical microscopy revealed that MY17 CMV invaded the SAM at 7 dpi, the same as Pepo CMV, but Y CMV did not, even at 21 dpi. Using RNA pseudorecombinants between Pepo and Y CMV, we found that Pepo RNA 2 affected the rate of SAM invasion, and Pepo RNA 3 was required for successful SAM invasion. Inoculation with RNA 1 and RNA 2 from Y CMV and RNA 3 containing the chimeric coat protein (CP) gene between Pepo and Y CMV or a Y RNA 3 point mutant containing a Ser-to-Pro substitution at position 129 in CP (Y129P) revealed that amino acid 129 of CP is the determinant for successful SAM invasion. The rate of SAM invasion of the pseudorecombinants and Y129P was consistent with the efficiency of cell-to-cell movement in the inoculated leaves, implying that SAM invasion by CMV strains may be due to efficient cell-to-cell movement.     

Seed disinfection treatments do not sufficiently eliminate the infectivity of Cucumber green mottle mosaic virus (CGMMV) on cucurbit seeds

Tobamoviruses induce crop diseases that are responsible for significant economic losses around the world. Like other tobamoviruses, Cucumber green mottle mosaic virus (CGMMV) forms highly stable particles that can persist for long periods on plant debris, in soil and on seed surfaces. These particles serve as a primary source of infection, infecting seedlings from which the virus can then be mechanically transmitted to other neighbouring plants. Contaminated seeds also provide a route for the movement of the virus between countries and its introduction into new areas. Effective seed disinfection treatments and the use of uncontaminated seed may reduce the global prevalence of this virus. Several treatments based on the use of heat or chemicals have been reported to effectively eliminate CGMMV and other tobamoviruses from seeds. An evaluation of these treatments on highly contaminated seed lots revealed inconsistent results, which encouraged the construction of a more accurate detection method that combines morphological, serological, molecular and biological analyses in one protocol. The detection of viable (infectious) viral particles in seed treated with heat, trisodium phosphate or a combined treatment, indicates that these treatments are insufficient. The serological detection of CGMMV in the inner parts of infected seeds provides a possible explanation for the inconsistent efficacy of these treatments.