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.     

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.     

An Analysis of Host Adaptation and Its Relationship with Virulence in Cucumber mosaic virus

ABSTRACT The host range of a pathogen can have special consequences on its evolution and the evolution of its virulence. For generalists, adaptation to different hosts may be conditioned by different trade-offs in the pathogen's life history and be affected by evolutionary processes that shape pathogen populations. We have examined adaptation of Cucumber mosaic virus (CMV) to different hosts, and analyzed the relationship between host adaptation and virulence. For this, six CMV isolates from central Spain from three different hosts were compared for the ability to multiply and to affect host growth. These analyses were done before and after an experimental evolution process consisting of 10 serial passages in the original host of the isolate. The differential capacity to infect different hosts was compatible with host adaptation. However, the capacity to multiply in different hosts did not provide evidence of host adaptation and was not improved after 10 passages, suggesting that fitness of the natural population of CMV was at, or near to, its maximum. No relationship was found between capacity of multiplication and virulence in any of the three different hosts. These results suggest that the "trade-off" model for the evolution of virulence may not apply to CMV.     

Transmission efficiency of Cucumber mosaic virus by aphids associated with virus epidemics in snap bean

Cucumber mosaic virus (CMV) is a major component of the virus complex that has become more pronounced in snap bean in the midwestern and northeastern United States since 2001. Multiple-vector-transfer tests were done to estimate the CMV transmission efficiencies (p) of the main aphid species identified in commercial snap bean fields in New York and Pennsylvania. The four most efficient vectors (p > 0.05) were Aphis gossypii, A. glycines, Acyrthosiphon pisum, and Therioaphis trifolii, which were all significant species in the migratory aphid populations in snap bean. Moderately efficient vectors (0.01 < p < 0.04) were A. spiraecola, A. craccivora, Macrosiphum euphorbiae, and Rhopalosiphum maidis. Poor vectors (p < 0.01) included A. fabae, Nearctaphis bakeri, and Myzus persicae. Only one species, Sitobion avenae, failed to transmit CMV in replicated tests. Estimates of p were consistent between different clones of the same aphid species and among three different field isolates of CMV tested. Single-vector-transfer test results for a subset of the species supported those obtained via the multiple-vector-transfer approach. Our results are consistent with the notion that A. glycines is a major vector of recent CMV epidemics in snap bean, but that species is only one of several that are involved.     

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。     

Identification and Subgrouping of Cucumber mosaic virus with Mouse Monoclonal Antibodies

ABSTRACT Using a mixture of isolates of Cucumber mosaic virus (CMV) from subgroups I and II as immunogens, 20 mouse hybridoma cell lines secreting monoclonal antibodies were produced. A reliable method for efficient detection and accurate subgrouping of CMV isolates has been developed. Tests with 12 well-characterized strains of CMV and other cucumoviruses demonstrated the presence of epitopes that were virus and subgroup specific. Analyses of 109 accessions of CMV isolates collected from various parts of the world revealed 70% were subgroup I, with 20% identified as subgroup II. Seven isolates (6%) did not react with group-specific antibodies but did react with antibodies that recognized all CMV isolates. Differential reactions among isolates suggested a total of 10 epi-topes were recognized. The antigenic diversity among subgroup II CMVs was greater than for the subgroup I isolates, even though fewer subgroup II isolates were tested.     

Cucumber mosaic virus Establishes Systemic Infection at Increased Temperature Following Viral Entrance Into the Phloem Pathway of Tetragonia expansa

ABSTRACT A potential regulatory site for Cucumber mosaic virus (CMV, pepo strain) movement necessary to establish systemic infection was identified through immunological and hybridization studies on Tetragonia expansa, which was systemically infected by CMV at 36 degrees C but not at 24 degrees C. In inoculated leaves, cell-to-cell movement of CMV was enhanced at 36 degrees C compared with that observed at 24 degrees C. CMV was distributed in the phloem cells of minor veins as well as epidermal and mesophyll cells at both 36 and 24 degrees C. CMV was detected in the petioles of inoculated leaves, stems, and petioles of uninoculated upper leaves at 36 degrees C, whereas CMV was detected only in the petioles of inoculated leaves and in stems at 24 degrees C. CMV moved into the phloem and was transported to the stem within 24 h postinoculation (hpi) at 36 degrees C. However, it did not accumulate in the petioles of the upper leaves until 36 hpi. In petioles of inoculated leaves at 24 degrees C, CMV was detected in the external phloem but not in the internal phloem. From these results, we conclude that systemic infection is established after viral entrance into the phloem pathway in T. expansa at 36 degrees C.     

Presence of Brome mosaic virus in Barley Guttation Fluid and Its Association with Localized Cell Death Response

ABSTRACT Water exits from inside the leaf through transpiration or guttation. Under conditions to promote guttation, surface fluid (guttation fluid) from Brome mosaic virus (BMV)-infected barley, wheat, and maize plants was analyzed for the presence of the virus by biological and serological assays. We also investigated the route by which BMV exited infected cells to the intercellular space of the barley leaf. BMV was detected in guttation fluid from systemically infected barley leaves when the initial viral symptoms were observed on these leaves. The virus was also detected in guttation fluid from systemically infected wheat leaves, but not in maize leaves showing either systemic necrosis or chlorotic streaks. Interestingly, in BMV-infected barley leaves, but not in maize leaves showing chlorotic streaks, cell death occurred within and adjacent to veins. Staining of xylem and phloem networks in infected barley leaves with fluorescent dyes showed that xylem, and to a lesser extent phloem, were severely damaged and thus became leaky for dye transport. No such damage was observed in BMV-infected maize leaves showing chlorotic streaks. We propose that in infected barley leaves, BMV exits from damaged vein cells (especially the xylem elements), accumulates in intercellular spaces, and then reaches the surface of the leaves through stomata during guttation or transpiration. In nature, BMV may be carried to adjacent plants and cause infection by movement of vertebrate and invertebrate vectors among infected plants exuding guttation fluid.     

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.     

Selective transmission of the seedling yellows-type genotypic variants of Citrus tristeza virus by Aphis gossypii in northern Iran

Citrus tristeza virus (CTV) has existed in northern Iran for more than five decades. The long-time interaction of different virus genotypes with Aphis gossypii, as the only aphid vector of CTV in northern Iran, has led to the emergence of highly virulent subpopulations, among others, in the established foci. Here, we studied the population structure of the originally established CTV isolates present in Satsuma mandarin (Citrus unshiu) trees imported from Japan, and subisolates thereof, formed following experimental transmission by A. gossypii, as well as those evolved through natural transmission by this aphid species in the groves. Symptoms of the naturally spread and the experimentally aphid-transmitted isolates were similar to those of the Satsuma CTV source isolates for all indicator plants except for sour orange (Citrus aurantium) and grapefruit (Citrus paradisi), with the aphid-transmitted isolates additionally inducing severe seedling yellows and stunting in these two indicators. Studies on the population heterogeneity of these isolates through comparison of their single-strand conformational polymorphism profiles and nucleotide sequences of the 25 kDa capsid protein gene from the predominant haplotypes, and dot-blot hybridization signals, revealed the presence of two major T36- and SY568- (or NUagA-) like genotypes along with a minor poorly characterized one in the originally infected Satsuma trees; in contrast, only a certain genomic variant having the highest similarity to the isolate SY568 (and NUagA) was predominant both in the naturally infected trees and in those infected experimentally by A. gossypii. It seems that transmission by A. gossypii to sweet orange (Citrus sinensis) has led to the preponderance of the CTV genomic variants inducing severe seedling yellows in northern Iran.     

2种蚜虫取食诱导棉花对棉长管蚜与棉蚜取食行为的影响

为研究棉长管蚜Acyrthosiphon gossypii 与棉蚜 Aphis gossypii 对蚜虫取食诱导棉花的取食适应性, 揭示取食诱导下两种蚜虫的取食竞争机制, 采用刺探电位图谱技术(electrical penetration graph, EPG)测定在健康棉花、棉长管蚜或棉蚜诱导的棉花上取食6 h的棉长管蚜和棉蚜的16个重要参数, 比较两种蚜虫的取食行为。研究发现:1) 取食健康棉花时, 棉蚜在木质部(G波)的取食持续总时间长于棉长管蚜，棉蚜在木质部的取食能力大于棉长管蚜，但二者在韧皮部的取食能力相差不大。2) 取食棉长管蚜诱导的棉花时, 棉蚜取食韧皮部(E2波)持续的总时间为(2 531.33±60.86)s, 显著大于棉长管蚜E2波持续总时间［(2 196.00±98.91)s］;取食棉蚜诱导的棉花时, 棉蚜取食韧皮部、木质部持续的总时间显著长于棉长管蚜(P＜0.05), 说明棉蚜对于棉蚜诱导棉花的取食耐受性大于棉长管蚜。棉蚜对两种蚜虫取食诱导棉花的适应性更强, 更易在竞争中取得优势。棉长管蚜与棉蚜取食棉长管蚜或棉蚜诱导棉花的F波持续总时间长于健康棉花, 说明经取食诱导的棉花具有一定的抗虫性。     

A Variant of Cucumber mosaic virus Is Restricted to Local Lesions in Inoculated Tobacco Leaves with a Hypersensitive Response

A variant of Cucumber mosaic virus, CMV(Y/GM2), was isolated from a tobacco plant with mild green mosaic symptoms that was regenerated in vitro from a yellow strain of CMV [CMV(Y)]-infected tobacco leaves by tissue culture. CMV(Y/GM2) has two amino acid substitutions at 36 and 111 positions in the coat protein encoded on RNA3. CMV, assembled by mixing in vitro transcribed CMV(Y) RNA1 and RNA2 plus infectious RNA3 transcribed in vitro from cDNA to RNA3 of CMV(Y/GM2), was prepared and designated as CMV(Y/GM2)tr. When tobacco (Nicotiana tabacum cv. Xanthi nc) plants were inoculated with CMV(Y/GM2)tr, large necrotic local lesions in which the virus was localized, developed on the inoculated leaves. This host response unique to CMV(Y/GM2)tr was similar to the hypersensitive response (HR), which is a common resistance response to avirulent pathogens and was observed in five cultivars of Nicotiana tabacum and eight Nicotiana species. The revertant virus, however, accumulated to quite different levels in the various hosts. CMV(Y/GM2)tr induced pathogenesis-related 1 (PR-1) protein accumulation and systemic acquired resistance (SAR) which were generally observed in the HR. However, when tobaccos were inoculated with CMV(S36P)tr and CMV(V111I)tr, which have an amino acid substitution at either the 36 or 111 position in the coat protein of CMV(Y), respectively, CMV(S36P)tr was restricted to the primary infection site without necrotic local lesion formation and PR-1 protein and SAR induction. CMV(V111I)tr, however, systemically spread and induced mild green mosaic symptoms, while the host had the HR to CMV(Y/GM2)tr. The localization of CMV(Y/GM2)tr at the primary infection site may not only be caused by the HR, but also by the restriction of virus systemic movement resulting from the amino acid substitution at position 36 in the coat protein of CMV(Y). Received 15 December 1999/ Accepted in revised form 18 April 2000     

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。     

用一步PCR法同时检测香蕉束顶病毒和香蕉花叶心腐病毒

采用改进的CTAB方法同时提取BBTV和CMV侵染的香蕉叶片的总核酸,然后同时加入BBTV和CMV的特异性引物进行扩增来检测BBTV和CMV。结果显示,虽然BBTV和CMV属于不同核酸性质的病毒,但可以通过一步RT—PCR的方法同时被检测出来,为实际检测香蕉组培苗中的这2种最主要的病毒提供了一个有效的检测方法。     

利用黄瓜花叶病毒M和Fny株系 的假重组体分析致病关键因子

 用RT-PCR扩增黄瓜花叶病毒M株系（CMV-M）全长基因组cDNA,成功构建CMV-M RNA2和RNA3侵染性克隆后,与CMV-Fny基因组RNA交换得到3个假重组型病毒 （F1M2F3、F1F2M3、F1M2M3）。用F1M2F3、F1F2M3、F1M2M3分别侵染白肋烟,产生坏死环斑、轻微绿斑驳、明脉、黄白化和叶尖线性化等症状。根据假重组型病毒和野生型病毒的表观症状,分析引起各种症状的关键因子,初步判定：CP基因是诱导花叶症状的关键因子,CMV-Fny RNA2是诱导叶尖线性化的关键因子,CMV-M RNA2是诱导叶尖坏死斑关键因子。实时荧光定量PCR结果显示：野生株CMV-M、CMV-Fny和假重组体F1M2F3、F1F2M3、F1M2M3侵染烟草后引起的症状差异与病毒基因组RNA累积没有直接关系。     

西瓜感染黄瓜绿斑驳花叶病毒后糖的变化与倒瓤关系的研究

 By high performance liquid chromatography (HPLC), the contents of glucose, fructose and sucrose, and the sweetness were analyzed in watermelon inoculated with Cucumber green mottle mosaic virus (CGMMV).The relationship between sugar change and blood-flesh of watermelon was determined. The results indicated that the content of glucose was apparently increased compared to that of the control before maturity (within 28 days after pollination), but reduced to 24.8% of the control after maturity (at 35 days after pollination). The content of fructose was higher than that of the control within 14 days after pollination, and then decreased with a significant difference. In all cases, the content of sucrose increased with the growth of watermelon. However, compared with the control, the sucrose content of watermelon inoculated with CGMMV was lower. The ratios of glucose, fructose and sucrose in the total sugar were abnormal. Coincidence with the changes of the total sugar, the fruit sweetness before maturity was higher than that of the control, whereas decreased sharply after maturity (lower than that of the control). The inner pulp of the mature fruit appeared to be water-soaked and dirty red with no edibility. In a word, after inoculation with CGMMV, the changes of sugars and sweetness affected the watermelon quality.