Coat protein-mediated transgenic resistance in tomato against a IB subgroup Cucumber mosaic virus strain

Transgenic tomato plants containing the coat protein (CP) gene of Cucumber mosaic virus (CMV) of subgroup IB were developed through Agrobacterium-mediated transformations. The progenies of transgenic plants showed the presence of transgene, its expression and translation of 26 KDa CP. The T₁ and T₂ generation plants were evaluated for resistance against challenge inoculations by a homologous strain of CMV. Visual observations of challenged transgenic plants categorized them into resistant, tolerant and susceptible as compared with untransformed control plants. Out of 33 plants of the T₁ generation, 36.3% showed resistance and remained symptomless throughout their life, 48.4% showed tolerance which developed delayed symptoms of mild mosaic, and 15.1% showed susceptibility to CMV which developed severe systemic mosaic and leaf distortion symptoms after 30?days of virus challenge. Out of 120 plants of the T₂ generation, 60% showed resistance, 26.6% were tolerant and only 13.3% were found susceptible to challenge inoculations of CMV. Resistant transgenic plants also showed less CP accumulation in systemic upper leaves as compared with challenged untransformed plants. In this study, CP of a CMV subgroup IB strain has demonstrated a significant level of resistance in transgenic tomato plants against the CMV strain. The strategy may be applied for better quality and productivity of tomato crops.     

我国部分地区常见农作物上黄瓜花叶病毒分离物核酸多样性分析

为明确我国黄瓜花叶病毒株系分化及系统进化基本情况,从湖南、新疆、青海和海南4省区采集1 367个样品对其进行酶联免疫和RT-PCR检测,并对分离获得的15个黄瓜花叶病毒(Cucumber mosaic virus,CMV)纯化分离物CP、MP、2b核苷酸序列进行相似性和进化树分析及生物学性状比较。结果表明,辣椒、龙葵和黄瓜的CMV阳性检出率较高,分别为54.13%、29.19%和18.46%。进化树分析显示CMV-Q5与CMV亚组II的亲缘性较高;CMV-N7为新发现的重组株系,其CP、2b基因属于CMV亚组IB,MP基因却属于CMV亚组II;其余13个分离物均属于CMV亚组IB。CMV-N7和CMV-Q5在系统寄主心叶烟和枯斑寄主苋色藜上引发的症状相似,但比对照株系CMV-P3613(IB)的发病时间要晚1~2 d,系统花叶较温和,枯斑较小。表明在以上4省区常见农作物上广泛流行的CMV存在分子变异。     

Phylogeny of Egyptian isolates of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV) and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) infecting <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.     

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     

Transgenic resistance to cucumber mosaic virus in tomato: blocking of long-distance movement of the virus in lines harboring a defective viral replicase gene

ABSTRACT Tomato breeding lines were transformed with a defective replicase gene from RNA 2 of cucumber mosaic virus (CMV). A total of 63 transformants from five tomato genotypes were evaluated for resistance to CMV strains. The responses of R1 transgenic offspring fit into three categories: fully susceptible lines (44%), fully resistant lines (8%), and an intermediate-type mixture of susceptible and resistant seedlings in variable proportions (48%). Further characterization of the response of two highly resistant lines was performed by mechanical inoculation, aphid transmission, or grafting experiments. No virus was detected in noninoculated leaves from these lines, although a low level of virus accumulated initially in the inoculated leaf. The homozygous R2 plants and further generations that were evaluated (up to R5) showed resistance to the Fny-CMV strain, two Israeli isolates tentatively classified as subgroup IA, and K-CMV (a representative of subgroup IB). These lines were partially resistant to LS-CMV (a representative of subgroup II) when a high-virus-titer inoculum was used. Expression of the viral transgene was verified in these lines; however, the expected translation product was not detectable. In grafting experiments, we demonstrated that CMV virions were blocked in their ability to move from infected rootstocks of nontransformed tomato or tobacco into the transgenic scions. Interestingly, virions could not move through a transgenic intersection into the upper scion. These results provide an additional indication that replicase-mediated resistance affects long-distance movement.     

Molecular characterization of Cucumber mosaic virus strain causing severe mosaic disease on petunia in India

Severe mosaic, yellowing and stunting symptoms were observed on petunia (Petunia hybrida L.) growing in pots at NBRI and in various gardens of Lucknow, India. The association of Cucumber mosaic virus (CMV) with the mosaic disease was detected based on positive bioassay on susceptible hosts, isometric cored virus particles of ~28?nm during electron microscopic observations in leaf dip preparations and positive amplification of expected size (~650?bp) during RT-PCR using coat protein gene specific primers. Further, the complete RNA 3 genomic fragment of virus isolate was amplified by RT-PCR using RNA 3 specific primers. The obtained amplicons of ~2.2 Kb were cloned and sequenced. The analysis of sequence data of RNA 3 revealed highest sequence identities (96%) with several CMV strains which belong to subgroup IB. The virus isolate also showed closest phylogenetic relationships with banana strain of CMV of subgroup IB (Acc. EF178298) reported from India. To the best of our knowledge, we report the first molecular characterization of CMV strain of subgroup IB causing severe mosaic disease on petunia in India.     

Characterization of symptom determinants in two mutants of cucumber mosaic virus Y strain,causing distinct mild green mosaic symptoms in tobacco

Two mutants of Cucumber mosaic virus, CMV(Y/GM1) and CMV(Y/GM2), which induced mild green mosaic symptoms in tobacco, were isolated from plants regenerated from tobacco leaves with yellow mosaic symptoms originally infected with the yellow strain of CMV [CMV(Y)]. Although the appearance of mild green mosaic symptoms in tobacco infected with CMV(Y/GM2) was unstable, CMV(Y/GM3) derived from CMV(Y/GM2) reproducibly induced mild green mosaic symptoms in tobacco similar to CMV(Y/GM1). A comparison of the deduced amino acid sequences of the coat proteins of CMV(Y), CMV(Y/GM1) and CMV(Y/GM3), showed single amino acid substitutions from Thr to Ile at position 124 in the CMV(Y/GM1) coat protein and from Val to Ile at position 111 in the CMV(Y/GM3) coat protein. When the amino acid at the 124 or 111 position in the CMV(Y) coat protein was changed to Ile at the cDNA level, CMV RNA3 transcribed in vitro from each cDNA induced mild green mosaic symptoms in tobacco after inoculation with in vitro transcribed CMV(Y) RNA1 and RNA2. The results indicated that amino acids at positions 111 and 124 in the coat protein were responsible for the phenotypic changes caused by the two CMV isolates.     

Evolutionary Characterization of Two Lily Isolates of Cucumber mosaic virus Isolated in Japan and Korea

We analyzed the evolutionary histories of two lily strains of Cucumber mosaic virus (CMV) isolated in Japan and Korea (HL- and Ly2-CMVs). They share common biological characteristics in that their host ranges are very restricted perhaps from a unique adaptation to lily plants. Although HL and Ly2 were isolated independently from different lily species in separate countries, their RNA3 sequences had a very high sequence similarity (97%). The evolutionary relationships between the two isolates were characterized by comparing their phylogenetic trees for the 3a and CP genes. The two lily CMVs always formed a distinct cluster within subgroup IB in 3a, but within IA in CP. Together, the phylogenetic tree topology and the sequence identity between the two lily CMVs suggest that they evolved from a common progenitor. Received 5 November 2001/ Accepted in revised form 11 January 2002     

Molecular characterization of a Cucumber mosaic virus isolate associated with mosaic disease of banana in India

Severe mosaic accompanied by leaf and fruit deformation symptoms was observed on banana plants growing in three banana farms of Uttar Pradesh, India. The disease incidence was approximately 18–25% at these locations during the three successive years from 2005 to 2007. The occurrence of Cucumber mosaic virus (CMV) was initially detected by bioassay, electron microscopic observations, Western blot immunoassay and RT-PCR. For molecular identification of virus, the RNA 1a, RNA 2b and RNA 3 genomic fragments were amplified by RT-PCR and sequenced. The sequence analysis of these genomic fragments revealed its highest identities and close relationships with Indian strains of CMV of subgroup IB; therefore, virus associated with the mosaic disease of banana was identified as an isolate of CMV of subgroup IB. In the limited reports existing from India, which provided preliminary serological or only coat protein-based identification of CMV infecting banana but the comprehensive studies were lacking. In the present communication, we present a detailed biological, serological and molecular characterization of CMV-Banana for the first time from India.     

Survey of viruses infecting open‐field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

The prevalence of viruses in pepper crops grown in open fields in the different agro‐ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed. The molecular diversity of CMV and PVMV were analysed from partial genome sequences. Despite the low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to subgroup IA and the other to reassortants between subgroups IA and IB. RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates, whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg‐ and coat protein‐coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance becomes more widely deployed.     

<Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> isolated from Amazon lily (<Emphasis Type="Italic">Eucharis grandiflora</Emphasis>)

Cucumber mosaic virus (CMV) was isolated from a mosaic diseased plant of Eucharis grandiflora. The virus caused mosaic symptoms on leaves and slight distortion of flower petals in E. grandiflora by either mechanical or aphid inoculation. The virus was identified as a strain of CMV subgroup I from its biological and serological characteristics.     

RNA 2 of Cucumber Mosaic Virus Subgroup I Strain NT-CMV is Involved in the Induction of Severe Symptoms in Tomato

A selection of cucumber mosaic virus (CMV) subgroup I strains originating from Asia and Fny-CMV isolated in USA were studied for their interaction with tomato plants. All strains caused mosaic, fernleaf expression and stunting of tomato plants. Symptom expression was relatively mild after infection with Fny-CMV, T-CMV, Le-CMV and MB-CMV, whereas strains PRC-CMV, NT-CMV and K-CMV caused more severe symptoms. Biologically active clones of NT-CMV RNAs 2 and 3 were generated to construct pseudorecombinant viruses with Fny-CMV to map the symptom determining RNA. The pseudorecombinant FNF-CMV (RNAs 1 and 3 from Fny-CMV, RNA 2 from NT-CMV) showed a similar phenotype on tomatoes to those caused by NT-CMV, whereas FFN-CMV (RNAs 1 and 2 from Fny-CMV, RNA 3 from NT-CMV) induced symptoms comparable to Fny-CMV. The data indicate that CMV RNA 2 of NT-CMV is involved in the induction of severe symptoms in tomato plants.     

Complementation of CMV Subgroup IA Strains in Replicase-mediated Resistant Tobacco Plants after Co-inoculation with Different Cucumoviruses

Replicase-mediated tobacco plants are highly resistant to the Fny strain of Cucumber mosaic virus (CMV) and closely related subgroup IA strains. Two of these subgroup IA strains, Fny- and M-CMV, were co-inoculated with different resistance breaking cucumoviruses to nontransformed and transformed tobacco plants. RT-PCR analyses of single CMV RNAs were performed to study potential complementation of the subgroup IA strains by the resistance breaking cucumoviruses. After co-inoculation of M-CMV with PII-CMV, RNAs 1, 2 and 3 from M-CMV were detected in systemically infected leaves of control plants, whereas in noninoculated parts of replicase-mediated resistant plants only M-CMV RNAs 1 and 3 were found. Western blot studies confirmed the expression of M-CMV coat protein after co-inoculation with PII-CMV in leaves of transgenic plants. These plants also exhibited M-CMV typical yellow spots. M-CMV/TAV co-inoculated transgenic plants contained only M-CMV RNA 3, but no M-CMV RNAs 1 and 2. No M-CMV typical yellow spots were observed in these plants. Our data suggest different types of complementation of M-CMV in replicase-mediated resistant plants by PII-CMV and TAV in trans potentially leading to new RNA combinations in transformed plants compared to nontransformed plants.     

A Cucumber mosaic virus isolated from Momordica charantia L.

Momordica charantia L. plants systemically infected with Cucumber mosaic virus (CMV) were found in Oita Prefecture. The virus isolated from the host plant was characterized by biological, serological, and molecular biological methods. The purified virus was used to mechanically inoculate the host and produced green mottle, green mosaic, and/or chlorotic spots in the noninoculated upper leaves of the host. The virus was identified as an isolate of CMV containing genomic RNA3 derived from subgroup IA by several lines of evidence based on electron microscopy, serological detection, host range, symptoms, and the entire nucleotide sequence of RNA3.     

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     

Cucumber mosaic cucumovirus incidence in open‐field tomato in the Olympia area and trap captures of alate aphids*

Cucumber mosaic cucumovirus (CMV) has been the most serious pathogen of tomato in Greece for the last 15 years, causing tomato shrinkage, tomato necrosis and tomato fruit necrosis. In an epidemiological study in Eleia county, one of the main centres of production of processing tomato in Greece and one most affected by CMV, it was confirmed that the virus had an extremely high frequency. Disease frequency and severity was found to have a patchy spatial and temporal distribution at county, zone and locality level, during the years and within 1998, the main year of experimentation (and a disastrous year for CMV). Great variation was found in the trends of infection frequencies during the growing season of 1998 in the 15 experimental fields but all were finally 100% or almost 100% infected. The trends of infection frequency in these 15 fields paralleled total captures of alate aphids by a Rothamsted‐type trap, whereas in one of these fields, with a Moericke‐type trap, these parallel captures were composed almost exclusively of Aphis spiraecola.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

Leonotis nepetaefolia: An important plant virus reservoir in central Mexico

The presence of viruses in the weedLeonotis nepetaefolia in central México is reported from two field surveys.L. nepetaefolia, with viral-like symptoms such as mosaic, leaf deformation and calico, was observed growing next to cultivated fields in the Valley of Atlixco, Puebla, an important agricultural region in Mexico. The viruses harbored by this plant were characterized biologically, serologically and by molecular methods. The viruses detected wereAlfalfa mosaic virus (AMV),Cucumber mosaic virus (CMV), a satellite RNA of CMV (CMV satRNA) andTobacco mosaic virus (TMV). This last one was detected only during the first survey. CMV was the predominant virus found in both surveys, and was associated mostly to mosaic symptom. Phylogenetic analysis based on the coat protein gene sequence of CMV indicated that this isolate belongs to subgroup IA and confirmed that it is a mosaic-inducing isolate, whereas AMV belongs to subgroup II. Finally, CMV satRNA was found to be a non-necrogenic ameliorative variant, both by symptomatology and by phylogenetic analysis. Our results suggest thatL. nepetaefolia is a reservoir for several viruses in central Mexico, and given its wide distribution in several parts of the world, its role as a virus reservoir could be more general. http://www.phytoparasitica.org posting Aug. 31, 2005.     

Induction of systemic resistance against Cucumber mosaic virus by Penicillium simplicissimum GP17‐2 in Arabidopsis and tobacco

The plant growth‐promoting fungus, Penicillium simplicissimum GP17‐2, was evaluated for its ability to induce resistance against Cucumber mosaic virus (CMV) in Arabidopsis thaliana and tobacco plants. Treatment with barley grain inoculum (BGI) of GP17‐2 significantly enhanced fresh weight, dry weight and leaf number of A. thaliana and tobacco plants 6 weeks after planting. Two weeks after CMV inoculation, all plants treated with BGI of GP17‐2 or its culture filtrate (CF) showed a significant reduction in disease severity compared with non‐treated control plants, which exhibited severe mosaic symptoms by the end of the experiment. The enzyme‐linked immunosorbent assay (ELISA) demonstrated that CMV accumulation was significantly reduced in plants treated with GP17‐2 or its CF relative to control plants. Based on RT‐PCR, plants treated with GP17‐2 (BGI or CF) also exhibited increased expression of regulatory and defence genes involved in the SA and JA/ET signalling pathways. These results suggested that multiple defence pathways in A. thaliana and tobacco were involved in GP17‐2‐mediated resistance to CMV, although neither the transgenic NahG line, nor the npr1, jar1 or ein3 mutants disrupted the response in A. thaliana. This is the first report to demonstrate the induction of systemic resistance against CMV by GP17‐2 or its CF.