Recovery from Cucumber Mosaic Virus Infection for ‘Calwonder’ Bell Pepper Plants Does not Counter Negative Impacts on Plant Growth

Cucumber mosaic virus (CMV) accumulation in leaves and stems of infected bell pepper plants at specific symptom stages was evaluated with an emphasis on the transition from full infection to recovery from Cucumber mosaic disease. Four symptom phases occurred in successive order, designated chlorosis (leaves 6–8), mosaic (leaves 9–11/12), leaf distortion (first series of leaves on secondary and tertiary branches) and recovery (progressive recovery with newly emerging leaves in tertiary and younger branches). In situ detection of CMV in leaf tissues revealed widespread occurrence in leaves expressing chlorosis and mosaic symptoms but reduced, localized occurrence in leaves in the recovery phase. Similarly, CMV accumulated to high levels throughout stems expressing chlorosis and mosaic symptoms but with dramatically reduced levels for plants in the recovery symptom phase. Stunting of internodes occurred at all locations above the inoculated leaves by the first expression of systemic symptoms, suggesting an impact on stem growth in response to initial virus invasion of young developing tissues of the stem. Despite the recovery from CMV infection, plant growth was negatively impacted early in the infection process and remained so through the course of the experiment.     

Responses of potato cultivars (Solanum tuberosum) to infection with Cucumber mosaic virus isolated from different host species

Eight isolates of Cucumber mosaic virus (CMV) isolated from seven different host species were tested for their virulence on potato cv. Desirée. Three isolates caused a systemic infection, of which one isolate from Asiatic lily (CMV-P26) and one from cucumber (CMV-J) appeared to be highly virulent, in contrast to the third isolate (CMV-M) that originated from cucumber and caused mild symptoms only. These three isolates were transmitted to 26 additional potato cultivars by mechanical inoculation in a greenhouse. All cultivars were infected with at least one CMV isolate and developed local chlorotic symptoms, but only 17 cultivars (including Desiree) developed primary systemic symptoms including necrosis, mosaic and/or malformation of leaves. Furthermore, in only five cultivars (including Desiree) CMV was transmitted to tubers and was subsequently detected in plants of the first and second vegetative progeny, the secondary symptoms of these plants being severe. The observed phenotypic responses of potatoes to CMV were not associated with the maturity type (early or late) or resistance to other viruses. Results of this study indicate a high level of biological variability among CMV isolates and that infection in potato depends on CMV isolate and potato cultivar.     

Serological detection of Cucumber mosaic virus on some important host crops in the north region of Iran

Plant virus diseases cause major losses in agricultural and horticultural products, especially in tropical and subtropical regions. The first step to manage these diseases is detecting, identifying and determining the pathogen characteristics. Cucumber mosaic disease is one of the most prevalent and damaging plant diseases in the world which is caused by Cucumber mosaic virus (CMV). Each year, this virus causes yield decreasing and substantial economic damages in its host plants worldwide including the north of Iran. In order to study and identify CMV, 935 leaf samples were collected based on typical symptoms from 10 crops (tomato, pea, tobacco, soybean, watermelon, broad bean, squash, cucumber, eggplant and lettuce) in different regions of Golestan and Mazandaran provinces (north of Iran) during 2009–2010. Suspicious samples were analysed by DAS-ELISA and polyclonal antibodies. The results showed that 275 samples (29.4%) were infected by CMV. Between these hosts, the highest and the lowest CMV infection was associated to watermelon (62.44%) and lettuce (0%), respectively. Among sampling locations, Behshahr (100%) and Minoodasht (3.47%) showed the maximum and minimum infection, respectively.     

Problems Encountered with the Selection of Cucumber Mosaic Virus (CMV) Isolates for Resistance Breeding Programs

Among the Chili breeding lines from the Asian Vegetable Research Center, two were chosen for the screening of a larger selection of Cucumber mosaic virus (CMV) isolates, mainly from Asian countries. The chili line (VC246) showed a resistance against several CMV‐isolates and was compared with chili line VC27a that was susceptible to CMV infection. Among the 28 CMV isolates, five were identified as resistance breaking (AN‐like) and non‐resistance breaking (P3613‐like) for the line VC246, whereas all isolates could establish a systemic infection on VC27a. However, further testing revealed that resistance in VC246 was also dependent on the way of inoculation and the inoculums itself. Graft inoculation could overcome the resistance, and the inoculation with isolated viral RNA resulted in no infection at all on the resistant chili line, independent of the virus isolate. Using a pseudo‐recombinant approach, we identified RNA2 of resistance breaking isolates as responsible for systemic infection and confined the area within RNA2 to the 3′ terminal part including the ORF 2b. Sequence alignments of that area revealed eight distinct mutations on amino acid level, which was present either in resistance or non‐resistance breaking isolates. A reversion from the P3613‐like to the AN‐like sequence of two of these mutations induced no effect on Capsicum sp., but induced symptoms on several tobacco species distinct from those induced by the wild‐type virus. However, pseudorecombinants, each generated from sets of two different AN‐like isolates, which were expected to infect VC246 systemically, did not indicating that probably RNA2 must be in a specific context to have the effect. In this case, a generalized attribution of functions to single amino acid exchanges might be impossible or at least extremely difficult.     

Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.     

Expression of coat protein gene of Cucumber mosaic virus (CMV-subgroup IA) Gladiolus isolate in Nicotiana tabacum

The coat protein (CP)-mediated resistance against Cucumber mosaic virus (CMV) subgroup IA was developed in transgenic lines of Nicotiana tabacum cv. Petit Havana using Agrobacterium tumefaciens-mediated transformation. Ten independently transformed lines have developed, four of which were tested for resistance against CMV using virus challenge inoculations. The transgenic lines exhibiting complete resistance remained healthy and symptomless in their life span and showed reduced or no virus accumulation in their systemic leaves after virus challenge inoculation. These transgenic lines also showed resistance against CMV strains which are not closely related to CMV-Gladiolus strains. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IA member isolated from India showing resistance to all CMV strains occurring in the same vicinity.     

Development of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> as a vector modifiable for different host species to produce therapeutic proteins

We have developed Cucumber mosaic virus (CMV) as a plant virus vector especially for production of pharmaceutical proteins. The CMV vector is a vector modifiable for different host plants and does not require further engineering steps. CMV contains three genomic RNA molecules (RNAs 1–3) necessary for infectivity. With this system, instead of creating different vector constructs for each plant we use, we take advantage of the formation of pseudrecombinants between two CMV isolates by simply reassembling a vector construct (RNA 2 base) and an RNA molecule containing the host determinant (mostly RNA 3). In this study, the gene for acidic fibroblast growth factor (aFGF), one of the human cytokines, was cloned under the control of the subgenomic promoter for RNA 4A of the CMV-based vector, C2-H1. Infected Nicotiana benthamiana plants produced aFGF at levels up to 5–8% of the total soluble protein. The tobacco-produced aFGF was purified, and its biological activity was confirmed. Using this system, which provides a versatile and viable strategy for the production of therapeutic proteins in plants, we also demonstrated a high level of aFGF in Glycine max (soybean) and Arabidopsis thaliana.     

Salicylic acid-induced resistance to Cucumber mosaic virus in squash and Arabidopsis thaliana: contrasting mechanisms of induction and antiviral action

Salicylic acid (SA)-induced resistance to Cucumber mosaic virus (CMV) in tobacco (Nicotiana tabacum) results from inhibition of systemic virus movement and is induced via a signal transduction pathway that also can be triggered by antimycin A, an inducer of the mitochondrial enzyme alternative oxidase (AOX). In Arabidopsis thaliana, inhibition of CMV systemic movement also is induced by SA and antimycin A. These results indicate that the mechanisms underlying induced resistance to CMV in tobacco and A. thaliana are very similar. In contrast to the situation in tobacco and A. thaliana, in squash (Cucurbita pepo), SA-induced resistance to CMV results from inhibited virus accumulation in directly inoculated tissue, most likely through inhibition of cell-to-cell movement. Furthermore, neither of the AOX inducers antimycin A or KCN induced resistance to CMV in squash. Additionally, AOX inhibitors that compromise SA-induced resistance to CMV in tobacco did not inhibit SA-induced resistance to the virus in squash. The results show that different host species may use significantly different approaches to resist infection by the same virus. These findings also imply that caution is required when attempting to apply findings on plant-virus interactions from model systems to a wider range of host species.     

Cucumber mosaic virus: viral genes as virulence determinants

TAXONOMIC RELATIONSHIPS: Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus in the family Bromoviridae, which also encompasses the Peanut stunt virus (PSV) and the Tomato aspermy virus (TAV). Nucleotide sequence similarity among these three cucumoviruses is 60%-65%. CMV strains are divided into three subgroups, IA, IB and II, based on the sequence of the 5' untranslated region of the genomic RNA 3. Overall nucleotide sequence similarity among CMV strains is approximately 70%-98%. GEOGRAPHICAL DISTRIBUTION, HOST RANGE AND SYMPTOMATOLOGY: CMV is distributed worldwide, primarily in temperate to tropical climate zones. CMV infects more than 1200 species of 100 plant families, including monocot and dicot plants. Symptoms caused by CMV infection vary with the host species and/or CMV strain, and include mosaic, stunt, chlorosis, dwarfing, leaf malformation and systemic necrosis. CMV disease is spread primarily by aphid transmission in a nonpersistent manner. PHYSICAL PROPERTIES: In tobacco sap, the thermal inactivation point of the viral infectivity is approximately 70 °C (10 min), the dilution end-point is approximately 10(-4) and viral infectivity is lost after a few days of exposure to 20 °C. Viral infectivity can be retained in freeze-dried tissues and in the form of virions purified using 5 mm sodium borate, 0.5 mm ethylenediaminetetraacetic acid and 50% glycerol (pH 9.0) at -20 °C. CMV particles are isometric, approximately 28-30 nm in diameter and are composed of 180 capsid subunits arranged in pentamer-hexamer clusters with T= 3 symmetry. The sedimentation coefficient (s(20) ,(w) ) is c. 98 S and the particle weight is (5.8-6.7) × 10(6) Da. The virions contain 18% RNA. The RNA-protein interactions that stabilize the CMV virions are readily disrupted by sodium dodecylsulphate or neutral chloride salts. GENOMIC PROPERTIES: The genomic RNAs are single-stranded messenger sense RNAs with 5' cap and 3' tRNA-like structures containing at least five open reading frames. The viral RNA consists of three genomic RNAs, RNA 1 (c. 3.3 kb), RNA 2 (c. 3.0 kb) and RNA 3 (c. 2.2 kb), and two subgenomic RNAs, RNA 4 (c. 1.0 kb) and RNA 4A (c. 0.7 kb). The 3' untranslated regions are conserved across all viral RNAs. CMV is often accompanied by satellite, noncoding, small, linear RNA that is nonhomologous to the helper CMV.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Virus diseases of garden lupin in Great Britain

Two viruses occur widely in lupins in Britain. Alfalfa mosaic virus (AMV), of which two strains were isolated, was found mainly in named Russell varieties. Lupin mottle virus (LMV), a previously undescribed strain of the bean yellow mosaic virus (BYMV) common pea mosaic virus (CPMV) complex, was found more commonly in seedling lupins. Cucumber mosaic virus (CMV) was isolated once. The AMV strains were differentiated by their reaction in Phaseolus vulgaris; they were serologically closely related. Both AMV and LMV were aphid transmitted but not transmitted in lupin seed. LMV was distantly serologically related to both BYMV and CPMV. It cross-protected against BYMV but not against CPMV and it differed from both these viruses in some host reactions. The CMV isolate from lupins was similar to type CMV. It was transmitted both mechanically and by aphid, easily from cucumber to cucumber, but with difficulty from cucumber to lupin.     

Differentiation of Bulgarian Isolates from Cucumber Mosaic Virus by Serological Methods

Cucumber mosaic virus (CMV) is of great importance to the Bulgarian economy and hence a detailed knowledge of its diversity under local geographic and climatic conditions is required. An extended study was carried out on CMV strains the currently occur in Bulgaria. Fifty-one isolates and strains found in different regions and various crops were biologically characterized and serologically differentiated into subgroups I and II using different variants of enzyme-linked immunosorbent assay (ELISA) [double antibody sandwich (DAS)-, antigen-coated plate (ACP)-, triple antibody sandwich (TAS)- with poly and monoclonal antibodies] and immunodiffusion tests. The ELISA modifications with monoclonal antibodies individually (ACP) or in combination with polyclonal antibodies (TAS-ELISA) are suitable for mass screening of CMV isolates. The hyperimmune sera against strains from CMV subgroups I and II were very efficient for use in isolate differentiation via gel double immunodiffusion. The results obtained correlated with the polymerase chain reaction and restriction fragment length polymorphism data reported by other authors. The majority of the isolates belonged to subgroup I, whereas 10, mainly from tomato and pepper, belonged to subgroup II. Most of the subgroup II isolates came from the north of Bulgaria. The results of the present study will help to clarify the virus epidemiology and to develop specific control measures.     

Occurrence, distribution and relative incidence of mosaic viruses infecting field--grown squash in Tehran province, Iran

Squash (Cucurbita pepo) belongs to Cucurbitaceae family. Every year Cucurbitaceae are planted world wide. They are one of the most important economic crops. Cucurbitaceae are threatened by viruses. Many viruses damage the plants of this family. Since nine viruses have been reported on squash from Iran. In this survey, during 2002--2003, to determine the distribution of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV), 466 samples were collected from squash field in Tehran province. Infected plants showing symptoms such as: mosaic, yellowing, deformation, shoestring of leaves and fruit deformation and yield reduction. Distribution of CMV, ZYMV and WMV were determined by DAS-ELISA. Thepercentage of ZYMV, WMV and CMV were 35.6, 26.1 and 25.1% respectively. Triple infection (CMV+ZYMV+WMV) were found in 6.4% of samples. ZYMV were found the most frequently the viruses. This is the first report of WMV on squash in Tehran province.     

Occurrence and distribution of lettuce mosaic disease in Tehran province from Iran

Lettuce mosaic virus (LMV) Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in fields of Tehran province. In this study 452 leaf samples were collected from the fields throughout the Tehran province during 2002 and 2003. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV)and Arabis mosaic virus (ArMV) was determined with DAS-ELISA. Percentage of single Infection to LMV. CMV or TSWV was 20.58, 15.93 and 9.96% respectively. Also 15.28% of samples were co- infected with LMV+CMV, 8.19% with LMV+TSMV and 7.74% with CMV+TSWV. 4.65% of samples were Infected to all of these three viruses. LMV was found in 48.69%, CMV in 43.59% and TSWV in 30.54% of samples totally. Therefore LMV is major dominant agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and first report of CMV and LMV in Tehran province.     

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum ‘Bukang’ cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.     

Serological diagnosis and host range studies of important viral diseases of a few cucurbitaceous crops in Maharashtra,India

Around 39 well characterised viruses affect cucurbits crops in developing countries and their viral diversity may be the consequence for genetic and ecological diversity of their hosts. Indeed, cucurbits are grown in variety of climatic, environmental and agricultural conditions, and this may provide more or less favourable conditions for the specific viruses or their hosts. The presence of various viral diseases caused by different viruses in Maharashtra was studied from the infected samples collected from cucurbits and melons during the survey conducted in 2009–2010 in different locations. The virus isolates collected from various cucurbitaceous crops were established and their host ranges were studied by sap transmission. The study revealed Cucumber Mosaic Virus (CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and Cucumber green mottle mosaic virus infections predominately found in Nashik region, and Watermelon bud necrosis virus (WBNV), CMV, ZYMV, WMV and Watermelon silver mottle virus (WSMoV) infections in Aurangabad and Paithan regions. In Sangamner region, the crop was mostly affected by WBNV, ZYMV and WSMoV, and CMV was found only in Sillod region. The protocols for performing sap transmission tests in assay hosts were standardised for ZYMV, CMV and WBNV. Using direct antigen-coating enzyme-linked immunosorbent assay, of all the plant parts, young leaves were found to have high concentration of virus and suitable for virus detection in screening programmes. CMV and ZYMV was found to have high concentration of virus and suitable for virus detection in screening programmes.     

Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic Disease

Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana.     

Isolates of cucumber mosaic virus from spontaneously infected plants ofChelidonium majus andImpatiens parviflora

Cucumber mosaic virus (CMV) isolates obtained from spontaneously infected (1)Chelidonium majus L. and (2)Impatiens parviflora DC. plants growing in the same part of a scree forest in central Bohemia showed differences in ten test plants, but not in further 26 plant species tested and in TIP. Slight quantitative differences between both isolates were obtained in serological tests. Properties of both isolates did not suit precisely any proposed CMV strain classification.     

Characterization, Genetic Diversity, and Evolutionary Link of Cucumber mosaic virus Strain New Delhi from India

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.