Coat protein-mediated resistance as an approach for controlling an Egyptian isolate of Cucumber mosaic virus (subgroup I)

Biologia ◽  
2008 ◽  
Vol 63 (5) ◽  
Author(s):  
Ali El-Borollosy ◽  
Sabry Mahmoud ◽  
Abdel-Sabour Khaled
Keyword(s):  
Coat Protein ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Restriction Enzymes ◽  
Significant Loss ◽  
Enzyme Linked Immunosorbent Assay ◽  
35S Promoter ◽  
Rt Pcr ◽  
Dot Blot ◽  
Egyptian Isolate

AbstractCucumber mosaic virus (CMV, cucumovirus) is the most important virus infecting cucurbit crops in Egypt and worldwide causing significant loss in yield quality and quantity. The main target of the present work was to establish a simple controlling system for an Egyptian isolate of such virus (belonging to the subgroup I) via production of tobacco transgenic plants expressing viral coat protein (CP). Coat protein gene (cp) was isolated and amplified using immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) and primers with add-on restriction sites for SmaI and SacI enzymes. The genes were cloned in pBI121 vector plasmid between the CaMV 35S promoter and the nos terminator after removing the Gus gene by restriction enzymes digestion. The new construct was used for Agrobacterium tumefaciens transformation, which was then used for tobacco transformation. Evaluation of transformation success and CP expression degree were confirmed using indirect enzyme-linked immunosorbent assay (I-ELISA) and dot blot immuno-binding assay (DBIA). PCR and RT-PCR were used to study the integration of cp within genetic plant system and to what extent this gene was transcript. It was concluded that in spite of integration success some transformed plants can transcript the gene more than the others do. Plants resistance was tested by challenging with CMV under study and remarkable success was obtained in plants with higher gene transcription and translation degree.

scholarly journals Allamanda Mosaic Caused by Cucumber mosaic virus in Taiwan

Plant Disease ◽  
2005 ◽  
Vol 89 (5) ◽  
pp. 529-529 ◽  
Author(s):  
Y. K. Chen ◽  
C. C. Yang ◽  
H. T. Hsu
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chenopodium Quinoa ◽  
Rabbit Antiserum ◽  
Nucleotide Sequence Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Indicator Plants

Allamanda (Allamanda cathartica L., family Apocynaceae) is native to Brazil and is a popular perennial shrub or vine ornamental in Taiwan. Plants showing severe mosaic, rugosity, and leaf distortion symptoms on leaves are common in commercial nurseries and private gardens. Examination of crude sap prepared from symptomatic leaves using an electron microscope revealed the presence of spherical virus particles with a diameter of approximately 28 nm. The virus was mechanically transmitted to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). The virus caused local lesions on inoculated leaves of Chenopodium quinoa and C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, Nicotiana benthamiana, N. glutinosa, N. rustica, and N. tabacum. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Tests of leaf sap extracted from naturally infected allamanda and inoculated indicator plants using enzyme-linked immunosorbent assay were positive to rabbit antiserum prepared to CMV. Viral coat protein on transblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis reacted with CMV subgroup I specific monoclonal antibodies (2). With primers specific to the 3′-half of RNA 3 (1), amplicons of an expected size (1,115 bp) were obtained in reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from infected allamanda and N. benthamiana. The amplified fragment (EMBL Accession No. AJ871492) was cloned and sequenced. It encompasses the 3′ part of the intergenic region of RNA 3 (158 nt), CP ORF (657 nt), and 3′ NTR (300 nt) showing 91.8–98.9% and 71.4–72.8% identities to those of CMV in subgroups I and II, respectively. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and the nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB, which is predominant on the island. To our knowledge, CMV is the only reported virus that infects allamanda and was first detected in Brazil (3), and this is the first report of CMV infection in allamanda plants occurring in Taiwan. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) H. T. Hsu et al. Phytopathology 90:615, 2000. (3) E. W. Kitajima. Acta. Hortic. 234:451, 1988.

scholarly journals First Report of Cucumber mosaic virus Subgroup II Infecting Lycopersicon esculentum in India

Plant Disease ◽  
2006 ◽  
Vol 90 (11) ◽  
pp. 1457-1457 ◽  
Author(s):  
N. Sudhakar ◽  
D. Nagendra-Prasad ◽  
N. Mohan ◽  
K. Murugesan
Keyword(s):  
Coat Protein ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Tamil Nadu ◽  
Indian Subcontinent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Leaf ◽  
Polymorphism Analysis ◽  
First Report ◽  
Subgroup Ii

During a survey in January 2006 near Salem in Tamil Nadu (south India), Cucumber mosaic virus was observed infecting tomatoes with an incidence of more than 70%. Plants exhibiting severe mosaic, leaf puckering, and stunted growth were collected, and the virus was identified using diagnostic hosts, evaluation of physical properties of the virus, compound enzyme-linked immunosorbent assay (ELISA) (ELISA Lab, Washington State University, Prosser), reverse-transcription polymerase chain reaction (RT-PCR), and restriction fragment length polymorphism analysis (DSMZ, S. Winter, Germany). To determine the specific CMV subgroup, total RNA was extracted from 50 infected leaf samples using the RNeasy plant RNA isolation kit (Qiagen, Hilden, Germany) and tested for the presence of the complete CMV coat protein gene using specific primers as described by Rizos et al. (1). A fragment of the coat protein was amplified and subsequently digested with MspI to reveal a pattern of two fragments (336 and 538 bp), indicating CMV subgroup II. No evidence of mixed infection with CMV subgroup I was obtained when CMV isolates representing subgroups I (PV-0419) and II (PV-0420), available at the DSMZ Plant Virus Collection, were used as controls. Only CMV subgroup I has been found to predominantly infect tomato in the Indian subcontinent, although Verma et al. (2) identified CMV subgroup II infecting Pelargonium spp., an ornamental plant. To our knowledge, this is the first report of CMV subgroup II infecting tomato crops in India. References: (1) H. Rizos et al. J. Gen. Virol. 73:2099, 1992. (2) N. Verma et al. J. Biol. Sci. 31:47, 2006.

scholarly journals Transfer and Expression of Cucumber Mosaic Virus Coat Protein Gene in the Genome of Cucumis sativus

1991 ◽  
Vol 116 (6) ◽  
pp. 1098-1102 ◽  
Author(s):  
Paula P. Chee ◽  
Jerry L. Slightom
Keyword(s):  
Coat Protein ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Cucumis Sativus ◽  
Blot Analysis ◽  
Binary Vector ◽  
Enzyme Linked Immunosorbent Assay ◽  
Embryogenic Calli ◽  
Mature Embryos ◽  
Npt Ii

Cotyledon explants of cucumber (Cucumis sativus L. cv. Poinsett 76) seedlings were cocultivated with disarmed Agrobacterium strain C58Z707 that contained the binary vector plasmid pGA482GG/cpCMV19. The T-DNA region of this binary vector contains plant-expressible genes for neomycin phosphotransferase II (NPT II), β -glucuronidase (GUS), and the coat protein of cucumber mosaic virus strain C (CMV-C). After infection, the cotyledons were placed on Murashige and Skoog medium containing 100 mg kanamycidliter. Putative transformed embryogenic calli were obtained, followed by the development of mature embryos and their germination to plants. All transformed RO cucumber plants appeared morphologically normal and tested positive for NPT IL Southern blot analysis of selected cucumber DNAs indicated that NPT II, GUS, and CMV-C coat protein genes were integrated into the genomes. Enzyme-linked immunosorbent assay and Western blot analysis indicated that the CMV-C coat protein is present in the protein extracts of progeny plants. These results show that the Agrobacterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for producing transgenic plants in Cucurbitaceae.

scholarly journals First Report of Tomato torrado virus Infecting Tomato in Single and Mixed Infections with Cucumber mosaic virus in Panama

Plant Disease ◽  
2009 ◽  
Vol 93 (2) ◽  
pp. 198-198 ◽  
Author(s):  
J. A. Herrera-Vásquez ◽  
A. Alfaro-Fernández ◽  
M. C. Córdoba-Sellés ◽  
M. C. Cebrián ◽  
M. I. Font ◽  
...  
Keyword(s):  
Coat Protein ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Potato Virus ◽  
Plant Viruses ◽  
Tomato Mosaic Virus ◽  
Rt Pcr ◽  
Tomato Production ◽  
Related Sequence ◽  
First Report

In February of 2008, in open-field-grown tomato crops (Solanum lycopersicum L.) from the central regions of Coclé, Herrera, Los Santos, and Veraguas of Panama, unusual disease symptoms, including deformation, necrosis, purple margins, interveinal yellowing, downward and upward curling of the leaflets alternately, necrotic lines in sepals and branches, fruits distorted with necrotic lines on the surface, and severe stunting, were observed. Tomato production was seriously damaged. To verify the identity of the disease, five symptomatic tomato plants from four fields of these regions were selected and analyzed by double-antibody sandwich (DAS)-ELISA using specific antibodies to Cucumber mosaic virus (CMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany), and Pepino mosaic virus (PepMV) (DSMZ, Braunschweig, Germany). Total RNA was extracted from all plants and tested using reverse transcription (RT)-PCR with three pairs of specific primers: one pair designed to amplify 586 bp of the coat protein gene of CMV (CMV-F 5′-CCTCCGCGGATGCTAACTT-3′ and CMV-R 5′-CGGAATCAGACTGGGAGCA-3′) and the other two pairs to Tomato torrado virus (ToTV) that amplify 580 and 574 bp of the polyprotein (4) and coat protein (Vp23) (3) region of RNA2, respectively; and by dot-blot hybridization with a digoxygenin-labeled RNA probe complementary to the aforementioned polyprotein. The serological analysis for PVX, PVY, ToMV, TSWV, and PepMV were negative. ToTV was detected in all samples analyzed. Three of these samples were also positive for CMV by serological and molecular analysis. No differences in symptom expression were observed between plants infected with both viruses or with ToTV alone. RT-PCR products were purified and directly sequenced. BLAST analysis of one CMV sequence (GenBank Accession No. EU934036) showed 98% identity with a CMV sequence from Brazil (most closely related sequence) (GenBank Accession No. AY380812) and 97% with the Fny isolate (CMV subgroup I) (GenBank Accession No. U20668). Two ToTV sequences were obtained (GenBank Accession Nos. EU934037 and FJ357161) and showed 99% and 98% identities with the polyprotein and coat protein region of ToTV from Spain (GenBank Accession No. DQ388880), respectively. CMV is transmitted by aphids and is distributed worldwide with a wide host range (2), while ToTV is transmitted by whiteflies and has only been reported in tomato crops in Spain and Poland and recently on weeds in Spain (1). To our knowledge, this is the first time ToTV has been detected in Panama and the first report of CMV/ToTV mixed infection. References: (1) A. Alfaro-Fernández et al. Plant Dis. 92:831, 2008. (2) A. A. Brunt et al. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Online Publication, 1996. (3) H. Pospieszny et al. Plant Dis. 91:1364, 2007. (4) M. Verbeek et al. Arch. Virol. 152:881, 2007.

scholarly journals First Report of Tomato torrado virus Infecting Tomato in Hungary

Plant Disease ◽  
2009 ◽  
Vol 93 (5) ◽  
pp. 554-554 ◽  
Author(s):  
A. Alfaro-Fernández ◽  
G. Bese ◽  
C. Córdoba-Sellés ◽  
M. C. Cebrián ◽  
J. A. Herrera-Vásquez ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Polyclonal Antibodies ◽  
Blot Hybridization ◽  
Sandwich Elisa ◽  
World Intellectual Property Organization ◽  
Tomato Mosaic Virus ◽  
Rt Pcr ◽  
Dot Blot ◽  
First Report

During the growing seasons of 2007 and 2008, in commercial greenhouses of tomato crops (Solanum lycopersicum L.) located in Szeged, Öcsöd, and Csongrád (southeastern regions of Hungary), unusual disease symptoms were observed, including necrotic spots in defined areas at the base of the leaflet, necrosis in the stems, and necrotic lines on the fruits surface. Affected plants appeared inside the greenhouses with a random distribution and the incidence recorded was at least 40%. These symptoms resembled those described for Tomato torrado virus (ToTV) infection in Spain (1) and Poland (3). To verify the identity of the disease, three symptomatic plants from commercial greenhouses of each geographic location were selected and analyzed by double-antibody sandwich-ELISA using polyclonal antibodies specific to Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany), and Pepino mosaic virus (PepMV) (DSMZ, Braunschweig, Germany). Total RNA was extracted and tested by reverse transcription (RT)-PCR with three pair of specific primers: one pair used to amplify the coat protein (CP) gene of PepMV (2) and the other two pairs specific to ToTV that amplify 580 bp of the polyprotein (4) and a fragment of 574 bp in the CP Vp23 (3). Nonisotopic dot-blot hybridization using a digoxygenin-labeled RNA probe complementary to the aforementioned fragment of the polyprotein was also performed. Tomato samples were negative for all the viruses tested by serological analysis and for PepMV by RT-PCR. However, all three samples were positive for ToTV by molecular hybridization and RT-PCR. RT-PCR products were purified and directly sequenced. The amplified fragments of the three Hungarian isolates, ToTV-H1, ToTV-H2, and ToTV-H3, for the polyprotein (GenBank Accession Nos. EU835496, FJ616995, and FJ616994, respectively) and the CP Vp23 (GenBank Accession Nos. FJ616996, FJ616997, and FJ616998, respectively) showed 99 to 98% nt identity with the polyprotein and the coat protein regions of ToTV from Spain and Poland (GenBank Accession Nos. DQ3888880 and EU563947, respectively). Whiteflies, commonly found in Hungarian greenhouses, have been reported to transmit ToTV (3), although the efficiency of transmission is unknown. To our knowledge, this is the first report of ToTV in Hungary. References: (1) A. Alfaro-Fernández et al. Plant Dis. 91:1060, 2007. (2) I. Pagán et al. Phytopathology 96:274, 2006. (3) H. Pospieszny et al. Plant Dis. 91:1364, 2007. (4) J. Van der Heuvel et al. Plant Virus Designated Tomato Torrado Virus. Online publication. World Intellectual Property Organization. WO/2006/085749, 2006.

Identification of Three Distinct Classes of Satellite RNAs Associated With Two Cucumber mosaic virus Serotypes from the Ornamental Groundcover Vinca minor

2012 ◽  
Vol 13 (1) ◽  
pp. 18 ◽  
Author(s):  
John R. Fisher
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Pcr Cloning ◽  
Cloning And Sequencing ◽  
First Report ◽  
Vinca Minor ◽  
Satellite Rnas

Cucumber mosaic virus (CMV) is a cosmopolitan virus which may also have small satellite RNAs (satRNA) associated with it affecting symptom development. Vinca minor (periwinkle) plants exhibiting subtle mosaic symptoms tested positive for CMV by enzyme linked immunosorbent assay (ELISA). Double-stranded ribonucleic acid (dsRNA) analysis of CMV-Vinca field isolates in Nicotiana tabacum ‘Glurk’ suggested two sizes of putative satRNA associated with CMV. Immunocapture RT-PCR, cloning, and sequencing of the movement protein, coat protein, and satRNAs demonstrated serogroup 1A and serogroup 2 CMV helper strains and three distinct classes of satRNAs of four sizes. Further, two classes of satRNAs could be distinguished by their necrosis domains. Previously CMV was reported in V. minor in New Jersey. This is the first report of CMV in V. minor in Ohio and the first report of satRNA associated with CMV in V. minor in the United States. Accepted for publication 1 February 2012. Published 12 April 2012.

RT-PCR-RFLP analysis for evaluating cross protection by an attenuated isolate of Cucumber mosaic virus

2005 ◽  
Vol 71 (3) ◽  
pp. 243-246 ◽  
Author(s):  
Eiko Nakazono-Nagaoka ◽  
Masako Suzuki ◽  
Yoshitaka Kosaka ◽  
Tomohide Natsuaki
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Rflp Analysis ◽  
Cross Protection ◽  
Rt Pcr ◽  
Pcr Rflp ◽  
Attenuated Isolate

scholarly journals Identification, Characterization, and Molecular Detection of Alfalfa mosaic virus in Potato

2006 ◽  
Vol 96 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
H. Xu ◽  
J. Nie
Keyword(s):  
Mosaic Virus ◽  
Gene Sequence ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Simple Method ◽  
Cp Gene ◽  
Nucleotide Sequence Alignment

Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

Prevalence and Molecular Characterization Coat Protein Gene of Tobacco streak virus Causing Peanut Stem Necrosis Disease in Coastal and Rayalaseema Districts of Andhra Pradesh, South-India

2021 ◽  
Author(s):  
K. Saratbabu ◽  
K. Vemana ◽  
A.K. Patibanda ◽  
B. Sreekanth ◽  
V. Srinivasa Rao
Keyword(s):  
Coat Protein ◽  
Molecular Characterization ◽  
Andhra Pradesh ◽  
Disease Incidence ◽  
Tobacco Streak Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Streak Virus ◽  
Rt Pcr ◽  
Cp Gene ◽  
Stem Necrosis

Background: Peanut stem necrosis disease (PSND) caused by Tobacco streak virus (TSV) is a major constraint for groundnut production in Andhra Pradesh (A.P.). However, studies on prevalence and spread of the disease confined to only few districts of A.P. with this background current study focused on incidence and spread of the disease in entire state of A.P. Further an isolate of TSV occurring in A.P. characterized on the basis of genetic features by comparing with other TSV isolates originated from different hosts and locations from world.Methods: Roving survey was conducted during kharif 2017-18 in groundnut growing districts of Andhra Pradesh (A.P.) for peanut stem necrosis disease incidence. Groundnut plants showing PSND symptoms were collected and tested with direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). Groundnut samples found positive by ELISA once again tested by reverse transcription polymerase chain reaction (RT-PCR). The representative TSV-GN-INDVP groundnut isolate from Prakasham district was maintained on cowpea seedlings by standard sap inoculation method in glasshouse for further molecular characterization. The Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny.Result: Thirty-eight (52.7%) of seventy-two groundnut samples collected from different locations in A.P were given positive reaction to TSV by DAC-ELISA. For the first time, PSND incidence observed in coastal districts (Krishna, Guntur, Sri Pottisriramulu Nellore, Prakasham) of A.P. Maximum PSND incidence recorded from Bathalapalli (22.2%) and the minimum incidence in Mulakalacheruvu (4.1%). The coat protein (CP) gene of TSV-GN-INDVP groundnut isolate was amplified by RT-PCR and it shared maximum per cent nucleotide identity (97.51-98.62%) with TSV isolates from groundnut and other different crops reported in India. All Indian isolates cluster together irrespective of crop and location based on the phylogenetic analysis.

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