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 Genetic Diversity Among Viruses Associated with Sugarcane Mosaic Disease in Tucumán, Argentina

2009 ◽  
Vol 99 (1) ◽  
pp. 38-49 ◽  
Author(s):  
M. F. Perera ◽  
M. P. Filippone ◽  
C. J. Ramallo ◽  
M. I. Cuenya ◽  
M. L. García ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Mosaic Virus ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Mosaic Disease ◽  
Rt Pcr ◽  
Rflp Profile ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Sugarcane Mosaic Disease

Sugarcane leaves with mosaic symptoms were collected in 2006–07 in Tucumán (Argentina) and analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) and sequencing of a fragment of the Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) coat protein (CP) genes. SCMV was detected in 96.6% of samples, with 41% showing the RFLP profile consistent with strain E. The remaining samples produced eight different profiles that did not match other known strains. SCMV distribution seemed to be more related to sugarcane genotype than to geographical origin, and sequence analyses of CP genes showed a greater genetic diversity compared with other studies. SrMV was detected in 63.2% of samples and most of these were also infected by SCMV, indicating that, unlike other countries and other Argentinean provinces, where high levels of co-infection are infrequent, co-existence is common in Tucumán. RFLP analysis showed the presence of SrMV strains M (68%) and I (14%), while co-infection between M and H strains was present in 18% of samples. Other SCMV subgroup members and the Sugarcane streak mosaic virus (SCSMV) were not detected. Our results also showed that sequencing is currently the only reliable method to assess SCMV and SrMV genetic diversity, because RT-PCR-RFLP may not be sufficiently discriminating.

scholarly journals Detección y caracterización de un aislamiento de Cucumber mosaic virus que infecta peperina, una especie nativa de Argentina.

Agriscientia ◽  
2013 ◽  
Vol 30 (2) ◽  
pp. 79-85 ◽  
Author(s):  
P. Rodríguez Pardina ◽  
M. Ojeda ◽  
E. Biderbost ◽  
L. Di Feo
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Rt Pcr ◽  
Pcr Rflp

Minthostachys mollis (Kunth.) Griseb., "peperina", un miembro de la familia Lamiaceae, es una especie aromática que se emplea en la farmacología moderna y en medicina. Está ampliamente distribuida en los Andes, desde Venezuela y Colombia hasta Argentina. En el último país, la principal área de explotación de peperina incluye el área serrana de la provincia de Córdoba, donde la especie es arrancada indiscriminadamente, lo que conlleva una pérdida irreversible de germoplasma. A los fines de preservar este recurso nativo y fuente regional de ingresos, la especie está siendo domesticada. Durante este proceso, se observó la aparición de síntomas de un conspicuo mosaico amarillo, típico de infección viral. Análisis biológicos, serológicos y moleculares (RT-PCR, RFLP, clonado y secuenciación) pusieron de manifiesto la presencia del subgrupo IA de Cucumber mosaic virus en las plantas domesticadas de peperina. El aislamiento viral estudiado está íntimamente relacionado con la raza Y previamente informada en Japón. Éste es el primer informe de un virus que infecta a la peperina.

scholarly journals Identification of Soybean mosaic virus Strains by RT-PCR/RFLP Analysis of Cylindrical Inclusion Coding Region

Plant Disease ◽  
2004 ◽  
Vol 88 (6) ◽  
pp. 641-644 ◽  
Author(s):  
Yul-Ho Kim ◽  
Ok-Sun Kim ◽  
Jae-Hwan Roh ◽  
Jung-Kyung Moon ◽  
Soo-In Sohn ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Soybean Mosaic Virus ◽  
Rflp Analysis ◽  
Cylindrical Inclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Strain Identification ◽  
Coding Region ◽  
Rt Pcr ◽  
Detection And Identification ◽  
Pcr Rflp

A reverse-transcriptase polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) was employed successfully for detection and identification of Soybean mosaic virus (SMV) strains. A primer pair amplifying a 1,385-bp fragment of the cylindrical inclusion (CI) coding region was used for RT-PCR and the RFLP profiles of the RT-PCR products were compared after restriction digestion with RsaI, EcoRI, or AccI restriction endonucleases. These enzymes were chosen based on the nucleotide sequences of SMV strains G2, G5, G5H, G7, and G7H in the CI coding region. These five strains, as well as seedborne SMV isolates from local soybean cultivars, could be differentiated by RT-PCR/RFLP analysis. The results correlated well with strain identification by symptom phenotypes produced on differential cultivars inoculated with strains and isolates. The sensitivity of RT-PCR enabled detection of SMV from plants with necrotic symptoms in which the number of virus particles was too low to be detected by enzyme-linked immunosorbent assay.

scholarly journals Isolado do vírus do mosaico do pepino obtido de bananeira no Estado de São Paulo pertence ao subgrupo Ia

2001 ◽  
Vol 26 (1) ◽  
pp. 53-59 ◽  
Author(s):  
MARCELO EIRAS ◽  
ADDOLORATA COLARICCIO ◽  
ALEXANDRE L.R. CHAVES
Keyword(s):  
Mosaic Virus ◽  
Vigna Unguiculata ◽  
Sao Paulo ◽  
São Paulo ◽  
Rt Pcr ◽  
Nicotiana Glutinosa ◽  
Musa Sp ◽  
De Se ◽  
Pcr Rflp ◽  
Das Elisa

Em 1996, foi feita a caracterização parcial de um isolado do vírus do mosaico do pepino (Cucumis mosaic virus, CMV) obtido de bananeira (Musa sp.) proveniente do município de Miracatu, SP. Com o objetivo de se determinar o subgrupo do isolado de CMV, recorreu-se às técnicas de ELISA, RT-PCR, RFLP e seqüenciamento de fragmentos de RNA genômico. Amostras de folhas infetadas, desidratadas com cloreto de cálcio e armazenadas à -20 °C desde 1994 na viroteca do Laboratório de Fitovirologia e Fisiopatologia, foram inoculadas em plantas de Nicotiana glutinosa. Dez dias após a inoculação, folhas apresentando mosaico foram utilizadas para DAS-ELISA e extração de RNAs totais. Em ELISA, houve reação apenas contra o anti-soro específico para CMV subgrupo I. Através de RT-PCR com primers desenhados para anelar em regiões conservadas da porção terminal 3' do gene da capa protéica, foi amplificado um fragmento de DNA com 486 pares de bases. O produto obtido via RT-PCR foi submetido à digestão com as enzimas EcoRI, HindIII, BamHI e MspI, obtendo-se um padrão de restrição esperado para o subgrupo I. Estes resultados foram confirmados através do seqüenciamento do produto de PCR, o qual apresentou homologia de 96% a 98% com os isolados do CMV pertencentes ao subgrupo I. Pelos sintomas observados na hospedeira diferencial Vigna unguiculata, o isolado foi confirmado como sendo do subgrupo Ia.

scholarly journals First Report of Natural Infection of Penstemon acuminatus with Cucumber mosaic virus in the Treasure Valley Region of Idaho and Oregon

Plant Disease ◽  
2009 ◽  
Vol 93 (7) ◽  
pp. 762-762 ◽  
Author(s):  
R. K. Sampangi ◽  
C. Almeyda ◽  
K. L. Druffel ◽  
S. Krishna Mohan ◽  
C. C. Shock ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Great Basin ◽  
Natural Infection ◽  
The United States ◽  
Native Plant ◽  
Rt Pcr ◽  
Cmv Infection ◽  
Spot Virus ◽  
First Report

Penstemons are perennials that are grown for their attractive flowers in the United States. Penstemon species (P. acuminatus, P. deustus, and P. speciosus) are among the native forbs considered as a high priority for restoration of great basin rangelands. During the summer of 2008, symptoms of red spots and rings were observed on leaves of P. acuminatus (family Scrophulariaceae) in an experimental trial in Malheur County, Oregon where the seeds from several native forbs were multiplied for restoration of range plants in intermountain areas. These plants were cultivated as part of the Great Basin Native Plant Selection and Increase Project. Several native wildflower species are grown for seed production in these experimental plots. Plants showed red foliar ringspots and streaks late in the season. Fungal or bacterial infection was ruled out. Two tospoviruses, Impatiens necrotic spot virus and Tomato spotted wilt virus, and one nepovirus, Tomato ring spot virus, are known to infect penstemon (2,3). Recently, a strain of Turnip vein-clearing virus, referred to as Penstemon ringspot virus, was reported in penstemon from Minnesota (1). Symptomatic leaves from the penstemon plants were negative for these viruses when tested by ELISA or reverse transcription (RT)-PCR. However, samples were found to be positive for Cucumber mosaic virus (CMV) when tested by a commercially available kit (Agdia Inc., Elkhart, IN). To verify CMV infection, total nucleic acid extracts from the symptomatic areas of the leaves were prepared and used in RT-PCR. Primers specific to the RNA-3 of CMV were designed on the basis of CMV sequences available in GenBank. The primer pair consisted of CMV V166: 5′ CCA ACC TTT GTA GGG AGT GA 3′ and CMV C563: 5′ TAC ACG AGG ACG GCG TAC TT 3′. An amplicon of the expected size (400 bp) was obtained and cloned and sequenced. BLAST search of the GenBank for related sequences showed that the sequence obtained from penstemon was highly identical to several CMV sequences, with the highest identity (98%) with that of a sequence from Taiwan (GenBank No. D49496). CMV from infected penstemon was successfully transmitted by mechanical inoculation to cucumber seedlings. Infection of cucumber plants was confirmed by ELISA and RT-PCR. To our knowledge, this is the first report of CMV infection of P. acuminatus. With the ongoing efforts to revegetate the intermountain west with native forbs, there is a need for a comprehensive survey of pests and diseases affecting these plants. References: (1) B. E. Lockhart et al. Plant Dis. 92:725, 2008. (2) D. Louro. Acta Hortic. 431:99, 1996. (3) M. Navalinskiene et al. Trans. Estonian Agric. Univ. 209:140, 2000.

scholarly journals Nuevos registros de aislamientos del Cucumber mosaic virus y su RNA satélite en Colima, México

2019 ◽  
Vol 37 (2) ◽  
Author(s):  
Pedro Valadez-Ramírez ◽  
Javier Paz-Román ◽  
Salvador Guzmán-González ◽  
Marco Tulio Buenrostro-Nava ◽  
Daniel Leobardo Ochoa-Martínez
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Solanum Lycopersicum ◽  
Capsicum Annuum ◽  
Catharanthus Roseus ◽  
Cucumis Melo ◽  
Rt Pcr ◽  
Impacto Económico

El <em>Cucumber mosaic virus</em> (CMV) ocasiona una de las enfermedades virales más importantes a nivel mundial en plantas silvestres y cultivadas. En México son pocos los estudios que se han abordado con este virus, y dada su amplia gama de hospedantes e impacto económico, es necesario contar con mayor información de su presencia y distribución en zonas de importancia agrícola como las del estado de Colima. En este trabajo, se reportan nuevos aislamientos del CMV identificados por RT-PCR, secuenciación de DNA y su análisis filogenético: CMV-Vin en vinca (<em>Catharanthus roseus</em>), CMV-Chi en chile jalapeño (<em>Capsicum annuum</em>) y CMV-Tom en tomate saladette (<em>Solanum lycopersicum</em>). Se confirmó, además, la presencia del CMV en melón cantaloupe (<em>Cucumis melo</em>) (CMV-Mel). Los aislamientos CMV-Vin, CMV-Chi y CMV-Mel agruparon en el subgrupo IB, mientras que CMV-Tom agrupó en el subgrupo IA de CMV. De estos aislamientos, sólo CMV-Vin evidenció la presencia de un RNA satélite (satRNA Vin) sin dominio necrogénico. Este es el primer reporte de la presencia del CMV en vinca, chile y tomate y de un RNA satélite en vinca en Colima, México.

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 Lamium maculatum is a Natural Host for Cucumber mosaic virus

Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 150-150 ◽  
Author(s):  
R. Bešta-Gajević ◽  
A. Jerković-Mujkić ◽  
S. Pilić ◽  
I. Stanković ◽  
A. Vučurović ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Impatiens Necrotic Spot Virus ◽  
Natural Occurrence ◽  
Rt Pcr ◽  
Natural Ecosystems ◽  
New Host ◽  
Total Rna ◽  
Negative Controls ◽  
Das Elisa

Lamium maculatum L. (spotted dead-nettle) is a flowering perennial ornamental that is commonly grown as a landscape plant for an effective ground cover. In June 2010, severe mosaic accompanied by reddish brown necrosis and leaf deformation was noticed on 80% of L. maculatum growing in shade under trees and shrubs in Sarajevo (Bosnia and Herzegovina). Leaves from 10 symptomatic L. maculatum plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Bioreba AG, Reinach, Switzerland) against Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV), the most important viral pathogens of ornamental plants (1,2). Commercial positive and negative controls and extracts from healthy L. maculatum leaves were included in each assay. All samples tested negative for TSWV and INSV and positive for CMV. The virus was mechanically transmitted to test plants and young virus-free plants of L. maculatum using 0.01 M phosphate buffer (pH 7). The virus caused chlorotic local lesions on Chenopodium quinoa, while systemic mosaic was observed on Capsicum annuum ‘Rotund,’ Nicotiana rustica, N. glutinosa, N. tabacum ‘White Burley,’ and Phaseolus vulgaris ‘Top Crop.’ The virus was transmitted mechanically to L. maculatum and induced symptoms resembling those observed on the source plants. Inoculated plants were assayed by DAS-ELISA and all five inoculated plants of each species tested positive for CMV. The presence of CMV in L. maculatum as well as mechanically infected N. glutinosa plants was further confirmed by RT-PCR. Total RNA from symptomatic leaves was isolated using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) following the manufacturer's instructions. The primer pair, CMVAu1u/CMVAu2d, that amplifies the entire coat protein (CP) gene and part of 3′- and 5′-UTRs was used for both amplification and sequencing (4). Total RNA obtained from the Serbian CMV isolate from pumpkin (GenBank Accession No. HM065510) and a healthy L. maculatum plant were used as positive and negative controls, respectively. All naturally and mechanically infected plants as well as the positive control yielded an amplicon of the expected size (850 bp). No amplicon was observed in the healthy control. The amplified product derived from isolate 3-Lam was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions and deposited in GenBank (JX436358). Sequence analysis of the CP open reading frame (657 nt), conducted with MEGA5 software, revealed that the isolate 3-Lam showed the highest nucleotide identity of 99.4% (99.1% amino acid identity) with CMV isolates from Serbia, Australia, and the USA (GQ340670, U22821, and U20668, respectively). To our knowledge, this is the first report of the natural occurrence of CMV on L. maculatum worldwide and it adds a new host to over 1,241 species (101 plant families) infected by this virus (3). This is also an important discovery for the ornamental industry since L. maculatum is commonly grown together with other ornamental hosts of CMV in nurseries and the urban environment as well as in natural ecosystems. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997. (3) M. Jacquemond. Adv. Virus Res. 84:439, 2012. (4) I. Stankovic et al. Acta Virol. 55:337, 2011.

Sign in / Sign up

Export Citation Format

Share Document