ABSTRACT
Color is an important indicator for evaluating the ornamental traits of horticultural plants, and plant pigments is a key factor affecting the color phenotype of plants. Plant pigments and their metabolites play important roles in color formation of ornamental organs, regulation of plant growth and development, and response to adversity stress. It has therefore became a hot topic in the field of plant research. Virus-induced gene silencing (VIGS) is a vital genomics tool that specifically reduces host endogenous gene expression utilizing plant homology-dependent defense mechanisms. In addition, VIGS enables characterization of gene function by rapidly inducing the gene-silencing phenotypes in plants. It provides an efficient and feasible alternative for verifying gene function in plant species lacking genetic transformation systems. This paper reviews the current status of the application of VIGS technology in the biosynthesis, degradation and regulatory mechanisms of plant pigments. Moreover, this review discusses the potential and future prospects of VIGS technology in exploring the regulatory mechanisms of plant pigments, with the aim to further our understandings of the metabolic processes and regulatory mechanisms of different plant pigments as well as improving plant color traits.
Subject(s)
Plant Viruses/genetics , Plants/genetics , Gene Silencing , Plant Development , Gene Expression Regulation, Plant , Genetic VectorsABSTRACT
BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.
Subject(s)
Aphids/pathogenicity , Pest Control, Biological , Pinellia/chemistry , Plant Viruses , Tobacco , Blotting, Southern , Polymerase Chain Reaction , Promoter Regions, Genetic , Plants, Genetically Modified , Plant Leaves/chemistry , Transgenes , Disease Resistance , Crop ProtectionABSTRACT
La punta morada es una enfermedad que afecta la producción de algunas especies de solanáceas como la papa y el tomate, causando enrollamiento en las puntas de las hojas con una marcada coloración morada, decaimiento temprano de la planta y en la papa se observa tuberización aérea. Como patógenos asociados a la enfermedad se consideran al fitoplasma BLTVA y la bacteria Candidatus Liberibacter solanacearum. Dada la similitud en la sin-tomatología foliar que generan ambos patógenos, es difícil precisar cuál de ellos está implicado en la enfermedad. En Guatemala, existen reportes de la sintomatología típica de punta morada en las principales zonas productoras de papa y tomate, desconociéndose el agente asociado. La investigación determinó cuál de los dos patógenos reportados está asociados a la enfermedad en 12 municipios productores de papa y/o tomate en el país. Se realizaron ampli-ficaciones de ADN con cebadores específicos para cada patógeno asociado a la enfermedad. Por la alta incidencia del fitoplasma BLTVA en las muestras de papa (73.9%), en comparación a C. Liberibacter solanacearum (26%), este es considerado como el patógeno asociado más importante en papa. En las muestras de tomate, la incidencia del fitoplasma BLTVA (29.8%) y C. Liberibacter solanacearum del (27.6%) fue similar. Además, sobresale el primer reporte de la detección del fitoplasma BLTVA afectando el cultivo de tomate en Guatemala. Se sugiere un monitoreo constante, mediante métodos moleculares, para un diagnóstico certero y establecer medidas de manejo de la enfermedad para evitar su diseminación hacia zonas aún no afectadas.
The potato purple top is a disease that affects the production of some solanaceous species such as potatoes and tomatoes, causing curl at the tips of the leaves with a marked purple coloration, early decay of the plant, and aerial tuberization is observed in the potato. BLTVA phytoplasma and Candidatus Liberibacter solanacearum are considered as pathogens associated with the disease. Given the similarity in foliar symptoms generated by both pathogens, it is difficult to determine which one is involved in the disease. There are reports of the typical potato purple top symptoms in the main potato and tomato producing areas in Guatemala, being unknown the associated agent. The research determined which of the two reported pathogens is associated with the disease in 12 potatoes and/or tomato producing areas in the country. We performed DNA amplification with specific primers for each disease-associated pathogen. Due to the high incidence of BLTVA phytoplasma in potato samples (73.9%), com-pared to C. liberibacter solanacearum (26%), this is considered the most important associated pathogen in potatoes. In tomato samples, the incidence of BLTVA phytoplasma (29.8%) and C. liberibacter solanacearum (27.6%) was similar. Besides, the first report of the detection of the BLTVA phytoplasma affecting tomato cultivation in Gua-temala stands out. Using molecular methods, constant monitoring is suggested for an accurate diagnosis and to establish management measures for the disease to prevent its spread to areas not yet affected.
Subject(s)
Solanum tuberosum/virology , Solanaceae/virology , Phytoplasma Disease/microbiology , Plant Viruses/isolation & purification , Crop Production , DNA, Plant/analysis , Liberibacter/pathogenicityABSTRACT
Plant virus is one of the most economical devastating microorganisms for global agriculture. Although several strategies are useful for controlling viral infection, such as resistant breeds cultivation, chemical bactericides treatment, blocking the infection source, tissue detoxification and field sanitation, viral disease is still a problem in agricultural production. Genetic engineering approach offers various options for introducing virus resistance into crop plants. This paper reviews the current strategies of developing virus resistant transgenic plants.
Subject(s)
Agriculture , Crops, Agricultural , Genetics , Virology , Genetic Engineering , Plant Diseases , Virology , Plant Viruses , Plants, Genetically Modified , VirologyABSTRACT
La producción del cultivo de papa en Colombia se puede afectar por infección con diferentes patógenos virales, entre ellos, el Potato yellow vein virus (PYVV) que puede reducir la producción entre el 30 % y 50%. PYVV se ha diagnosticado molecularmente usando RT-PCR convencional en hojas sintomáticas y no sintomáticas. Sin embargo, no hay reportes sobre la detección y distribución viral en diferentes órganos infectados por PYVV en las plantas que expresan síntomas y sin síntomas. El objetivo de esta investigación, fue detectar a PYVV por RT-PCR convencional con cebadores específicos y por qRT-PCR (tiempo real) utilizando Sondas TaqMan® y analizar la distribución viral en plantas de S. tuberosum grupo Phureja cv. Criolla Colombia (papa criolla). Se logró la detección del virus en todos los órganos analizados (foliolo, peciolo, tallo aéreo y subterráneo, pedúnculo floral, pétalo y antera) mediante ambas técnicas, sin embargo, qRT-PCR fue 100 veces más sensible que la técnica convencional. Adicionalmente, se realizó la cuantificación absoluta del gen de la proteína mayor de la cápside de PYVV (CP). Los resultados indican que cuando la planta no expresa síntomas (NS), hay una distribución homogénea del virus, con un promedio del número de copias del gen CP de 4.09×107±2.35×107; mientras que en plantas sintomáticas el título viral es mayor (6.82×108±1.74×108) y la distribución heterogénea en los órganos, con mayor acumulación en órganos de la zona aérea. Este es el primer informe sobre la detección de PYVV en diferentes órganos de papa por medio de tiempo real, incluyendo las anteras y pedúnculo floral. La información debe ser de utilidad para el diagnóstico de PYVV y para adelantar estudios sobre la biología del virus y la relación con el huésped y el vector. La información suministrada debe ser valiosa para agricultores y fitomejoradores, además para programas de indexado de plantas contra PYVV y en la certificación de semilla.
Potato yield in Colombia could be affected by the infection with different viral pathogens, among which, Potato vein yellow virus (PYVV) could reduce potato production by 30% to 50%. PYVV has been diagnosed molecularly in symptomatic and symptomless leaves samples by conventional RT-PCR. However, the PYVV detection and distribution in different organs of symptomatic and symptomless plants have not been reported until now. The aim of this research was to detect and analyze PYVV distribution in different organs of infected S. tuberosum group Phureja cv. Criolla Colombia (papa criolla) plants using conventional and real time qRT-PCR usindTaqMan® probes. It was achieved to detect the virus in all analyzed organs (leaflets, petiole, peduncle, anther, petals, aerial and underground stem) by both techniques; however, qRT-PCR was 100 times more sensitive than the conventional technique. Additionally, the absolute quantification of coat major protein gene (CP) was determined. The results shown that in non symptomatic plants (NS), PYVV was distributed homogenously with an average CP gene copy number of 4.09 × 107 ± 2.35 × 107, while in symptomatic moderate and severe plants (M) or (S) the viral load was greater (6.82×108±1.74×108) with an heterogeneous distribution regarding the organ and with greater accumulation in the aerial organs. The results presented in this study will be important for PYVV detection and further studies on the virus biology, host and vector relations. The information should be useful to farmers, breeders, indexing and seed certification programs.
Subject(s)
Crinivirus , Plant Diseases , Plant Viruses , Solanum tuberosum , Plants , Plants, EdibleABSTRACT
The aim was to establish an effective screening microarray at genus level for Pospiviroid. We analyzed nucleotide sequences from Pospiviroid viroid and designed 19 probes with genus identification characteristics. The standards of these probes included the characters of (i) a GC content between 40 and 60%, (ii) less than 50% of single nucleotide, (iii) less than 4 continuous mononucleotides, and (iv) less than 6 nucleotides in the inner hairpin. We synthesized microarrays by using these probes on glass slides. The validation results of microarray probes show effective signals from chrysanthemum stunt viroid and tomato planta macho viroid standard samples hybridization. The sensitivity results show that the microarray detected 200 pg/microL of total RNA. The microarray can be used to screen Pospiviroid viroid.
Subject(s)
Base Composition , Base Sequence , Microarray Analysis , Nucleic Acid Hybridization , Plant Diseases , Virology , Plant Viruses , Classification , RNA , Viroids , ClassificationABSTRACT
Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.
Subject(s)
Blotting, Northern , Genome , Genome Size , Plant Viruses , Product Packaging , RNA , RNA, Viral , Tobacco , TymovirusABSTRACT
Grape virus diseases are a serious problem in Iran. Leaves and fruits of grape have been used for different purposes like cooking in Iran. The present investigation was carried out to study on the cytotoxic-activities of extracts of fruits and leaves of Vitis vinifera from both virus-free and virus-infected grape cultivars against breast cancer cell line [MDA-MB-231] and human embryonic kidney normal cell line [HEK 293]. In this experimental study, the considered grape cultivars were as follows: Rish Baba Sefid, Shahani Ghasre Shirin, Rotabi Zarghan, Asgari Najaf Abad, Fars, Kaj Angor Bojnord, Sarkesh Shiraz and Siahe Zarqan. A real-time multiplex polymerase chain reaction [real-time Multiplex PCR] assay was applied to detect virus infected cultivars. The cytotoxic effect of the methanol extracts of different Vitis vinifera varieties on cultured cells was monitored using [3- [4, 5-Dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide [MTT] assay at different concentrations [62.5, 125, 250, 500, 750, 1000 micro g mL[-1]]. Among these cultivars, Grapevine fanleaf virus [GFLV] along with related symptoms was detected in Siahe Zarqan and Fars. Methanolic extracts of leaves and fruits of Vitis vinifera from both virus free and virus infected cultivars showed a range of limited to moderate cytotoxic activity. However, methanol extract of leaves belonged to virus infected cultivars was found to have strong cytotoxic effect against MDA-MB-231 at different concentrations. Grapevine fanleaf virus [GFLV] can potentially increase the cytotoxicity of grape cultivars
Subject(s)
Antineoplastic Agents , Fruit , Plant Leaves , Plant Extracts , Plant VirusesABSTRACT
<p><b>OBJECTIVE</b>To test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana.</p><p><b>METHODS</b>Seven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement.</p><p><b>RESULTS</b>The 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression.</p><p><b>CONCLUSION</b>Transient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.</p>
Subject(s)
Genetic Vectors , Green Fluorescent Proteins , Genetics , Plant Viruses , Genetics , Plants, Genetically Modified , Genetics , Metabolism , Potexvirus , Genetics , RNA Interference , Tobacco , Genetics , MetabolismABSTRACT
The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5’ end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.
Subject(s)
Circular Dichroism , Elettaria/metabolism , Genome, Viral/genetics , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Models, Molecular , Mosaic Viruses/genetics , Mosaic Viruses/metabolism , Plant Viruses/genetics , Plant Viruses/metabolism , Potyvirus/genetics , Potyvirus/metabolism , Protein Refolding , Protein Structure, Secondary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21-23 nt RNA molecules. During infection, RNAi can act as an innate immune system to defend against viruses. As a counter-defensive strategy, silencing suppressors are encoded by viruses to inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and act via different mechanisms. In this review, we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses. We also review the modes of action proposed for some silencing suppressors.
Subject(s)
Animals , Humans , Gene Expression Regulation, Viral , Gene Silencing , Host-Pathogen Interactions , Mammals , Virology , MicroRNAs , Genetics , Metabolism , Plant Viruses , Physiology , Plants , Virology , RNA, Small Interfering , Genetics , Metabolism , Repressor Proteins , Genetics , Metabolism , Viral Proteins , Genetics , Metabolism , VirusesABSTRACT
Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA genome. Information for TYMV replication is limited, except that the 3'-terminal sequence and 5'-untranslated region are required for genome replication. When a foreign sequence was inserted at the position upstream of the coat protein (CP) open reading frame (ORF), replication of the recombinant TYMV was comparable to wild type, as long as an RNAi suppressor was provided. In contrast, when the foreign sequence was inserted between the CP ORF and the 3'-terminal tRNA-like structure, replication of the recombinant virus was not detected. This result suggests that the CP ORF contains an essential replication element which should be appropriately spaced with respect to the 3'-end. Analysis of TYMV constructs containing a part or a full additional CP ORF indicates that the 3' quarter of the CP ORF is required for TYMV replication.
Subject(s)
Animals , Brassica napus , Ecthyma, Contagious , Genome , Open Reading Frames , Plant Viruses , RNA , Tymovirus , VirusesABSTRACT
The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck 'Pêra'. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 μm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.
A doença leprose dos citros tem etiologia viral sendo o citrus leprosis virus seu agente etiológico. Demonstrou-se que há dois vírus distintos que causam sintomas de leprose em ci-tros: citoplasmático (CiLV-C) e o nuclear (CiLV-N). O objetivo desse estudo foi comparar as diferenças morfo-anatômicas nas lesões causadas por CiLV-C e por CiLV-N em laranjeira doce (Citrus sinensis (L.) Osbeck) 'Pêra'. As lesões foliares e dos frutos foram coletadas em Piracicaba/SP (tipo citoplas-mático) e em Monte Alegre do Sul/SP e Amparo/SP (tipo nuclear). As lesões foram fotografadas e em seguida fixadas em solução Karnovsky, desidratadas em série etílica, incluídas em historesina e secionadas em micrótomo rotativo. As lâminas foram coradas, analisadas e fotografadas. As lesões foliares e do fruto causadas pelos dois vírus eram morfologicamente distintas. Somente a lesão causada por CiLV-N apresentou três regiões bem definidas. Em ambas as lesões ocorreu acúmulo de substâncias lipídicas nas áreas necrosadas que se achavam envoltas por células com conteúdo protéico amorfo ou em gotas. Somente as lesões da folha e do fruto causadas pelo CiLV-N exibiram ductos traumáticos gomosos nos feixes vasculares.
Subject(s)
Citrus sinensis/virology , Plant Diseases/virology , Plant Viruses/classification , Citrus sinensis/ultrastructure , Microscopy, Electron, TransmissionABSTRACT
Plant Dicer-like (DCL) and Argonaute (AGO) are the key enzymes involved in anti-virus post-transcriptional gene silencing (AV-PTGS). Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates. In comparison with genome sequences of dicot-infecting Turnip mosaic virus (TuMV) and monocot-infecting Cocksfoot streak virus (CSV), viral-derived small interfering RNAs (vsiRNAs) displayed positive correlations between AV-PTGS efficiency and G+C content (GC%). Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases. This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations, and AGO associated Arabidopsis endogenous siRNA populations, indicating that plant AGOs operated with G-preference. We further propose a hypothesis that AV-PTGS imposes selection pressure(s) on the evolution of plant viruses. This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination, suggesting that plant virus evolution to have low GC% genomes would have a unique function, which is to reduce the host AV-PTGS attack during infections.
Subject(s)
Arabidopsis , Genetics , Virology , Base Composition , Dactylis , Genetics , Virology , Genes, Plant , Genes, Viral , Models, Genetic , Mustard Plant , Genetics , Virology , Plant Diseases , Genetics , Virology , Plant Proteins , Metabolism , Plant Viruses , Genetics , Virulence , Plants , Genetics , Virology , Potyvirus , Genetics , Virulence , RNA Interference , RNA, Plant , Genetics , RNA, Small Interfering , Chemistry , Genetics , Metabolism , RNA, Viral , Chemistry , Genetics , Metabolism , RNA-Induced Silencing Complex , Metabolism , Ribonuclease III , Metabolism , Selection, Genetic , Substrate SpecificityABSTRACT
Viruses form a major threat to the strawberry industry in Egypt causing severe economic losses. Rapid and simple methods for the detection of the major strawberry viruses are absent due to the lack of sensitive diagnostic tools. Plants showing virus-like symptoms [VLS] were collected from the field and subjected to indirect enzyme-linked immunosorbent assay [I-ELISA] tests using the polypeptide CP3 antiserum specific for whitefly transmitting geminivirus [WTG] in addition to Tomato yellow leaf curl geminivirus [TYLCV] polyclonal antiserum. In addition, plants were subjected to PCR as a molecular diagnosis test for further confirmation. Experiments proved that the virus could be transmitted mechanically, by viruliferous whiteflies and by grafting. Inoculated strawberry plants with viruliferous whiteflies showed curling and upward cup shape of the leaves. Primers specific for whitefly transmitted geminivirus were used in PCR diagnosis of the inoculated plants. Based on the positive molecular and serological diagnosis results, we concluded that the virus belongs to WTGs. PCR was also carried out for the inoculated plants using primers specific for TYLCV, however negative amplification was obtained indicating that the virus under this study is not a TYLCV. Electron microscopy of purified virus preparation showed the presence of geminate virus particles about 18x20 nm. Antiserum was raised against the purified virus and used for indirect-ELISA to measure the antigenicity of the raised antibodies. Western blot analysis was also used for confirmation of the specificity of the raised antiserum. The isolated virus was given the name strawberry leaf curl geminivirus [StLCV] and it represents the first record of WTG that infect strawberry plants in Egypt
Subject(s)
Plants, Edible , Plant Viruses/genetics , Base Sequence , Polymerase Chain Reaction , Plant Viruses/isolation & purification , Blotting, Western , Serologic TestsABSTRACT
Se describe el desarrollo de un ensayo de diagnóstico del tipo DAS-ELISA para la detección del virus de la hoja blanca del arroz (RHBV) en Venezuela. El virus fue aislado y caracterizado parcialmente a partir de material vegetal infectado proveniente de los estados Portuguesa y Guárico. Se obtuvieron inmunoglobulinas (IgGs) altamente específicas que reaccionaron contra el virus en diluciones de 1:1000 en los inmunoensayos. De acuerdo con los análisis serológicos, la incidencia del virus en las principales regiones productoras de arroz durante los años muestreados fue muy baja (11 por ciento), posiblemente debido a la siembra de líneas de arroz resistentes al virus en los estados encuestados y/o a la baja incidencia del insecto vector (Sogatodes orizicola) durante las estaciones de colecta. El desarrollo de este método de diagnóstico permitirá en el futuro la certificación fitosanitaria de material vegetal libre de RHBV.
Subject(s)
Insect Vectors , Oryza , Plant Leaves , Plant Viruses , Biology , VenezuelaABSTRACT
The sequences encoding the mouse heavy-chain [V[H]] and light-chain [V[L]] variable region genes were isolated by PCR and joined into a single-chain Fv [scFv] DNA by using a DNA linker encoding [Gly4Ser][3] peptide. The scFv DNA fragment was cloned into the phagemid pCANTAB5E and expressed in Escherichia coli as a fusion protein with M13 phage p3 polypeptide and E tag. The scFv fusion protein was displayed on the surfaces of recombinant M13 phages in the presence of the helper phage M13K07. High-affinity scFv phage-bodies against citrus tristeza virus [CTV] were enriched through affinity selection on immobilized recombinant CTV coat protein preparations. The selected recombinant phages were used to infect Escherichia coli HB2151 for the production of soluble scFv antibodies. One selected clone in HB2151 secreted a soluble scFv antibody that detected CTV in extracts of infected citrus plants with a sensitivity comparable to that of a commercial monoclonal antibody. The nucleotide sequence of the light-chain and heavy-chain portions were closely related to other published scFv against CTV. The potential of this scFv was demonstrated in routine field testing using an inexpensive tissue print-ELISA
Subject(s)
Animals, Laboratory , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Polymerase Chain Reaction , Immunologic Tests , Plant VirusesABSTRACT
Grapevine leafroll-associated virus 1 [GLRaV-1] was detected in the grapevine plants collected from different cultivated areas in Egypt and tested using different serological and molecular tools. Double-antibody sandwich ELISA [DAS-ELISA] was successfully carried out using GLRaV-1 polyclonal antibodies to detect infected plants. PCR with primers designed at the heat shock protein 70 [HSP70] gene region, a fragment of 271 bp of GLRaV-1, was used. Molecular hybridization with non-radioactive probes was used to detect the presence of virus particles. The partial sequence of HSP70 fragment from the Egyptian isolate of GLRaV-1 was performed and showed high identity [95%] with the Australian isolate of GLRaV-1 sequence. The molecular methods used for viral diagnosis showed a higher sensitivity in the detection of GLRaV-1 compared to DAS-ELISA. These procedures may serve as an alternative method for GLRaV-1 detection, due to the weak sensitivity of ELISA test to differentiate between the different isolates
Subject(s)
Plants, Edible , Plant Viruses/genetics , Enzyme-Linked Immunosorbent Assay , Polymerase Chain Reaction , Cloning, MolecularABSTRACT
A survey was conducted in 30 fields located at three different altitudes in Cartago, Costa Rica's main potato producing area. Twenty plants were sampled per farm, for a total of 600 samples with 200 samples per altitude. ELISA was used with commercial reagents to independently test for PVX, PVY, PVM, PVA, PVS, PLRV, PMTV, PAMV, PVV, PVT, APLV, APMoV and TRSV. The presence of the following viruses was determined: PVX (77 %), PAMV (62 %), PLRV (42 %), TRSV (42 %), PVT (39 %), PVV (37 %), PMTV (31%), PVY (30 %), PVS (19 %), PVM (13 %), PVA (8 %), and APMoV (8%). APLV was not detected in any sample. This is the first report in Costa Rica of the presence of the viruses PMTV, PAMV, PVV, PVT and APMoV. A high viral incidence in the tuber seed production area as well as a high rate of mixed infections is reported.
En Cartago, la zona productora de papa más importante de Costa Rica, se realizó un muestreo en 30 fincas ubicadas a tres altitudes. Se recolectaron 20 plantas por finca y 200 muestras por altitud. Todas las muestras se analizaron independientemente mediante ELISA, para PVX, PVY, PVM, PVA, PVS, PLRV, PMTV, PAMV, PVV, PVT, APLV, APMoV y TRSV, utilizando reactivos comerciales. Se identificó la presencia de PVX (77 %), PAMV (62 %), PLRV (42 %), TRSV (42 %), PVT (39 %), PVV (37 %), PMTV (31 %), PVY (30 %), PVS (19 %), PVM (13 %), PVA (8 %), y APMoV (8 %). No se detectó APLV en ninguna de las muestras analizadas. Se informan por primera vez la presencia en Costa Rica de los virus PMTV, PAMV, PVV, PVT y APMoV. Se informa la alta incidencia viral en la zona dedicada a la producción de tubérculos como semilla y la alta tasa de infecciones mixtas.
Subject(s)
Altitude , Plant Diseases/virology , Solanum tuberosum/virology , Plant Viruses/classification , Enzyme-Linked Immunosorbent Assay , Costa Rica/epidemiology , Plant Diseases/statistics & numerical data , Incidence , Prevalence , Plant Viruses/isolation & purificationABSTRACT
Avaliou-se a transmissão de Passion fruit woodiness virus (PWV) por Aphis gossypii (Glover). Em dois experimentos independentes, o afídeo transmitiu o PWV para maracujazeiros com taxas de 75 por cento e 100 por cento, ao se depositarem oito e doze afídeos virulíferos por planta, respectivamente. No final dos testes, observaram-se, em algumas plantas de maracujá, formas ápteras e ninfas de A. gossypii, sugerindo a colonização dessas plantas pelo afídeo. Esse parece ser o primeiro relato da colonização de Passiflora edulis f. flavicarpa (Deneger) por uma espécie de afídeo.