Molecular diagnosis of grapevine fleck virus

A digoxigenin-labelled riboprobe was developed for the detection of grapevine fleck virus (GFkV) in infected tissues of grapevine leaves, roots and canes. The probe was GFkV-specific and was successfully used for virus identification both with dot spot assays, using alkali-treated crude sap, and tissue blot assays, using cross and longitudinal sections of leaf petioles. Primers designed for the amplification by reverse transcription-polymerase chain reaction of a viral genome fragment 243 nucleotides in size, gave also positive and repeatable results. These newly developed molecular-based detection tools extend the range of available procedures for the sensitive identification of GFkV in naturally infected hosts

A survey of grapevine fanleaf nepovirus isolates for the presence of satellite RNA

Grapevine fanleaf virus (GFLV) isolates from different geographical origins were surveyed for natural occurrence of satellite RNA. The results of molecular hybridization assays indicated that 5 isolates out of 34 tested, support the multiplication of a satellite RNA, both in Chenopodium quinoa and grapevine. The satellite molecules appear to have a high degree of sequence homology with, and the same size of, the satellite RNA supported by GFLV-F13 strain, isolated and characterized in France

Non-radioactive molecular probes for the detection of three filamentous viruses of the grapevine

Digoxigenin-labelled riboprobes (DIG-RNA) were developed for the detection in infected tissue extracts of grapevine trichovirus A (GVA), grapevine trichovirus B (GVB) and grapevine leafroll-associated closterovirus III (GLRaV III). The probes were virus-specific and could be used for the identification of the respective viruses in sap expressed from infected Nicotiana species (GVA and GVB) and in total nucleic acid extracts from infected grapevines (GVA, GVB and GLRaV III). The efficiency of detection was the same as (GLRaV III), or slightly less than (GVA), with ELISA. No difference was found in detection efficiency between DIG-RNA and cDNA radioactive probes

Grapevine virus diseases: economic impact and current advances in viral prospection and management.

Grapevine (Vitis spp.) is a major vegetative propagated fruit crop with high socioeconomic importance worldwide. It is susceptible to several graft-transmitted agents that cause several diseases and substantial crop losses, reducing fruit quality and plant vigor, and shorten the longevity of vines. ] The vegetative propagation and frequent exchanges of propagative material among countries contribute to spread these pathogens, favoring the emergence of complex diseases. Its perennial life cycle further accelerates the mixing and introduction of several viral agents into a single plant. Currently, approximately 65 viruses belonging to different families have been reported infecting grapevines, but not all cause economically relevant diseases. The grapevine leafroll, rugose wood complex, leaf degeneration and fleck diseases are the four main disorders having worldwide economic importance. In addition, new viral species and strains have been identified and associated with economically important constraints to grape production. In Brazilian vineyards, eighteen viruses, three viroids and two virus-like diseases had already their occurrence reported and were molecularly characterized. Here, we review the current knowledge of these viruses, report advances in their diagnosis and prospection of new species, and give indications about the management of the associated grapevine diseases. Index terms: Vegetative propagation, plant viruses, crop losses, berry quality, next-generation sequencing. VIROSES EM VIDEIRAS: IMPACTO ECONÔMICO E RECENTES AVANÇOS NA PROSPECÇÃO DE VÍRUS E MANEJO DAS DOENÇAS DE ORIGEM VIRAL A videira (Vitis spp.) é propagada vegetativamente e considerada uma das principais culturas frutíferas por sua importância socioeconômica mundial. Ela é suscetível a vários agentes transmitidos por meio da enxertia, os quais causam diversas doenças e significativas perdas na produtividade e produção, redução na qualidade dos frutos, no vigor da planta e na longevidade dos vinhedos. A propagação vegetativa e o frequente intercâmbio de material propagativo entre países contribuem para a disseminação destes patógenos, favorecendo a emergência de doenças complexas. Seu ciclo de vida perene acelera ainda mais a mistura e a introdução de vários agentes virais em uma mesma planta. Atualmente, aproximadamente 65 vírus pertencentes a diferentes famílias foram reportados infectando videiras, embora nem todos causem doenças economicamente relevantes. As viroses do enrolamento da folha, complexo do lenho rugoso, degenerescência e mancha-das-nervuras da videira são as quatro principais desordens que têm importância econômica mundial. Além disso, novas espécies e estirpes virais foram identificadas e associadas a limitações economicamente importantes para a produção de uvas. Em vinhedos brasileiros, dezoito espécies virais, três viroides e duas doenças semelhantes a viroses já tiveram sua ocorrência reportada e foram molecularmente caracterizados. Aqui, nós revisamos o conhecimento atual dessas viroses, os recentes avanços na diagnose e prospecção viral, e fornecemos recomentações sobre o manejo das viroses da videira. Termos para indexação: Propagação vegetativa, vírus de plantas, redução da produtividade e produção, qualidade das bagas, sequenciamento de nova geração

Complete nucleotide sequence and genome organization of Grapevine fleck virus

The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3′-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5′ and 3′ ends, respectively. ORF 1 potentially encodes a 215·4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24·3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3′ end of the viral genome and encode proline-rich proteins of 31·4 kDa (p31) and 15·9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus

Xylella fastidiosa in Olive in Apulia: Where We Stand

A dramatic outbreak of Xylella fastidiosa decimating olive was discovered in 2013 in Apulia, Southern Italy. This pathogen is a quarantine bacterium in the European Union (EU) and created unprecedented turmoil for the local economy and posed critical challenges for its management. With the new emerging threat to susceptible crops in the EU, efforts were devoted to gain basic knowledge on the pathogen biology, host, and environmental interactions (e.g., bacterial strain(s) and pathogenicity, hosts, vector(s), and fundamental drivers of its epidemics) in order to find means to control or mitigate the impacts of the infections. Field surveys, greenhouse tests, and laboratory analyses proved that a single bacterial introduction occurred in the area, with a single genotype, belonging to the subspecies pauca, associated with the epidemic. Infections caused by isolates of this genotype turned to be extremely aggressive on the local olive cultivars, causing a new disease termed olive quick decline syndrome. Due to the initial extension of the foci and the rapid spread of the infections, eradication measures (i.e., pathogen elimination from the area) were soon replaced by containment measures including intense border surveys of the contaminated area, removal of infected trees, and mandatory vector control. However, implementation of containment measures encountered serious difficulties, including public reluctance to accept control measures, poor stakeholder cooperation, misinformation from some media outlets, and lack of robust responses by some governmental authorities. This scenario delayed and limited containment efforts and allowed the bacterium to continue its rapid dissemination over more areas in the region, as shown by the continuous expansion of the official borders of the infected area. At the research level, the European Commission and regional authorities are now supporting several programs aimed to find effective methods to mitigate and contain the impact of X. fastidiosa on olives, the predominant host affected in this epidemic. Preliminary evidence of the presence of resistance in some olive cultivars represents a promising approach currently under investigation for long-term management strategies. The present review describes the current status of the epidemic and major research achievements since 2013

First report on the occurence of grapevine rupestris stem pitting-associated virus in Moroccan grapevines

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Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae

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Propuesta de trabajo interdisciplinario: ¿hay buenos aires en buenos aires? La enseñanza de estadística en un contexto de biología

La siguiente es una experiencia didáctica sobre el uso de la estadística para el análisis de los líquenes como bioindicadores de contaminación atmosférica. Se propuso la realización de un trabajo de modelización intermatemática que permitiese al alumno de 1º Año de la Escuela Secundaria la aplicación de la estadística sobre datos obtenidos en el muestreo de líquenes epífitos y su posterior análisis como bioindicadores de la calidad del aire. Se buscó favorecer de esta manera el análisis crítico del uso de la estadística por parte de los alumnos. Los resultados mostraron eficiencia en los alumnos al momento de tomar criterio por las herramientas estadísticas pertinentes para realizar el análisis de datos obtenidos y poder tomar así conclusiones válidas con respecto a las hipótesis planteadas.Facultad de Humanidades y Ciencias de la Educació

Serological detection of Grapevine rupestris stem pitting-associated virus (GRSPa V) by a polyclonal antiserum to recombinant virus coat protein

The coat protein gene of Grapevine rupestris stem pitting-associated virus (GRSPaV) was amplified with primers based on the completely sequenced Californian GRSPaV isolate, The protein expressed in Escherichia coli was used to raise an antiserum in rabbit. This antiserum was successfully used to detect virus coat protein in infected grapevine extracts either spotted on polyvinyl difluoride membranes (dot immunobinding) or blotted on membranes after gel separation (Western blot). The antiserum titre was 1:5,000 in Western blot. GRSPaV was detected in leaf petioles and cortical scrapings from dormant canes during the whole vegetative season. Several accessions of Vitis rupestris, currently used as presumptive virus-free indicators of Rupestris stem pitting, were found to be infected by this virus. While the application of the antiserum in ELISA was ineffective, the availability of similarly simple and effective serological tools, such as dot immunobinding, may allow a wide survey for GRSPaV