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

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

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

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

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

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

CITRUS TRISTEZA VIRUS RESISTANCE GENE LOCUS: SMALL RNA PROFILE AND PRELIMINARY EPIGENETIC STUDIES

Small interfering RNAs (siRNAs), play a vital role in epigenetics of plant virus-host plant interactions. It has been extensively studied at both the transcriptional and post-transcriptional levels. In plants, siRNAs initiate and manage gene silencing by directing DNA methylation and/or histone methylation. In Arabidopsis, the ~24 nt siRNAs directs DNA methylation (RNA-directed DNA methylation, RdDM) and chromatin remodeling at their target loci. Recent advances in highthroughput sequencing techniques has enabled thorough exploration of small RNAs populations and allow rapid analysis of massive datasets to assemble complete full-length genome sequence for different plant species. This large database of sequence information also allows identification of genome regions specifically matched by siRNAs that likely differ among tolerant, resistant or susceptible hosts and advance epigenetic studies on diseased plants. Resistance to Citrus tristeza virus (CTV), the most severe virus affecting Citrus spp., associated with a single dominant gene locus Ctv occurring in Poncirus trifoliata while all Citrus spp. are considered susceptible. This locus contains 22 putative genes, but their regulation and mechanism for resistance remains unknown. In our study, CTV was graft-inoculated on Carrizo citrange (Poncirus trifoliata x C. sinensis (I think) ) and C. aurantium (sour orange) seedlings, and the population of siRNA characterized by high-throughput sequencing using an ILLUMINA platform. The Ctv-derived siRNA (~2% of the total short reads) were dominated in both hosts by the 24-nt. However, CTV infection caused an increase in accumulation of 24-nt siRNA sequences homologous to the Ctv gene in Carrizo but it decreased in sour orange. Distribution of the 24nt along the Ctv gene locus (282Kb) had a clearly different distribution between the two host. The predominant hot spot of siRNA in Carrizo mapped in the putative gene Ctv-20, whereas in sour orange it associated to the intergenic region between the putative genes Ctv-11 and Ctv-12, where a Copia-like retrotransposon C is located. This distribution profile was conserved for each species between CTV-infected and uninfected plants but, as previously mentioned, the frequency of the 24nt siRNAs was altered by the presence of the virus. We supposed that the different profile of 24nt between the two host in the locus ctv is due to RdDM mechanisms. To demonstrate the methylation status of the resistance locus we performed a bisulfite treatment of DNA. in which unmethylated cytosine was converted to uracile, while methylated cytosine did not react. A methylcytosines mapping was carried out on Ctv-11 and Ctv-12 sequences. By specific software were found 5 different CpG islands in the Copia-likeretrotransposon sequence and 42 primer pair were designed. The PCR analyses have been carried out using MSP and BSP primers followed by combined bisulfite restriction analysis (COBRA)

Draft Genome Sequence of CO33, a Coffee-Infecting Isolate of Xylella fastidiosa

The draft genome sequence of Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%

Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca

Background: The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. Results: A global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. Conclusions: Collectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response