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PubMed2005

A RT/PCR-partial restriction enzymatic mapping (PREM) method for the molecular characterisation of the large satellite RNAs of Arabis mosaic virus isolates.

The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV.

Amino Acid Sequence↗
PubMed2006

Efficient methods for sample processing and cDNA synthesis by RT-PCR for the detection of grapevine viruses and viroids.

Template preparation is important in reverse-transcription polymerase chain reaction (RT-PCR)-based detection methods. A TissueLyser with tungsten carbide beads was used for simultaneous processing of up to 48 samples under the same conditions in the development of a simple and rapid procedure to prepare a plant extract for RT reaction. A sandpaper method was also developed by which wood tissue of dormant cuttings could be macerated easily to process with minimal time and effort. It was also demonstrated that the combination use of random primers and oligo dT primer in an RT reaction was efficient for simultaneous cDNA synthesis of viral and viroid RNAs in plant extracts. These template preparation methods were used for the amplification of Grapevine leafroll-associated virus-1,-2, and -3; Grapevine virus A and B; Grapevine rupestris stem pitting-associated virus; Grapevine fleck virus; and Grapevine fanleaf virus. All these viruses tested in this study were reliably detected up to a 10(3)-fold or higher dilution of the original extract. Besides, Hop stunt viroid and Grapevine yellow speckle viroid 1 were well amplified in the same manner as the template preparation and following PCR for virus detection. These methods would contribute to cost-effective testing of a large number of samples through the year and help to detect viral pathogens in grapevine.

DNA Primers↗
PubMed2006

Development of degenerate and species-specific primers for the differential and simultaneous RT-PCR detection of grapevine-infecting nepoviruses of subgroups A, B and C.

Based on the nucleotide sequence homology of RNA-1 and RNA-2 of nepoviruses isolated from grapevines, three sets of degenerate primers, one for each of the three subgroups of the genus (A, B and C), were designed and proved effective for RT-PCR detection of subgroups in infected grapevines and herbaceous hosts. Primers designed specifically for detecting subgroup A species amplified a fragment of 255 bp from samples infected by Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV) and Grapevine deformation virus (GDefV), but not from samples infected by other nepovirus species. Similarly, primers for detection of subgroup B nepoviruses amplified a 390 bp product from samples infected by Grapevine chrome mosaic virus (GCMV), Tomato black ring virus (TBRV), Grapevine Anatolian ringspot virus (GARSV) and Artichoke Italian latent virus (AILV). The third set of primers amplified a 640 bp fragment, only from samples infected by subgroup C nepoviruses, i.e Tomato ringspot virus (ToRSV) Grapevine Bulgarian latent virus (GBLV), and Grapevine Tunisian ringspot virus (GTRSV). These primers were able to detect simultaneously all viral species belonging to the same subgroup and to discriminate species of different subgroups. Multiplex-PCR detection of subgroup A and B nepoviruses was obtained using a specific primer (sense for subgroup A and antisense for subgroup B) for each of the species of the same subgroup in combination with the degenerate subgroup-specific primers. In this way it was possible to detect four different viral species in single samples containing mixtures of viruses of the same subgroup. In particular, for viruses of subgroup A (TRSV, GFLV, ArMV and GDefV) amplicons of 190, 259, 301 and 371 bp were obtained, whereas amplicons of 190, 278, 425 and 485 bp, respectively, were obtained from samples infected with viruses of subgroup B (GCMV, AILV, GARSV and TBRV).

Amino Acid Sequence↗
PubMed2007

Real-time RT-PCR (TaqMan) assays for the detection of Grapevine Leafroll associated viruses 1-5 and 9.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Grapevine Leafroll associated viruses 1-5 and 9 (GLRaV-1-5 and -9). The assays were evaluated against a wide range of geographically distributed isolates. Geographical locations included South Africa, Europe, Australia, Asia, Latin America and the United States. Sequences were piled up from the most conserved regions of these geographically diverse isolates and TaqMan primers and probes were designed, targeting the regions with 100% sequence identity. Improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control to validate the quality of the extracted RNA proved to generate better diagnostic assays. The real-time TaqMan RT-PCR assays were compared to the conventional RT-PCR assays for the detection of viruses using purified total RNA as well as crude extract. The data showed that when using total RNA extracted either by the Qiagen RNeasy method or by an ABI automated system more isolates were detected in comparison to crude extract. The optimum volume of crude extract prepared in GES for use in real-time TaqMan RT-PCR cocktail was determined to be 1 microl per reaction. In addition this report showed that TaqMan RT-PCR was more sensitive than conventional one-step RT-PCR for testing different isolates of these viruses either using RNA or crude tissue extract.

Closteroviridae↗
PubMed2007

A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll-associated virus 2 variants in grapevine.

Grapevine leafroll syndrome is caused by a complex of up to nine different Grapevine leafroll-associated viruses (GLRaV-1-9) with GLRaV-2 being reported as one of the most variable species of this group. Many methods, including indexing, serological and molecular procedures, have been developed for the detection of GLRaV-2. However, due to the low concentration of the virus in plants and the high variability of GLRaV-2, a method with improved sensitivity and with the capacity to detect of all known variants is required. Such improvement is essential for grapevine rootstocks, as these are suspected to harbour frequent GLRaV-2 infections difficult to detect, thus contributing to the spread of the leafroll disease. The development of new universal primers is described using a target sequence located in the 3' end of the virus genome. These primers were combined with a one-step SYBR Green real-time RT-PCR assay to achieve quantitative detection. All 43 GLRaV-2 isolates tested in this study were identified readily and reproducibly, regardless of their geographical origin or variety of grapevine. Using the procedure developed in this study, the sensitivity was increased 125 times compared to a conventional single-tube RT-PCR. This real-time method opens new perspectives for the sanitary selection of grapevine and in leafroll 2 disease monitoring.

3' Flanking Region↗
PubMed2004

Neuroprotective effects of grape seed extract on neuronal injury by inhibiting DNA damage in the gerbil hippocampus after transient forebrain ischemia.

Grape seed extract (GSE) possess cardioprotective abilities by functioning as in vivo antioxidants and by virtue of their ability to directly scavenge ROS including hydroxyl and peroxyl radicals. In the present study, we investigated the neuroprotective effects of grape seed extract (GSE) in the gerbil hippocampus after 5 min transient forebrain ischemia. Neuronal cell density in GSE-treated ischemic animals was significantly increased as compared with vehicle-treated ischemic animals 4 days after ischemic insult. In the GSE-treated groups, about 60% of pyramidal cells of the sham-operated group were stained with cresyl violet 4 days after ischemic insult. In this study, we found that GSE had neuroprotective effects on neuronal injury by inhibiting DNA damage in the CA1 region after ischemia. In vehicle-treated groups, 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivity was significantly changed time-dependently, whereas the immunoreactivity in the GSE-treated group was similar to the sham-operated group. In addition, we confirmed that astrocytes and microglia did not show significant activation in the CA1 region 4 days after ischemia-reperfusion, because many CA1 pyramidal cells were not damaged. Therefore, these results suggest that GSE can protect ischemic neuronal damage by inhibiting DNA damage after transient forebrain ischemia.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2005

Effects of black grape extract on activities of DNA turn-over enzymes in cancerous and non cancerous human colon tissues.

Effects of extract of dried whole black grape including seed on adenosine deaminase (ADA), 5' nucleotidase (5'NT) and xanthine oxidase (XO) enzymes were investigated in cancerous and non-cancerous human colon tissues. Enzyme activities were measured in 20 colon tissues, 10 from cancerous region and 10 from non cancerous region with and without pre incubation with black grape extract. ADA and 5'NT activities were found increased and that of the XO decreased in the cancerous tissues relative to non cancerous ones. After incubation period with black grape extract for 12 h, ADA and 5'NT activities were found to be significantly lowered but that of XO unchanged in both cancerous and non cancerous tissues. Results suggest that ADA and 5'NT activities increase but XO activity decreases in cancerous human colon tissues, which may provide advantage to the cancerous tissues in obtaining new nucleotides for rapid DNA synthesis through accelerated salvage pathway activity. Black grape extract makes significant inhibition on the ADA and 5'NT activities of cancerous and non cancerous colon tissues, thereby eliminating this advantage of cancer cells, which might be the basis for the beneficial effect of black grape in some kinds of human cancers.

5'-Nucleotidase↗
PubMed2006

Proteomics analysis of the actions of grape seed extract in rat brain: technological and biological implications for the study of the actions of psychoactive compounds.

Grape seed extract (GSE) is a commonly available dietary supplement taken for the anti-oxidant activity that's attributed to its proanthocyanidin (oligomers of monomeric polyphenols) content. Similar polyphenol-enriched preparations from blueberries and soy have shown protection against ovariectomy-induced or age-related cognitive deficits, suggesting that the molecular changes induced by these polyphenol preparations correlated with behavioral benefit. We hypothesized that ingestion of polyphenol-enriched preparations such as GSE would be manifested as protein changes that would be consistent with neuroprotection. Proteomics technology, namely 2D gel electrophoresis and mass spectrometry, identified quantitative changes in specific proteins induced in adult rat brain following ingestion of a powdered preparation of GSE. As recently reported [Deshane, J., Chaves, L., Sarikonda, K.V., Isbell, S., Wilson, L., Kirk, M., Grubbs, C., Barnes, S., Meleth, S. and Kim, H., 2004. Proteomics analysis of rat brain protein modulations by grape seed extract. Journal of Agricultural and Food Chemistry 52, 7872-7883.], the direction of change for the majority of the affected proteins was opposite to the direction the proteins were changed in either Alzheimer disease or in transgenic mouse models of dementia. A conservative conclusion is that GSE has neuroprotective activity, by affecting specific proteins in particular ways. In this chapter, elements of proteomics-type analysis are discussed that demonstrate the power of the technology to enable discovery of proteins involved in the response of the brain to a stimulus whether it be a dietary supplement, or a psychoactive drug. The fact that GSE affects proteins implicated in cognitive disorders suggests moreover that GSE may have impact on the actions of psychoactive drugs by maintaining an overall viability of the nervous system.

Animals↗
PubMed2006

Grape phytochemicals: a bouquet of old and new nutraceuticals for human health.

Health benefits associated with Mediterranean diets are due to the significantly large intake of functional plant foods and beverages, i.e., fruits, vegetables, cereals, legumes, nuts, wine, beer, and olive oil, containing a great array of bioactive phytochemicals or nutraceutical compounds. Therefore, the low risk of chronic diseases, such as coronary hearth disease and certain cancers, observed in some population groups, results from a diversified eating style, either in term of foods and food components. The paradigm of the relationship between the chemical diversity of a particular food and the array of its biological activities may be symbolized by grape. Despite the extensive knowledge about phenylpropanoids, principally polyphenols (stilbenes and anthocyanins) and condensed tannins (proanthocyanidins), in grape and wine, little it is known about the other compounds, such as tetrahydro-beta-carbolines. Recently, it has been attached importance to the dietary indoleamines, melatonin, and serotonin, in different plant foods, including grape, thus further supporting the hypothesis that health benefits, associated with Mediterranean dietary style, are due to plant food chemical diversity.

Fruit↗
PubMed2005

Detection and characterization of protease secreted by the plant pathogen Xylella fastidiosa.

Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a copolymerized substrate. Three major protein bands were produced by the citrus strain with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9,713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) activity gel indicated that the protease of Xylella fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30 degrees C with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and beta-1,3-glucanase activities were also detected in cultures of Xylella fastidiosa (citrus). From these results, it is suggested that proteases produced by strains of Xylella fastidiosa from citrus and grape, belong to the serine- and metallo-protease group, respectively.

Chitinases↗
PubMed2006

Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries.

Brettanomyces bruxellensis spoilage is a serious problem for the wine industry. Mainly, by producing 4-ethylphenol and 4-ethylguaiacol, it confers off-odors to the wine and changes its aromatic quality. The presence of B. bruxellensis cells on the berry was speculated but it had never been clearly demonstrated. On grape berries, the microbial ecosystem is highly diverse and the population of B. bruxellensis can be very small. The aim of our study was to reveal and confirm the presence of B. bruxellensis on the surface of grape berries. We developed an enrichment medium for B. bruxellensis in order to overcome the detection limit of the molecular methods (species-specific PCR, ITS-RFLP PCR, PCR-DGGE). This medium, named EBB medium, made it possible to detect B. bruxellensis after 10 days of culture. For the first time, the presence of B. bruxellensis has been clearly established in several vineyards and at different stages of the grape development after the veraison. This work led to the conclusion that the grape berry is the primary source of B. bruxellensis. Grape growers and winemakers should take these results into account when deciding on the treatment to apply in the vineyards and the must. With the information provided here, B. bruxellensis prevention could start in the vineyard.

Colony Count, Microbial↗
PubMed2006

Antifungal activity of diketopiperazines extracted from Alternaria alternata against Plasmopara viticola: an ultrastructural study.

Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10(-3), 10(-4), 10(-5) and 10(-6)M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.

Alternaria↗
PubMed2006

Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues.

Plasmopara viticola, the causal agent of grapevine downy mildew, is an obligate biotrophic oomycete that grows in the intercellular spaces of host tissues and develops haustoria in the cells. Histological observations are the most effective methods to visualize and quantify the development of the infection structures. We chose two staining techniques leading to high resolution and contrast between parasite structures and host-plant tissues with a minimum of sample preparation: Blankophor and KOH-aniline blue fluorescent stainings. Blankophor (50 ppm in water or 15% KOH) staining was used to study the zoospore encystement on the leaf surface after release from sporangia. The aniline blue dye (0.05% in 0.067 M K(2)HPO(4), pH 9-9.5, after hot KOH whitening) was used to observe the invasive structures inside host tissues that lead to the production of sporangiophores and infectious sporangia. We tested modifications of some parameters of the procedures to determine the most appropriate for high throughput analyses adapted to our pathosystem and equipment facilities.

Aniline Compounds↗
PubMed2005

Improvement of the cDNA-AFLP method using fluorescent primers for transcription analysis in bacteria.

Here the cDNA-fluorescent amplified fragment length polymorphism (cDNA-FAFLP) technique, an improvement of cDNA-AFLP method, was used in order to analyse expression profiling in bacteria. The obtained results were validated by real-time PCR. This is the first report that validates the foreseen transcription pattern by cDNA-FAFLP with the application of real-time PCR in bacteria. This new protocol offers the possibility of quick and reliable analysis of transcription profiles, also avoids the problem linked with the use of radioisotopes, and allows a quick identification of genes differentially expressed in bacteria.

Bacteria↗
PubMed2007

A new TaqMan method for the identification of phytoplasmas associated with grapevine yellows by real-time PCR assay.

Three real-time PCR systems for direct detection of phytoplasmas associated to Flavescence dorée (FD), Bois noir (BN) and aster yellows (AY) diseases were developed. TaqMan probes and primers were designed on the 16S ribosomal RNA sequences of phytoplasma genome. A further TaqMan assay, targeting a grapevine gene encoding for the chloroplast chaperonin 21, was developed in order to check the DNA quality and to verify the absence of PCR inhibition. A comparison between real-time PCR and conventional nested-PCR methods for phytoplasma detection was carried out on several reference samples from grapevine, periwinkle, other host plants and insect species. Detection of FD, BN and AY phytoplasma DNA on infected specimens was rapid, specific and reproducible. Sensitivity was as high as nested-PCR assay. The two procedures were then used on about 450 samples collected from grapevines showing yellows symptoms. The results showed that real-time PCR approach for phytodiagnostic purposes was more advantageous than nested-PCR method with regard to rapidity of the assay and reduced risk of sample cross contamination. These new protocols represent an improvement of existing analytical methods and could be used as a reliable diagnostic procedure in certification and control programs.

Animals↗
PubMed2006

Influence of the region of origin on the mycobiota of grapes with emphasis on Aspergillus and Penicillium species.

A three-year study was undertaken to investigate the fungal species present on the surface of grape berries from Portuguese vineyards in four winemaking regions. Emphasis was given to Aspergillus and Penicillium species due to their relevance for mycotoxin production. From the 3517 fungal strains detected 27 genera were identified. The region of origin markedly influenced the spoilage fungal population to which berries are exposed. The main differences found were in the incidence of A. niger aggregate, Botryis cinerea and Penicillium species (P. brevicompactum, P. citrinum, P. glabrum/spinulosum, P. expansum, P. implicatum and P. thomii). In more humid climates, Botrytis seems to be the main pathogen and spoiling agent, and the incidence of black Aspergillus is minimal. The most important mycotoxin-producing species found was A. carbonarius, which is an ochratoxin A producer. The present study provides a detailed description of the fungi found on the berry surface of Portuguese grapes and shows the Aspergillus and Penicillium species, which vary significantly by geographic origin. This is of crucial importance to understand fungal hazards for grapes and wine and to the knowledge of field ecology of the species.

Aspergillus↗
PubMed2006

Detection and quantification of Erysiphe necator DNA in wine grapes and resultant must and juice.

Powdery mildew of grapevines is difficult to assess visually at the weighbridge, particularly in large consignments of machine-harvested fruit. To facilitate accurate methods for the detection and quantification of the disease in grape samples obtained from both the vineyard and winery, we developed a DNA probe for the pathogen Erysiphe necator. The E. necator-specific 450 bp DNA fragment pEnA1, targets highly repetitive sequences and was isolated from a partial genomic library. In screening for species specificity, clone pEnA1 was used in slot-blot hybridization and detected E. necator DNA from grapes and resultant must and juice, but not from clarified juice and wine. The detection threshold was approximately 50 pg of E. necator DNA per 100 ng total DNA of grape sample and was equivalent to 1-5% of a grape bunch visually affected by powdery mildew. Disease severity, expressed as the percentage of surface area of a bunch with powdery mildew, and E. necator DNA content were highly correlated, r2=0.955, P<0.001. The DNA-based hybridization assay has the potential to predict the severity of powdery mildew in grape samples from the vineyard and in must and juice samples at the winery. The DNA sequence of clone pEnA1 was used to design species-specific primers, the results maintaining the same specificity patterns observed in the initial hybridization assays. The PCR-based assay was sensitive enough to detect approximately 1 pg DNA, being equivalent to 1 conidium per sample. This is the first report to date of the detection of all known phenetic groups of E. necator DNA and of the quantification of DNA from grape samples at the winery. Accurate information on the extent of powdery mildew contamination of grape lots would enable wineries to make more informed decisions about the use of fruit and must.

Ascomycota↗
PubMed2005

Intracellular oxidative activity and respiratory burst of leukocytes isolated from multiple sclerosis patients.

Oxidative damage induced by free radicals and reactive oxygen species (ROS) have been suggested to play an important role in the development of autoimmune diseases such as multiple sclerosis (MS) disease and it has been hypothesised that oxidative injury could mediate demyelination and axonal injury in MS subjects. In our study, we compared intracellular oxidative activity and the respiratory burst activity in MS patients (n=20) and healthy controls (n=15) using leukocytes as cellular model. At this purpose, intracellular ROS levels were evaluated by fluorometric assay using the 2'-7'-dichlorodihydrofluorescin diacetate probe (H(2)DCFDA) in untreated or in leukocytes stimulated with phorbol-12-myristate-13-acetate (PMA). Our results demonstrate that the intracellular spontaneous ROS production in leukocytes from MS patients was higher with respect to cells from control subjects (p<0.001). PMA addition induced a higher formation of ROS both in leukocytes from MS patients and controls (p<0.001). The PMA-induced production of ROS was significantly higher in leukocytes from MS with respect to controls (p<0.001). Significant positive correlations were established between intracellular spontaneous or PMA-induced production of ROS in leukocytes isolated from MS patients and the clinical parameters used to evaluate disease disability such as expanded disability status scale (EDSS), brain lesions evaluated by MRI and visual evoked potential (VEP) (p<0.001). In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects confirming the role of oxidative stress in multiple sclerosis.

Adult↗