Grapevine Tissues Research Articles

Seasonal Distribution of Micronutrients in Mature ‘Concord’ Grape: Boron, Iron, Manganese, Copper, and Zinc

This study investigated the distribution of the micronutrients boron (B), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) in 42-year-old ‘Concord’ grapevines (Vitis labruscana Bailey) grown in a calcareous soil to understand seasonal partitioning and distribution of micronutrients throughout various grapevine tissues. In 2006 and 2007, four vines each were excavated at winter pruning, budbreak, the three- to four-leaf stage, bloom, veraison, harvest, and postharvest. Separated plant organs were measured for biomass and analyzed for B, Fe, Mn, Cu, and Zn. The results showed that seasonal patterns of micronutrient concentrations varied considerably with respect to organ and growth stage. Leaf blades, shoot tips, and petioles had the highest concentration of B at bloom and Mn at harvest, whereas Fe, Cu, and Zn concentrations were highest in fine roots but values varied over time each year. Whereas seasonal patterns of Fe, Cu, and Zn contents differed year by year, B and Mn contents had a similar pattern over both years. Translocation of B and Mn from woody tissue to actively growing organs occurred at the beginning of the season. The majority of B uptake occurred between bloom and veraison, whereas that of Mn occurred between bloom and harvest. There were similar B concentrations in shoot tips and leaf blades. Boron remobilization to woody tissues from the leaves occurred between veraison and harvest, suggesting moderate, late-season, phloem mobility of B in ‘Concord’ grapevines. Microsite differences in soil pH likely contribute to variable nutrient availability around the root system, demonstrated by high variability of Fe, Cu, and Zn contents in different vine organs.

Effect of osmotic stress and post-stress recovery on the content of phenolics and properties of antioxidants in germinating seeds of grapevine Vitis californica

The tested material consisted of grapevine <em>Vitis californica</em> stratified seeds germinated under optimum conditions (+25°C in water), under osmotic stress (-0.2 MPa in PEG solution) and submitted to recovery after stress (+25°C in water). The germinating seeds were determined to contain tannins, catechins and the following phenolic acids: gallic, caffeic, <em>p</em>-coumaric and ferulic. The acids occurred in free, ester- and glycoside-bound forms. The dominant form of phenolic acids was the ester-bound fraction. Gallic acid was the most abundant phenolic acid in germinating seeds, while ferulic acid appeared in the smallest amounts. Our analysis of tannins demonstrated that osmotic stress depressed their concentration. Presence of catechin group compounds such as catechin and epicatechin was also determined. In each sample epicatechin was dominant. The total concentration of catechin increased under stress conditions and declined during post-stress recovery. Catechins are a constituent of tannins and their increase under osmotic stress is most probably caused by the breakdown of some tannins in seeds germinating under stress conditions. Samples submitted to osmotic stress were also found to contain less of total phenolic compounds, whereas in samples which underwent post-stress recovery the total level of phenolic compounds increased. Compared to extracts from seeds germinating under optimum conditions, osmotic stress depressed the capacity of extract to scavenge DPPH<sup>●</sup> (2,2-diphenyl-1-picrylhydrazyl) and ABTS<sup>●+</sup> – 2,2-Azino-bis (3-etylbenzothiazoline-6-sulfonic acid) free radicals, but the antioxidant activity rose in seeds submitted to recovery after stress. Positive correlation was therefore demonstrated between the total content of phenolic acids in germinating grapevine seeds and the reducing power of extracts obtained from these seeds and their free radical scavenging activity. The results suggest that osmotic stress inhibits the activity of antioxidizing enzymes in germinating grapevine seeds. Thus, the antioxidative defence system is largely blocked under osmotic stress. It seems that a very high oxidoreductive potential in grapevine tissues prior to occurrence of osmotic stress is essential for maintaining proper homeostasis of oxidation and reduction reactions.

Detection of Botryosphaeriaceae species within grapevine woody tissues by nested PCR, with particular emphasis on the Neofusicoccum parvum/N. ribis complex

Several species of Botryosphaeriaceae and Phaeomoniella chlamydospora are common agents of grapevine decline worldwide. Currently, the use of culture independent PCR based techniques for detection of Botryosphaeriaceae within grapevine tissues has been limited to Botryosphaeria dothidea. In the present study, two Botryosphaeriaceae specific nested PCR assays were developed. One with a narrow target range, to detect Neofusicoccum parvum and the closely related species complex (Neofusicoccum parvum/N. ribis sensu Pavlic et al. Molecular Phylogenetics and Evolution 51:259–268, 2009) and another, with a wider range, to detect all 17 species of Botryosphaeriaceae which have been reported as potential wood pathogens of grapevine. The effectiveness of these assays was validated in vivo on naturally infected wood samples collected from standing vines and dormant grafted rooted cuttings commercialized in Italy by different nurseries in different years. All samples were also screened by means of a previously published nested PCR assay specific for Phaeomoniella chlamydospora. It was found that: 1) propagation material may play an important role as source of primary inoculum, not only of Phaeomoniella chlamydospora, as previously reported, but also for members of the Botryosphaeriaceae, among which Neofusicoccum parvum, Botryosphaeria dothidea and Diplodia seriata are the most common, and 2) multiple infections by different species belonging to Botryosphaeriaceae and/or Phaeomoniella chlamydospora occur frequently both in standing vines and propagation material. This last finding supports the hypothesis that at least some of the non-specific symptoms of grapevine decline may be due to the presence of different pathogens within host tissues.

A Simple Microplate Assay to Quantify Nonstructural Carbohydrates of Grapevine Tissues

Knowledge of total nonstructural carbohydrates in plant material is useful for understanding the effects of environmental conditions and vineyard management decisions on grapevine physiology. While many techniques are described in the literature, varying in degree of sophistication, few have been validated in grapevines. We describe the miniaturization of a simple extraction method and microplate based colorimetric assay for the measurement of nonstructural carbohydrates in a range of grapevine tissues. The method provides quantitative results for soluble carbohydrates and starch in samples containing as low as 20 μg carbohydrate.

Deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses

We have characterized the virome in single grapevines by 454 high-throughput sequencing of double-stranded RNA recovered from the vine stem. The analysis revealed a substantial set of sequences similar to those of fungal viruses. Twenty-six putative fungal virus groups were identified from a single plant source. These represented half of all known mycoviral families including the Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and Totiviridae. Three of the mycoviruses were associated with Botrytis cinerea, a common fungal pathogen of grapes. Most of the rest appeared to be undescribed. The presence of viral sequences identified by BLAST analysis was confirmed by sequencing PCR products generated from the starting material using primers designed from the genomic sequences of putative mycoviruses. To further characterize these sequences as fungal viruses, fungi from the grapevine tissue were cultured and screened with the same PCR probes. Five of the mycoviruses identified in the total grapevine extract were identified again in extracts of the fungal cultures.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-010-0869-8) contains supplementary material, which is available to authorized users.

Expression and tissue and subcellular localization of anthocyanidin synthase (ANS) in grapevine

Anthocyanidin synthase (ANS) is one of the key enzymes in the biosynthesis of both anthocyanins and proanthocyanidins in grapevine. Although substantial researches have investigated ANS gene expression and regulation at the transcriptional level, little is yet known about protein expression and distribution in grapevine. Here, the expression and tissue and subcellular localization of ANS in different Cabernet sauvignon grapevine tissues were investigated by using the techniques of Western blotting, immunohistochemical localization, immuno-electron microscopy, and confocal microscopy. The results showed that the ANS was expressed in the grape berries, leaves, stems, petioles, and leaf buds. In grape berry skin and flesh, ANS expression is developmental dependent. Immunohistochemical analysis revealed that ANS is primarily distributed in the exocarp, mesocarp, and seed of the fruit; in palisade and spongy tissues of the leaves; in the primary phloem and pith ray in the stems; and in the growth point and leaf primordium of the leaf buds. Furthermore, at the subcellular level, the ANS was mainly localized in the cytoplasm regardless of cell types and some ANS were also found in the nucleus in the mesocarp vascular bundle and leaf bud cells. This research will give further insight for the biosynthesis and regulation of different flavonoid compounds in grapevine.

Quantificational analysis of NPT-II protein from genetically modified Vitis vinifera L.

Widely distributed inhibitors in grapevine extracts make it difficult to improve analytical procedures for protein detection. In this study, acidity in grapevine extracts was one of the major factors inhibiting the detection of neomycin phosphotransferase II via enzyme-linked immunosorbent assay. Leaf and berry extracts with low pH (3.0 – 4.0) strongly inhibited NPT-II detection, while root and xylem sap extracts (normally pH 5.5 to 7.0) allowed the successful detection of NPT-II. The other inhibitory effect against the detection was successfully solved by heat treatment to samples extracted. Boiling leaf extract prior to ELISA, in conjunction with pH adjustment (to 7.0) was essential to improve NPT-II detection, while with berry extracts only pH adjustment was required. In the basis of above results, NPT-II protein contents in transgenic grapevine tissues possessing a NPT-II gene were successfully measured. The results here may be useful to help in evaluation of the bio-safety whether the transgenic grapevines were released or contaminated on the grapevine cultivation area by NPT-II protein detection.

RELATIONSHIPS BETWEEN SOIL CHARACTERISTICS AND LEAF NUTRIENT CONCENTRATIONS IN GRAPEVINE

The results of a study investigating the relationships between different soil chemical-physical characteristics and nutrient concentrations in leaf tissues of grapevine (Vitis vinifera L.) are reported. The main feature of the experimental site, its high soil spatial variability, allowed examination of the variables of interest while keeping constant crop variety, plant age, climatic conditions and agronomic techniques management, the variability of which is often cause of data misinterpretation. The research was carried out on a winegrape vineyard, cv. Trebbiano Toscano grafted on 1103P rootstock, in Southern Italy. Ten areas characterised by different soil average fertility were selected within the experimental field and, within each area, two representative plants were chosen as test plants. Soil samples at three depths and leaf blades at three sampling times (end of bloom, fruit set and veraison) were collected. In the soil, total N content was positively correlated with organic C, available P and exchangeable K and inversely related to total CaC0 3 content. Among the leaf mineral nutrients, P showed the highest variation in relation to soil parameters (positive correlation with N content, organic C, available P; negative with CaC0 3 ) at the end of bloom and at veraison. Total leaf N was positively correlated with soil organic C and available P only at veraison. Moreover, in this stage, a higher Ca and Mg leaf content was observed in the areas characterised by a lower chemical fertility (lower total N and organic C; higher CaCO 3 ). Principal component analysis clearly discriminated areas of different soil fertility and underlined the variation in tissue nutrient concentrations as a function of soil chemical and physical properties.

Analysis ofS-3-(Hexan-1-ol)-Glutathione andS-3-(Hexan-1-ol)-l-Cysteine inVitis viniferaL. cv. Koshu for Aromatic Wines

<i>S</i>-3-(hexan-1-ol)-Glutathione (3MH-<i>S</i>-glut) and <i>S</i>-3-(hexan-l-ol)-l-cysteine (3MH-<i>S</i>-cys) concentrations in grape berry from <i>Vitis vinifera</i> cv. Koshu grown in four vineyards by liquid chromatography-tandem mass spectrometry were analyzed. Commercial yeast strains converted 3MH-<i>S</i>-glut and 3MH-<i>S</i>-cys into 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), suggesting that both 3MH-<i>S</i>-glut and 3MH-<i>S</i>-cys are the flavor precursors of 3MH. Experiments conducted with a fermentation model showed that 3MH-S-cys was more effective in releasing volatile thiols than 3MH-<i>S</i>-glut. 3MH-<i>S</i>-glut and 3MH-<i>S</i>-cys were scarcely accumulated in stem and seed, but were accumulated in leaf and berry from 11 weeks postflowering (wpf). Leaf had the highest 3MH-<i>S</i>-glut and 3MH-<i>S</i>-cys contents among the grapevine tissues tested throughout development. In Koshu grape juice, the accumulation of both compounds peaked at 16 to 18 wpf and decreased thereafter. Cultivation conditions also affected 3MH-<i>S</i>-glut and 3MH-<i>S</i>-cys concentrations in grape berry. The accumulation of 3MH-<i>S</i>-glut and 3MH-<i>S</i>-cys in grape berry was the most rapid in the Kofu vineyard, which has the lowest elevation, and the slowest in Nirasaki vineyard, which has the highest elevation. These findings are expected to contribute to improving viticultural practices with the aim of harvesting grape berries having high concentrations of flavor precursors that are responsible for enhancing varietal aroma of Koshu wine.

The development of a method for the extraction of carotenoids and chlorophylls from grapevine leaves and berries for HPLC profiling

Background and Aims: Carotenoids and chlorophylls perform a number of essential roles in plants making their accurate quantification important to a variety of studies. We aimed to develop an extraction protocol to accurately determine the photosynthetic pigments in grapevine leaf and berry tissue, specifically focusing on limiting the degradation of these pigments. Methods and Results: An extraction protocol for grapevine leaf and berry tissue was systematically optimised by identifying a number of critical parameters. Extracted pigments were analysed using Reversed Phase-High Performance Liquid Chromatography (RP-HPLC). Specific parameters that were optimised included avoiding freeze-drying the material; the volume of acetone and the time required to extract all the pigments from the tissue; the addition of 0.1% (v/v) N-ethyldiisopropylamine to berry extracts to minimise pigment degradation during the extraction procedure; and avoiding concentration of the extracts that otherwise resulted in differential degradation of pigments. Additionally, the method of extraction and normalisation with an internal standard was adapted and improved for accuracy. The optimised protocol was validated using authentic standards and its utility shown by analysing the pigment content of berries and leaves at different growth stages. Conclusions: A method has been developed that is able to extract and accurately quantify, by means of HPLC profiling, the levels of photosynthetic pigments from grape berries and leaves. The method avoided any degradation of the pigments during the extraction and was applicable to both berries and leaves in different stages of growth and development, indicating its general usefulness to vegetative and reproductive organs, even if their metabolic states are very different. Significance of Study: The divergence of methods used for photosynthetic pigment analysis in plants, each with specific advantages and disadvantages were considered and used to optimise a number of parameters in a single method that proved to be applicable to plant organs in different developmental stages. The method is fast, applicable to vegetative and reproductive grapevine tissues, avoids degradation of pigments and ensures maximum accuracy when quantifying these important pigments.

Effects of adjuvants on deposition efficiency of fenhexamid sprays applied to Chardonnay grapevine foliage

Adequate spray deposition on susceptible grapevine tissue is an essential requirement for effective chemical control of economically important diseases, such as grey mould, powdery mildew and downy mildew. The objective of this study was to evaluate the potential of some agricultural adjuvants to improve foliar spray deposition. Deposition quantity and quality was assessed by means of a spray assessment protocol using fluorometry, photomicrography and digital image analyses. The visual droplet rating technique developed by G Furness, Australia, was also included in initial assessments. Both assessment protocols showed that spray deposition quantity increased with increasing spray volume applications of 27 l ha −1 to 581 l ha −1 with a motorised backpack mistblower, but decreased at 698 l ha −1, possibly due to run-off. Addition of selected spray adjuvants at 526 l ha −1 volume demonstrated improved deposition quantity and quality. Agral 90 (ethoxylated alkylphenol), BB5 (acidifier), Nu-film-P (terpene oil), and Solitaire (silicone/plant oil) significantly improved deposition on upper and lower leaf surfaces compared with the fenhexamid-only and water sprayed control. Break-thru S240 (organosilicone) and Villa 51 (alkylpolyethylene glycol ether) did not improve deposition quantity, although remarkably better deposition quality was obtained. An adjuvant concentration effect (within the registered concentration range) was evident at spray volumes of 502 l ha −1, especially those retained on the upper leaf surfaces. Agral 90 and Nu-film-P effected significant improvement of spray deposition at the higher concentrations (18 ml and 50 ml hl −1, respectively), but not at the lower concentrations (6 ml and 20 ml hl −1, respectively). Solitaire improved deposition at the lower concentration tested (50 ml hl −1), whereas reduced deposition at the higher concentration (100 ml per hl −1) was attributed to excessive spray run-off. No significant improvement of spray deposition was observed for both concentrations tested with Villa 51 (50 and 100 ml hl −1). Spray mixtures with adjuvants Agral 90 and Solitaire yielded similar deposition values at 500 l ha −1 compared with the fenhexamid-only control at 720 l ha −1, but reduced deposition at the higher spray volume, possibly due to spray run-off. This study clearly demonstrated the potential of adjuvants to improve deposition quantity and quality, but highlights the necessity to match adjuvant concentrations and application volumes on the spray target to achieve maximum spray deposition.

Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

Identification of grapevine microRNAs and their targets using high throughput sequencing and degradome analysis

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

DNA-Dependent Detection of the Grapevine Fungal Endophytes Aureobasidium pullulans and Epicoccum nigrum.

Aureobasidium pullulans and Epicoccum nigrum are frequently reported as endophytes of various crops, including grapevine (Vitis vinifera). Because of their potential role as biological control agents against grapevine pathogens, we examined the occurrence of A. pullulans and E. nigrum in two grapevine varieties (Merlot and Prosecco) in Italian vineyards where spontaneous recovery from phytoplasma disease is recurrent. Species-specific primers for A. pullulans and two genetically distinct strains of E. nigrum were designed in variable regions of ITS1 and ITS2. Primer specificity was confirmed by polymerase chain reaction using purified DNA from other fungal endophytes that are usually encountered during isolation attempts from grapevine tissues and from several other strains of A. pullulans and E. nigrum isolated from other sources. In order to determine the occurrence of the two endophytes in grapevine plants, DNA was extracted from shoots of 44 grapevines collected in six vineyards from different localities of northeast Italy. Both endophytes were detected and their identity was confirmed by restriction fragment length polymorphism (RFLP) patterns obtained from reference strains. RFLP analyses confirmed the presence of two E. nigrum strains belonging to different RFLP groups in grapevine. The molecular methods described allowed a sensitive, specific, and reliable identification of the two endophytes in grapevine.

Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR

Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

Improvement of an RNA purification method for grapevine (Vitis vinifera L.) suitable for cDNA library construction

A method is described, which consistently yields high quality total RNA from grapevine. Dissolving of crude RNA pellets in borate-containing buffer, instead of normally used water before a selective lithium chloride precipitation, was found to be a critical step, leading to a 2.5-fold increase of yield. The resulting RNA preparations were suitable for standard downstream applications and also for cDNA library construction. The method worked efficiently and reproducibly and could easily be scaled from milligram to gram quantities of plant material grown in hydroponic culture, sandy soil or Perlite. It was applied to different kinds of grapevine tissues (leaves, stem) and, after additional adaptation of the protocol, to roots.

Real-time RT-PCR (TaqMan ®) assays for the detection of viruses associated with Rugose wood complex of grapevine

Real-time TaqMan ® RT-PCR (TaqMan ® RT-PCR) assays were developed to detect the viruses associated with Rugose wood complex of grapevines. The viruses detected were Rupestris stem pitting-associated virus in the genus Foveavirus, Grapevine virus A, Grapevine virus B and Grapevine virus D in the genus Vitivirus. The coat protein was found to be the most conserved gene within the viral species, therefore, the primers and probes for TaqMan ® RT-PCR assays were designed from the multiple alignment of the coat protein sequence of various isolates of each virus. Comparisons were also made between the conventional one step RT-PCR and TaqMan ® RT-PCR for the detection of these viruses using four-fold serial dilutions of both purified RNA and crude extract prepared from grapevine tissue. Results showed that TaqMan ® RT-PCR was more sensitive and could detect viruses at 32- and 256-fold higher dilutions for purified RNA and crude extract, respectively, compared to RT-PCR. The use of an internal control (18S rRNA) allowed comparison of sample preparation protocols and amplification efficiencies between samples.

The herbicide flumioxazin stimulates pathogenesis‐related gene expression and enzyme activities in Vitis vinifera

In this work, the capacity of the soil-applied herbicide flumioxazin (fmx) to trigger defence mechanisms was assessed using 6-week-old in vitro grown Vitis vinifera L. plantlets. Time-course studies demonstrated that the herbicide induced the expression of basic beta-1,3-glucanase (Vvglu), basic chitinase (Vvchit1b) and PR10 (VvPR10.3) genes encoding three pathogenesis-related (PR) proteins involved in grapevine defence against pathogens. Thus, all transcripts accumulated in grapevine tissues to reach maximum values after 24-72 h of herbicide exposure, except for VvPR10.3 gene expression, which was induced in roots and stems but not in leaves. Induction of PR genes was observed to a greater extent in roots and leaves, and its intensity diminished in the stems although still remained noteworthy. The activities of beta-1,3-glucanase and chitinase enzymes significantly increased in the whole plant after herbicide exposure and were still stimulated 21 days after the beginning of treatments. Similarly, the most remarkable effect occurred in roots. However, all enzyme activities tested were stimulated in the upper aerial tissues as well, indicating that fmx or a derived product acts systemically, likely via root uptake.

Fungicide resistance and genetic diversity of Botrytis cinerea isolates from a vineyard in Germany

To evaluate the genetic diversity and the possibility of changes in the population of the grey mould fungus in a single vineyard, a total of 209 Botrytis cinerea isolates were collected from grapevine tissues in spring and autumn of 2004. The isolates revealed low frequencies of resistance against the fungicides fenhexamid (1.9%), fludioxonil (0%), carbendazim (3.4%), cyprodinil (3.8%) and tolylfluanid (0%). Two isolates belonged to the genetically isolated, fenhexamid-resistant B. cinerea group I. Two other isolates showed reduced sensitivities against all tested fungicides, indicating a multi-drug resistance mechanism. The majority of isolates (62.7%) harboured the transposable elements boty and flipper (transposa group), while smaller populations contained only boty (23.5%) or neither boty nor flipper (14.4%; vacuma group). Genotypic characterisation of the isolates with different molecular markers revealed a high degree of genetic diversity of the population, with most of the fungal isolates being different haplotypes. Spring and autumn populations were different, but did not show significant differences in their fungicide resistance patterns or in the distribution of transposa and vacuma isolates. Taken together, the study revealed a great diversity of genotypes in a chemically treated vineyard, but no evidence for large-scale fungicide resistance or seasonal changes in the grey mould population.

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.