Phenolic compounds involved in grafting incompatibility of Vitis spp: development and validation of an analytical method for their quantification.

INTRODUCTION: Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment. OBJECTIVE: To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion-rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4). METHODS: Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile. RESULTS: Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility. CONCLUSION: The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage.

Occurrence of grapevine leafroll-associated virus 5 in Portugal: genetic variability and population structure in field-grown grapevines.

A comparison of 15 field isolates of grapevine leafroll-associated virus 5 (GLRaV-5) was conducted, based on the analysis of nucleotide sequences of two viral ORFs: the coat protein (CP) and the heat shock protein 90 homolog (HSP90h). After amplification and cloning, the population of viral sequences was analyzed for each isolate, revealing the within-isolate presence of sequence variants for both genes, with one or more major CP variants. Phylogenetic analysis showed the gene sequence variants detected to be exclusive for each isolate. These data, together with estimates of genetic diversity and positive selection, did not reveal evidence of vector-mediated transfer of GLRaV-5. Conversely, a strong effect of host vegetative propagation on divergence dynamics of GLRaV-5 variants was suggested by the isolates from this work The phylogeny of the CP gene further revealed clustering of GLRaV-5 isolates into eight lineages, four of which were detected in our study, revealing a higher diversity than previously described. The information gathered also contributes to firmly establishing GLRaV-5 as a cohesive taxonomic group within the ampeloviruses.

Five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from Portuguese grapevine varieties.

The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure. This tool was used to assess the prevalence of each phylogenetic group among the infected grapevine varieties. Although most of the isolates harbour variants from groups 1 and 2, variants from the remaining three groups exist in a number of varieties, reinforcing the notion that they are genuine genomic variants and are not isolated, atypical cases.

Using Rapid Chlorophyll Fluorescence Transients to Classify Vitis Genotypes.

When a dark-adapted leaf is illuminated with saturating light, a fast polyphasic rise of fluorescence emission (Kautsky effect) is observed. The shape of the curve is dependent on the molecular organization of the photochemical apparatus, which in turn is a function of the interaction between genotype and environment. In this paper, we evaluate the potential of rapid fluorescence transients, aided by machine learning techniques, to classify plant genotypes. We present results of the application of several machine learning algorithms (k-nearest neighbors, decision trees, artificial neural networks, genetic programming) to rapid induction curves recorded in different species and cultivars of vine grown in the same environmental conditions. The phylogenetic relations between the selected Vitis species and Vitis vinifera cultivars were established with molecular markers. Both neural networks (71.8%) and genetic programming (75.3%) presented much higher global classification success rates than k-nearest neighbors (58.5%) or decision trees (51.6%), genetic programming performing slightly better than neural networks. However, compared with a random classifier (success rate = 14%), even the less successful algorithms were good at the task of classifying. The use of rapid fluorescence transients, handled by genetic programming, for rapid preliminary classification of Vitis genotypes is foreseen as feasible.

Label free DNA-based optical biosensor as a potential system for wine authenticity.

The diversity found among the Vitis vinifera L. species allows the production of wines with very different characteristics. The development of platforms suitable for food composition analysis is currently an emerging area. Among these, DNA biosensors have been developed for a wide variety of applications, ranging from food safety to authenticity. The main aim of this work was to study the detection capacity of the DNA-based optical biosensor using different V. vinifera matrices (leaf, must and wine). Genomic DNA was extracted from leaf, must and wine of three V. vinifera varieties and was tested on the long-period grating (LPG) DNA-based biosensor developed within our group. The biosensor was able to distinguish the varieties even using DNA extracted from complex matrices, revealing its potential to be applied in wine authenticity.

High Resolution Melting (HRM) applied to wine authenticity.

Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes.

Molecular data mining to improve antibody-based detection of Grapevine leafroll-associated virus 1 (GLRaV-1).

Testing for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.