Different DNA extraction methods can cause different AFLP profiles in grapevine (Vitis vinifera L.)

Amplified fragment length polymorphism (AFLP) is widely used for DNA fingerprinting and it has been broadly applied in population genetics. Since it is based on restriction digestion and PCR-based amplification it can be influenced by different chemical compounds commonly found in the isolated DNA. DNA extraction procedures may alter the AFLP banding profiles through DNA quality. Hence the DNA extraction method is crucial to produce reproducible AFLP-banding profiles.In this work two sets of AFLP analyses were performed on 62 Pinot noir, 6 Pinot blanc and 4 Pinot gris (Vitis vinifera L.) clones, and profiles obtained after three different DNA extraction methods were compared. AFLP profiles were different for the same genotypes due to the DNA extraction method used.

Comparison of bioassays to biotype grape phylloxera (Daktulosphaira vitifoliae Fitch) on Vitis ssp.

Grape phylloxera biotypes exist throughout viticultural regions causing substantial economic losses. In the past different biotyping assays were employed to determine host adaption and potential harm of phylloxera strains or field populations. Standardised and efficient laboratory assays are required to define biotypes according to their aggressivity as well as to make accurate pest management and quarantine decisions. We aim to provide information on the consistency of the three most commonly used assays to accurately identify grape phylloxera biotype. Two phylloxera biotypes (A, C) were tested on two host plants (rootstock 'Teleki 5C' V. berlandieri x V. riparia and V. vinifera 'Riesling') using three assays: Simple isolation chamber, excised root bioassay and aseptic dual culture bioassay. Insect number, life table and plant-based response parameters (root galling) were compared. The simple isolation chamber and aseptic dual culture bioassay produced consistent results, whereas the excised root bioassay did not. We demonstrated that biotype results depend on whether the technique used is tuberosity- or nodosity-based. Pest management decision based on a single assay may inaccurately assess the phylloxera aggressivity potential. Thus, we recommend using two assay types which allows comparison of both root gall types

Aseptic dual culture of grape (Vitis spp.) and grape phylloxera (Daktulosphaira vitifoliae FITCH)

An aseptic dual culture of grape phylloxera (Daktulosphaira vitifoliae FITCH) and grape vine (Vitis spp.) was developed. This method permits continuous observation of phylloxera feeding and the whole plant response on a dynamic basis. The plant/parasite interaction of three testplants (V. vinifera L., var. Riesling, SO 4 (V. berlandieri PLANCH. X V. riparia L.) and V. riparia, var. Gloire de Montpellier) are demonstrated by observing post-infectious reactions of the host- and population dynamics of the parasite. Different stages of phylloxera could be observed including nymphs, winged phylloxera (alatae) and sexual male phylloxera. Several potential applications for this aseptic dual culture are demonstrated

Application of current in situ hybridization techniques for grape phylloxera (Daktulosphaira vitifoliae, Fitch) and grapevine (Vitis spp. L.)

In situ hybridization and in situ PCR directly localize specific DNA and RNA sequences in tissues. To exactly focus on the processes occurring on cell- or tissue level, in situ techniques can be efficiently employed. Recent advances in viticultural research in the fields of genomics, proteomics and metabolomics are likely to employ these techniques to link DNA- or mRNA sequence information to physiological traits and processes occurring in the grapevine. In this paper, we present a range of possibilities for in situ techniques that can be applied in grapevine research. Two examples covering in situ PCR of grapevine roots and in situ hybridization of grape phylloxera will be given for illustration. Moreover, key steps of the techniques are discussed, which may be helpful to researchers aiming to employ in situ hybridization or in situ PCR.

Karyotype studies on grape phylloxera (Daktulosphaira vitifoliae FITCH)

A cytogenetic technique was developed to produce suitable chromosome spreads for phylloxera karyotype analysis. The karyotype for pathogenetic phylloxera was 2 n = 10. Karyotypes from haploid sex cells were found to vary between n = 5 and n = 6, the latter possibly indicating an aneuploidic aberration. Tetra- and polyploid cells were detected in somatic trophocytes. Preparation of phylloxera sex and somatic cells for chromosomal analysis reported here enables the study of genetic variation on a chromosomal scale

Histochemistry and anatomy of phylloxera (Daktulosphaira vitifoliae) nodosities on young roots of grapevine (Vitis spp).

Phylloxera (Daktulosphaira vitifoliae FITCH) induce galls (nodosities) on young grapevine roots. Histological and histochemical methods were applied to study the gall's morphology and enzyme activities (peroxidases, leucine aminopeptidases and acidic phosphatases). Susceptible V. vinifera cv. Cabernet Sauvignon was compared to the resistant rootstock 5 BB (V. berlandieri x V. riparia) using aseptic dual culture conditions. The gall induction phase was analyzed before visible signs of potential resistance responses were detected. Elevated metabolic activity has been found in nodosities compared to uninfected roots. Starch granule incorporation was detected in young galls and was highest at the feeding site. As galls mature, the starch density decreased at the feeding site and increased towards the periphery of the gall. Peroxidase, acidic phosphatase and leucine aminopeptidase activities were highest at the incision. No differences in enzyme activities could be detected between the two cultivars tested.

An experimental design applied to vineyards for identifying spatially and temporally variable crop parameters

Harvesting uniform batches of grapes is required to optimize must quality as one prerequisite for premium wine production. The definition of sub-units of vineyards based on within-field variation allows unitbased vineyard management during cultivation and harvest. Essential for such vineyard management is the definition of sub-units that correspond with uniform batches of quality parameters of the fruit (e.g. berry residual sugar, anthocyanin content) at harvest time or with physiological parameters measuring the vine during berry development until ripeness. The definition requires geo-referenced sampling and parameter analysis, usually in combination with interpolation and kriging methods employed to describe spatial vineyard variation.In an attempt to develop an assay for within-variation in vineyards physiological parameters assessed through chlorophyll fluorescence measurements and leaf temperature were assessed at bloom, veraison and post veraison in a randomized block design in two vineyards of Lower Austria. A statistical model based on a repeated measurement ANOVA was developed and showed suitability for the detection and monitoring of vineyard variability throughout the vegetation period based on the maximum quantum yield of photosystem II (Fv/Fm), the maximum leaf temperature (maxTleaf) and malic acid. These parameters allow the prospective classification of sub-units according to the vine’s vitality and may be adopted for scientific experimentation and for practical viticulture.

First European leaf-feeding grape phylloxera (Daktulosphaira vitifoliae Fitch) survey in Swiss and German commercial vineyards

Recent observations report the worldwide incidence of leaf-feeding grape phylloxera in formerly resistant scions of commercial vineyards. To analyze the genetic structure of leaf-feeding phylloxera, we performed an extensive sampling of leaf-feeding phylloxera populations in seven regions (“cantons”) in Switzerland and Germany. The use of polymorphic microsatellite markers revealed presence of 203 unique grape phylloxera multilocus genotypes. Genetic structure analyses showed a high genetic similitude of these European samples with phylloxera samples from its native habitat on Vitis riparia (northeastern America). Nevertheless, no genetic structure within the European samples was observed, and neither host, geography nor sampling date factors caused clear effects on phylloxera genetic stratification. Clonality was high in commercial vineyards and leaf-feeding grape phylloxera strains were found to be present in scion leaves and rootstock roots in the same vineyard, potentially indicating migration between both habitats. We found indications of sexual reproduction, as shown by high degrees of genetic variation among collection sites

A multivariate approach for the ampelographic discrimination of grapevine (Vitis vinifera) cultivars: application to local Syrian genetic resources

Due to its unique historical and geographical emplacement, grapes have been cultivated in the Syrian Arab Republic for more than 5000 years, so the characterization of its local genetic resources is paramount for understanding grapevine natural diversity. In this work, different local Syrian table grape cultivars were characterized for 42 traits related to plant phenology, shoot, leaf, cluster, berry and juice composition. A series of multivariate analyses were sequentially performed, and five highly-discriminant traits were identified as the most discriminant ones (shoot internode length, berry weight, berry elongation, 100-seed weight and juice titratable acidity). The clustering of the cultivars according to these five traits revealed that some local Syrian cultivars share similitude with some worldwide grown cultivars, suggesting their potential as new genetic resources for the development of new high-quality table grape varieties, and indicating the needing of specific preservation programs aimed to avoid the loss of endangered genetic local resources. Besides, the statistical multivariate pipeline followed in this work is proposed as an efficient one for the selection of ampelographic traits for the discrimination of grapevine cultivars.(VLID)233169