R. van Heeswijck

Identification and characterization of a fruit-specific, thaumatin-like protein that accumulates at very high levels in conjunction with the onset of sugar accumulation and berry softening in grapes.

The protein composition of the grape (Vitis vinifera cv Muscat of Alexandria) berry was examined from flowering to ripeness by gel electrophoresis. A protein with an apparent molecular mass of 24 kD, which was one of the most abundant proteins in extracts of mature berries, was purified and identified by amino acid sequence to be a thaumatin-like protein. Combined cDNA sequence analysis and electrospray mass spectrometry revealed that this protein, VVTL1 (for V. vinifera thaumatin-like protein 1), is synthesized with a transient signal peptide as seen for apoplastic preproteins. Apart from the removal of the targeting signal and the formation of eight disulfide bonds, VVTL1 undergoes no other posttranslational modification. Southern, northern, and western analyses revealed that VVTL1 is found in the berry only and is encoded by a single gene that is expressed in conjunction with the onset of sugar accumulation and softening. The exact role of VVTL1 is unknown, but the timing of its accumulation correlates with the inability of the fungal pathogen powdery mildew (Uncinula necator) to initiate new infections of the berry. Western analysis revealed that the presence of thaumatin-like proteins in ripening fruit might be a widespread phenomenon.

Clonal reproduction and population genetic structure of grape phylloxera, Daktulosphaira vitifoliae, in Australia

The grape phylloxera, Daktulosphaira vitifoliae, is a viticultural pest that in the past has devastated vineyards worldwide, yet little is known about this insects biology. The genetic structure of Australian populations of grape phylloxera and its mode of reproduction were studied following the development of four polymorphic microsatellite loci. Insects were collected from 28 vineyards, with a total of 361 insects included in the study. The majority of vineyards were infested by functionally parthenogenetic lineages of grape phylloxera that inhabit the root system and there was little support for the traditionally described holocyclic life cycle for this species. Clonal diversity was limited in all of the vineyard regions, with the exception of the Rutherglen region. A multiple founder scenario or occasional sex may contribute to diversity within the Rutherglen region. Leaf galling populations comprised classes distinct from the common genotypic classes identified on the roots, suggesting limited exchange between these groups. Implications for the management of D. vitifoliae are discussed.

Characterisation of Fusarium oxysporum isolated from carnation in Australia based on pathogenicity, vegetative compatibility and random amplified polymorphic DNA (RAPD) assay

Isolates ofF. oxysporum collected from symptomless carnation cuttings from Australian carnation growers properties, together with isolates from national collections, were screened for pathogenicity and grouped according to vegetative compatibility and random amplified polymorphic DNA (RAPD) patterns. The collection of 82 Australian isolates sorted into 23 different vegetative compatibility groups (VCGs). Of 69 isolates tested for pathogenicity, 24 were pathogenic to carnations, while the remaining 45 were non-pathogenic. All pathogenic isolates were within two VCGs, one of which was also compatible with an isolate obtained from an international culture collection, and which is known to represent VCG 0021 and race 2. Race status of the two pathogenic VCGs remains unknown. The RAPD assay revealed distinct DNA banding patterns which could distinguish pathogenic from non-pathogenic isolates as well as differentiate between isolates from the two pathogenic VCGs.

Pathogenesis Related Proteins — Their Accumulation in Grapes during Berry Growth and Their Involvement in White Wine Heat Instability. Current Knowledge and Future Perspectives in Relation to Winemaking Practices

Grape growing represents one of the world’s major horticultural enterprises with the vast majority of the production destined for further value-adding through winemaking. An important consideration of grape production is therefore how key constituents of the berry contribute positive and negative attributes to the final bottled wine product. One feature of wine, which is important to the perception of quality, is brilliance and clarity. The presence of “unstable” proteins ranges from 50 to 100 mg/L but occasionally up to several hundred mg/L (Koch and Sajak, 1959; Bayly and Berg, 1967; Somers and Ziemelis, 1973a; Hsu and Heatherbell, 1987a; Murphey et al., 1989; Yokotsuka et al.,1991; Dorrestein et al.,1995; Santoro, 1995; Pocock et al.,1998 a,b; Pocock et al.,1999), and their aggregation to form haze in white wine is a ubiquitous concern for winemakers. Protein haze formation can occur after bottling and is almost certainly due to the slow denaturation of wine proteins. This, in turn, leads to protein aggregation and formation of unattractive suspensions as well as amorphous cloudy precipitates (Paetzold et al., 1990; Waters et al., 1991). Regardless of the taste, consumers will in general reject wines containing hazes or cloudy precipitates due to fears of microbial spoilage having occurred.

Reduced sensitivity of Uncinula necator to sterol demethylation inhibiting fungicides in southern Australian vineyards

Isolates of Uncinula necator with reduced sensitivity to triadimenol and, to a lesser extent, fenarimol have been detected in Australian vineyards. Grapevine leaves and berries affected by powdery mildew were collected from a home garden and 13 vineyards in nine viticultural regions in Australia between 1993 and 1998. Single-spore isolates of U. necator established from vines, either not exposed to DMIs (‘unexposed’ collection) or treated with DMIs (‘selected’ collection), were maintained on micro propagated grapevines in vitro. A bioassay for fungicide sensitivity was used to test 60 single-spore isolates of U. necator for sensitivity to triadimenol. Of these, 34 were tested for sensitivity to fenarimol. Mean EC50 values for the 12 unexposed isolates were 0.07 and 0.08 mg/L for triadimenol and fenarimol, respectively. For the selected isolates, mean EC50 values were 0.83 mg/L for triadimenol and 0.19 mg/L for fenarimol. In comparison with the unexposed population, there appeared to have been a shift in the selected population towards reduced sensitivity. Examination of more isolates from vineyards not exposed to DMIs may strengthen this conclusion. Cut-off EC50 values, used to define individual isolates as having reduced sensitivity, were 0.42 mg/L (resistance factor, RF = 6) for triadimenol and 0.12 mg/L (RF = 1.5) for fenarimol. The findings were consistent with the existence of cross-resistance between triadimenol and fenarimol.

Evidence for host-associated clones of grape phylloxera Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae) in Australia

Grape phylloxera, Daktulosphaira vitifoliae Fitch, is an important pest of grapevines (Vitis vinifera L.) (Vitaceae). Using microsatellite DNA markers it was demonstrated strong associations can exist between D. vitifoliae asexual lineages and vine host type within a vineyard. Also, in excised root bioassays, D. vitifoliae collected from three regions where different genotypic classes predominated showed host-specific differences in life table parameters of reproductive rate and intrinsic rate of increase. Lastly, comparisons of mitochondrial DNA (cytochrome oxidase I) sequences revealed that D. vitifoliae in Australia have paraphyletic origins and fall into two clades partially related to vine host usage. These findings indicate introduction of separate lineages of D. vitifoliae which have close host associations and as such, have important implications for management of this pest in Australia.

Molecular Biology and Biochemistry of Proline Accumulation in Developing Grape Berries

In recent years, the value and competitive nature of the grape and wine industry has led to an increasing emphasis on producing quality fruit for the production of quality wines. In general terms the “quality” of a berry is determined by its composition. The reviewed research in this paper investigates an important aspect of grape composition, which is the accumulation of nitrogen in the form of free amino acids1, with an emphasis on the molecular biology and biochemistry of the accumulation of free proline.

Enhancement of grape berry weight induced by an ethanol spray four weeks before harvest and effects of a night spray at an earlier date

Spraying aqueous solutions of ethanol (between 2.5 and 10%, v/v) 1 month before harvest has been shown to increase berry weight of Cabernet Sauvignon by about 10% at harvest compared with controls sprayed with water. These observations were made in France and in Australia. The increase in berry weight was not associated with significant changes in total soluble solids. Additional measurements showed that spraying aqueous solutions of ethanol onto the berry surface 1 month before harvest had only small effects on the colour of berry skin extracts. In another trial, spraying ethanol at night 1 month earlier than other trials lead to a slight increase in the colour of the skin extracts. Optimisation of the spraying conditions is discussed.