Cultivar, site or harvest date: the gordian knot of wine terroir.

INTRODUCTION: The complex interactions of vine cultivars, and localised regional climate associated with specific vineyard sites are important attributes to the concept of terroir and significant contributors to grape maturity and wine sensory profiles. An improved understanding of the influence of each factor and their interactions is a challenging conundrum, and will enable more efficient production targeting specific wine styles. OBJECTIVES: To characterise the metabolic flux of grape berries and resulting wines to characterise the relative impact of site specific climate, cultivar, and grape maturity based upon berry sugar accumulation models that consistently target specific wine styles. METHODS: A spatial and temporal study of grape and wine composition was undertaken for two important cultivars in two distinct regions of New South Wales. Measures of composition and wine sensory ratings were simultaneously analysed using a multiblock algorithm taking advantage of the ANOVA framework to identify important contributions to wine style arising from grape maturity, vineyard site and cultivar. RESULTS: A consistent flux of grape and wine constituents is evident for wine made from sequentially harvested grapes from the same vineyard with increasing levels of grape maturity. Contributions of region and vineyard site to wine style could also be elucidated. Differences in metabolite flux in grapes and resulting wines between cultivars growing in similar conditions are evident. CONCLUSIONS: The combination of a metabolomics and multiblock data decomposition approach may be successfully used to profile and elucidate the contribution of abiotic factors to grape and wine composition and provide improved understanding of the terroir concept.

Aureobasidium pullulans volatilome identified by a novel, quantitative approach employing SPME-GC-MS, suppressed Botrytis cinerea and Alternaria alternata in vitro.

Volatile organic compounds (VOCs) produced by Aureobasidium pullulans were investigated for antagonistic actions against Alternaria alternata and Botrytis cinerea. Conidia germination and colony growth of these two phytopathogens were suppressed by A. pullulans VOCs. A novel experimental setup was devised to directly extract VOCs using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) from antagonist-pathogen culture headspace. The proposed system is a robust method to quantify microbial VOCs using an internal standard. Multivariate curve resolution-alternating least squares deconvolution of SPME-GC-MS spectra identified fourteen A. pullulans VOCs. 3-Methyl-1-hexanol, acetone, 2-heptanone, ethyl butyrate, 3-methylbutyl acetate and 2-methylpropyl acetate were newly identified in A. pullulans headspace. Partial least squares discriminant analysis models with variable importance in projection and selectivity ratio identified four VOCs (ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol), with high explanatory power for discrimination between A. pullulans and pathogen. The antifungal activity and synergistic interactions of the four VOCs were evaluated using a Box-Behnken design with response surface modelling. Ethanol and 2-phenylethanol are the key inhibitory A. pullulans VOCs against both B. cinerea and A. alternata. Our findings introduce a novel, robust, quantitative approach for microbial VOCs analyses and give insights into the potential use of A. pullulans VOCs to control B. cinerea and A. alternata.

Improving the detection of significant factors using ANOVA-PCA by selective reduction of residual variability.

Selective elimination of residual error can be used when applying Harrington's ANOVA-PCA in order to improve the capabilities of the method. ANOVA-PCA is sometimes unable to discriminate between levels of a factor when sources of high residual variability are present. In some cases this variability is not random, possesses some structure and is large enough to be responsible for the first principal components calculated by the PCA step in the ANOVA-PCA. This fact sometimes makes it impossible for the interesting variance to be in the first two PCA components. By using the proposed selective residuals elimination procedure, one may improve the ability of the method to detect significant factors as well as have an understanding of the different kinds of residual variance present in the data. Two datasets are used to show how the method is used in order to iteratively detect variance associated with the factors even when it is not initially visible. A permutation method is used to confirm that the observed significance of the factors was not accidental.

Study of the influence of micro-oxygenation and oak chip maceration on wine composition using an electronic tongue and chemical analysis.

The influence of micro-oxygenation (MOX) and maceration with oak chips treatments on wine was studied on wine samples from three vintages produced in the Yarra Valley, Australia. A full factorial design was employed where two factors (MOX and oak chips treatments) had two levels and one factor (vintage) had three levels. Three replicated treatments were run for each factor's setting. Wine samples were analysed using conventional laboratory methods with respect to the phenolic wine compounds and colour attributes since the phenolic fraction of wine is most affected by both MOX and oak maceration treatments. The same wine samples were measured with an electronic tongue based on potentiometric chemical sensors. The significance of treatments and vintage effects on wine phenolic compounds was assessed using ANOVA and ANOVA-Simultaneous Component Analysis (ASCA). Cross-validation was used for the ASCA sub-model optimisations and permutation test for evaluations of the significance of the factors. Main effects of vintage and maceration with oak chips were found to be significant for both physicochemical and the ET data. Main effect of MOX treatment was also found significant for the physicochemical parameters. The largest effect on the phenolic composition of wine was due to its vintage, which accounted for 70% and 33% of total variance in the physicochemical and ET data respectively. The ET was calibrated with respect to the total phenolic content, colour density and hue and chemical ages 1 and 2 and could predict these parameters of wine with good precision.

Antigen recognition by rainbow trout (Oncorhynchus mykiss) of whole cell proteins expressed by Lactococcus garvieae when obtained directly from fish and under iron limited culture conditions.

Infection by Lactococcus garvieae has become a widely recognised problem associated with intensively cultured fish. Long-term control of fish infections may be possible by vaccination providing a suitable and efficacious epitope is expressed during production of cells used for vaccine preparation. The identification of novel vaccine candidates must, therefore, consider how the host species recognises and responds to bacterial cell components. L. garvieae was cultured in iron deficient, limited and haem iron enriched media and the whole cell proteins expressed under these conditions were compared with those expressed in bacteria extracted with Percoll gradients directly from spleen tissue of infected rainbow trout (Oncorhynchus mykiss). SDS-PAGE of the cell proteins showed the existence of several different electropherotypes according to the iron status of the culture media. Only minor differences in cell protein profile were detected in bacteria obtained directly from fish spleens, but when the electropherograms were analysed by Western blots using L. garvieae hyperimmune fish sera, several proteins could be identified that were expressed only when L. garvieae was growing in vivo. Siderophore could be detected in culture supernatant of iron deficient, limited and haem iron enriched media but not in media with higher nutrient concentrations. The siderophore could not be identified as a type of catechol or hydroxymate. Rainbow trout recognise proteins in the range of approximately 50-80 kDa for bacterial cells obtained without subculture from infected fish and culture conditions can influence protein profiles for this pathogen.

Induction, characterisation and pathogenicity in rainbow trout Oncorhynchus mykiss (Walbaum) of Lactococcus garvieae L-forms.

Difficulties with induction and cultivation of L-forms, particularly those derived from Gram positive parent cells, have constrained to some degree the ability to evaluate the pathogenicity of these morphotypes. Induction of L-forms of Lactococcus garvieae was undertaken using either charcoal or inactivated horse serum media supplemented with ampicillin, benzylpenicillin or erythromycin, the drug of choice for treatment of infections in rainbow trout, Oncorhynchus mykiss, (Walbaum), and NaCl as an osmotic stabiliser. Lysozyme treated cells could be cultured in a cell wall deficient state using media consisting of charcoal, NaCl and either ampicillin or benzylpenicillin. The influence of some amino acids for induction of L-forms was assessed by disc diffusion and combined interaction. Analysis of variance of colony counts indicated that the amino acids glycine, DL-methionine, L-threonine and L-serine (P<0.03), and the presence of charcoal were beneficial and that inactivated horse serum was detrimental to L-form development. Electron microscopy revealed that the cell wall of L-forms was missing and this cell had a greatly expanded volume compared to parent cells. Electrophoresis of whole cell proteins showed some variation of electropherotype between parent and L-form cells. L-forms expressed greater quantities of proteins with molecular mass of 36 and 66 kDa and parent cells contained greater quantities of proteins of molecular mass 29, 43 and 60 kDa. Additional proteins of molecular mass 32, 44 and 53 kDa were present in L-form extracts, and in parent cells of 34, 38, 40, 42, 85 and 123 kDa which may represent cell wall associated proteins or alterations in expression due to different growth rates. Intraperitoneal challenge of rainbow trout with L-forms failed to produce overt infection even in immune-suppressed fish, but L-forms were shown by indirect fluorescent antibody test to remain inkidney tissue. Fish were susceptible to infection when challenged with parent cells of L. garvieae.

Characteristics of Vagococcus salmoninarum isolated from diseased salmonid fish.

Isolates of the salmonid pathogen Vagococcus salmoninarum were recovered from Atlantic salmon, rainbow trout and brown trout with peritonitis. The phenotypes of these isolates and the type strain of Vag. salmoninarum NCFB 2777 were determined by morphological, biochemical and physiological tests and whole cell protein profiles by SDS-PAGE. There was a high level of phenetic similarity between the salmonid isolates and the type strain. The species forms short Gram-positive rods, hydrolyses L-pyrrolidonyl-beta-naphthylamide, is alpha-haemolytic on sheep's blood agar, grows at pH 9.6 and 10 degrees C but not at 40 degrees C or in 6.5% NaCl and is catalase-negative; a Lancefield group N antigen is not present. Vagococcus salmoninarum can be distinguished phenetically from similar fish pathogens including Carnobacterium piscicola, Enterococcus seriolicida and Lactococcus piscium.