Expression of Anti-Microbial Genes in Transgenic Grapevines for Enhanced Disease Resistance

AVF Proj. ID: 256 / /Cat.: ,
Primary Investigator(s): Bruce I. Reischby

Grapevines are susceptible to numerous diseases that devour both plants and profits. Trangenic grapevines that resist disease would provide better disease control as well as economic benefits from the reduction in spray applications. Our overall goal has been to research and develop methods to create transgenic selections of elite cultivars with improved resistance to diseases. The transgenic stragegy is especially appropriate for clonally-propagated crops, such as grapevines, where the wine industry is rooted in tradtional European Grapes with strong name recognition and very high disease susceptibility. This project has focused on cultivars Chardonnay and Merlot. Two classes of anti-microbial genes (endochitinase adn magainin-types) have been inserted into these varieties. Initial tests indicate varying degrees of improvement in resistance to powdery mildew, Botrytis bunch rot, and Crown gall disease. During two years of field testing, 3 of 4 high endochitinase expressing Chardonnay lines have exhibited resistance to Botrytis bunch rot. In addition, preliminary tests in 2003 showed reduced Botrytis infection in 3 of 5 endochitinase expressing lines of Merlot. A total of 21 lines of Chardonnay tranformed with magainin-type genes showed improved resistance to crown gall disease when inoculated with two strains of the casual bacterium. Five magainin-transgenic lines showed moderate resistance to powdery mildew disease following artificial application of a highly concentrated inoculum. A relationship was observed between magainin gene transcription and disease resistance in this group of transformed lines. Further tests are needed to ascertain the relationship between gene expression and disease resistance.

Improving Grape and Wine Quality in the San Joaquin Valley

AVF Proj. ID: 258 / /Cat.: ,
Primary Investigator(s): Robert L. Wample, L.E. Williams, M.W. Fidelibus, N.K. Dokoozlianby

Three irrigation (-1.2, -1.4 or -1.6MPa) and two pruning (hand pruned (HP) or machine pruned (MP)) treatments were applied to approximately 10 acres of a commercial Syrah vineyard near Fresno CA. Each treatment was replicated five times. Data was collected to verify treatments effects on vine growth, water relations, soil moisture levels, fruit development, fruit quality and wine quality. Irrigation treatments were achieved, with anticipated effects on fruit size and development. Data indicate MP vines had fewer berries per cluster, smaller berry size, more clusters per vine than HP vines. These results reflect the combined effects of irrigation and pruning. Brix levels were generally higher in HP vines than in MP vines regardless of irrigation. This seems to be the result of lower yields for HP vines which ranged from 8.1 to 9.4 tons/acre. Yields for MP vines ranged from 10.1 to 13.2 tons/acre). In both HP and MP vines, the clear trend was for higher yields as the amount of irrigation was increased and stress was lower. Reduced stress resulted in larger berries and contributed significantly to yield. Within the MP vines, vines that received less water early in the season (-1.6 MPa) had higher Brix at harvest. MP vines also had fruit with lower pH, titratable acidity and color intensity than HP vines. The effects of irrigation were not as apparent on these parameters. On two occasions, wine makers tasted the fruit prior to harvest and in both instances, the preferred fruit came from the MP vines with the -1.6MPa irrigation treatment. It is also interesting that the MP vines at -1.4 MPa were considered to be the least desirable. The MP vines at -1.4 MPa received irrigation nearly two weeks sooner than those at -1.6 MPa, and suggests that timing of irrigation might be an important factor in managing fruit flavor and quality development.

The number of nodes retained per vine at pruning was nearly three times greater for machine pruning (136 nodes) compared to hand pruning (48 nodes). The mean number of shoots per vine was significantly less for hand pruned vines compared to machine pruned vines, while average shoot length and weight at dormancy was greater for hand pruned vines. Hand and machine pruned vines produced similar sized canopies, but partitioning between primary and lateral leaf area differed significantly between the two systems. Hand pruned vines had significantly larger primary and lateral leaves, and greater primary leaf area, compared to machine pruned vines. Sunlight within the fruiting zone was significantly greater in machine pruned vines. These differences resulted from changes in canopy growth characteristics, particularly the reduced primary shoot length and decreased primary and lateral leaf sizes of the machine pruned vines.

Total yields per acre ranged between 10.6 and 4.7 tons for hand pruned vines and 12.7 and 5.4 tons per acre for machine pruned vines. Machine pruned vines produced smaller clusters and berries than hand pruned vines. Preliminary color measurements indicate that wines from machine pruned vines had more color than wines from hand pruned vines. This is likely a result of their improved fruit zone light environment, as well as their decreased berry size. Crop load seemed to have less effect on wine color than pruning method, although significant increases in wine color were noted when crop load was reduced to less than 7 tons per acre in both methods.

While it is too soon to draw conclusions about the effects of pruning, irrigation and crop load treatments on quality of Syrah grapes in the San Joaquin Valley, but there is every indication that these treatments can cause changes in vine and fruit development and quality. Hence it is now our goal to determine the best combination of these factors to achieve better quality in an economically viable manner.

Wines made from the 2003 growing season are being finished and will be used to do taste panel analysis and for grower demonstrations of the effects of pruning and irrigation on grape and wine quality.

The Biology and Control of Botrytis Bunch Rot

AVF Proj. ID: 260 / /Cat.: ,
Primary Investigator(s): Wayne F. Wilcoxby

Botrytis bunch rot observed at harvest is often the result of infections initiated at bloom. Typically, the vast majority of such early infections remain latent (dormant) through harvest, and never cause disease. However, a fraction of these latent infections can become active as the crop matures, causing affected berries to rot and subsequently serve as sources for disease spread during the preharvest period. The factor or factors that promote activation of latent infections are poorly understood. In the research conducted last year, we found that by increasing the nitrogen content of berries after veraison, we increased the likelihood that latent infections initiated at bloom will transition to rotten berries at harvest. Higher N content also appeared to increase the susceptibility of berries to disease resulting from post-veraison infections via berry-to-berry contact within the cluster or air-blown spores from external sources. Confirming anecdotal observations, we furthermore determined that activation of latent infections was promoted significantly by periods of high humidity during the 10-day period before harvest, and that the effect was directly proportional to the duration of the humid period. In contrast, similar humid periods at veraison had no effect in this experiment. In field trials, we found that both Elevate and Vangard provided limited but significant postinfection control of latent infections when these fungicides were applied during the first few days after flowers were inoculated with spores of the Botrytis fungus. We also found that sprays designed to provide protective activity when applied before infection were equally effective when sprayed before or after cap fall. If substantiated, this finding suggests that the pedicel (berry stem), rather than stamens (flower parts), may be an important court for the establishment of latent infections. On a practical level, it also would be significant in by providing an element of flexibility in the timing of such sprays.

Spatial Variation in Soil Chemical and Physical Properties and Precision Agriculture in Two Carneros Pinot Noir Vineyards.

AVF Proj. ID: 261 / /Cat.: ,
Primary Investigator(s): David R. Smart, Kerri L. Steenwerth, Richard Plant, Nick Dokoozlian, Jean-Jacques Lambertby

In 2003 a limited amount of funding was obtained to initiate a research program that would be used to establish a Precision Viticulture Research Group at UCDavis. The goals outlined for our first year of support were to document the existence of spatial variation in fruit ripening and other indicators of vine performance, and to ascertain whether such variation correlated with soil heterogeneity. Spatial non-uniformity in winegrape vineyards is recognized, and its influence on production practices is the subject of much discussion. Nonetheless, the extent of such variation, its documentation and quantitative influence on vineyard production, and information or analysis linking such variation to specific soil properties is scarce. The extreme spatial variability that exists in soils is the major reason for the existence of the science of Precision Agriculture. How the principles of Precision Agriculture will be applied to viticulture practices, and what resources and vine parameters will be important to assess, will form the basis of our work.

We made substantial progress in 2003 using the support we received. Sampling grids were established in both of the targeted vineyard sites. In one such site, over one thousand observations concerning ripening uniformity, fruit quality, vine size, yields and pruning weights were acquired. The data revealed large differences in grape ripening (brix, range = 17.3-24.2, on August 12, 2003). Brix did not vary randomly from vine to vine, rather, it varied in a discernable pattern. Pruning weights appeared to correlate with ripening. Higher Brix readings were observed in the same vineyard locations where higher pruning weights were found. At the second vineyard site, parallel information was acquired with the exception of the fruit quality and yield information, and our efforts there were limited by funding restrictions and the fact that sampling protocols had to be established. In the coming year we will continue these investigations focusing on soils sampling and mapping, along with the collection of more fruit and vine data.

The Chemical Evolution and Preservation of Color in Red Wine Aging

AVF Proj. ID: 40 / /Cat.: ,
Primary Investigator(s): Andrew L. Waterhouseby

During the last year we have been working on the production of labeled malvidin 3-glucoside. Using unlabeled material, we have been able to optimize the conditions of enzymatic synthesis as well as the purification of malvidin-3-glucoside from starting material and side-products so that the reaction will yield adequate material to continue the project. Labeled malvidin-3-glucoside is not commercially available and there is no published method to produce it, which made the synthesis particularly difficult. An alternative procedure based on chemical labeling, which was tried first, did not work due to the ionization of anthocyanins, but it has produced other anthocyanins not described in the literature, and so these products may be interesting to analyze at some point later on. We have also advanced the understanding of red colored polymeric structures and the analytical procedures to measure them. We have found a poor agreement between two analytical procedures, the traditional method of SO2 bleaching and the newly developed Normal Phase – HPLC methodology. It is necessary to resolve the causes of these differences and also to compare them with the Adams’ assay (based on tannin precipitation). This wine study will unequivocally show the origin of the pigmented polymers and clarify the best method to measure them.

Chemical Characterization of Small Polymeric Pigments in Wines and Red Grape

AVF Proj. ID: 6 / /Cat.: ,
Primary Investigator(s): Douglas O. Adamsby

Polymeric pigments are important because they are the stable form of color in wines. They are thought to be formed by reaction of monomeric anthocyanin pigments with tannins or flavan-3-ols, such as catechin or epicatechin. During the 1999 season we observed a class of low molecular weight polymeric anthocyanin pigments in wines. In new wines these pigments were a large percentage of the anthocyanin color not bleached by SO2, thus classifying them as polymeric pigments. On the other hand they were not precipitated by protein, which suggested that they had a low molecular weight compared to typical tannins. This observation raised many practical questions, but also brought up several important questions regarding the chemical nature of the small polymeric pigments (SPP), which we addressed in this project during the 2000 season. One of the objectives of the work was to devise a purification scheme that would permit separation of small polymeric pigment (SPP) from monomeric anthocyanins and large polymeric pigment (LPP). We were successful in developing a procedure to purify SPP based on column chromatography on a Toyopearl HW-40(F) column and eluting the monomeric anthocyanins and the tannin fraction separately with different solvents. Using standard SO2 bleaching to assay polymeric pigments, we were able to show that the SPP actually resides in the monomeric fraction from the column. In the course of this work we discovered that the SPP could be partially bleached by SO2. This was an unexpected result, but indicates that further work needs to be done with polymeric pigments to determine the extent to which they are affected by SO2 bleaching. This is important because most assays for polymeric pigment rely on SO2 bleaching to distinguish them from monomeric anthocyanins. If polymeric pigments are indeed bleached by SO2, it means that such assays give an underestimate of the amount present, and that monomeric anthocyanins are overestimated. A major accomplishment in this work during the past year has been the synthesis of an SPP dimer containing catechin as the extension unit and malvadin-3-glucoside as the terminal unit. The availability of this dimer by an unambiguous chemical synthetic route will enable us to confirm the structure of the naturally occurring dimer found in grape skin extracts and wine. The availability of the synthetic dimer will also enable us to determine if anthocyanin pigments having that configuration (catechin-malvadin-3-glucoside) are affected by SO2 bleaching. The chemical reaction by which we created the dimer may also point the way to understanding how polymeric pigments are formed in wines during aging.

Identification of Character Impact Compounds of Chardonnay Which Determine

AVF Proj. ID: 9835 / /Cat.: ,
Primary Investigator(s): Ann C. Noble, Susan E. Ebelerby

Experiments have been conducted to optimize the Gas-Chromatograph – Olfactometry (GC-O) system. Peaks eluting from injection of headspace samples of wines were detected by sniffing. However further refinements of volatile collection protocols and of the GC-0 system are necessary to provide more sensitive “sniffing” of the eluting compounds. At present some volatiles detected by the flame ionization detector can be smelled by the judges, but many compounds detected by FID cannot be detectable by sniffing. It is recognized that some of these volatiles have no aroma, but the relatively low number of compounds which can be smelled indicate that an increase in the concentration of the volatiles or alternative optimization of the system to better focus the peak and reduce turbulence is needed. Alternative extraction/recovery methods are now being explored.

Studies on the Interaction of Flavor Compounds with Nonvolatile Components of Wine

AVF Proj. ID: 9744 / /Cat.: ,
Primary Investigator(s): Susan Ebeler, Linda Bissonby

Objectives: We proposed to investigate the effects that polyphenols, important nonvolatile constituents of wine, have on the volatility and aroma intensity of selected aroma compounds through: a) use of model odorant compounds to provide a systematic exploration of the effect odorant structure has on interactions with polyphenols; b) application of sensitive gas chromatographic headspace procedures to measure changes in volatility of odorants in the presence of polyphenols; c) correlation of instrumental results with measurements of sensory intensity to understand the effect interactions may have on flavor perception, and utilization of NMR technology to provide an understanding of the mechanisms of polyphenol and odorant interactions. Summary: During previous years of this proposal we focused on developing sensitive gas chromatographic headspace procedures for quantifying odorant/polyphenol interactions in model solutions. We also developed and evaluated sensory procedures for measuring these interactions. Using a time-intensity procedure and model solutions, we showed that retronasal aroma perception is significantly affected by the chemical/physical nature of the aroma compound, by the nature of the matrix, and by individual judge factors such as salivary flow rate. This work was recently published (Mialon and Ebeler, 1997). During the past year we have largely focused on optimizing NMR techniques to study flavor odorant interactions. We have observed that the structure of both the polyphenol and the odorant are critical for determining the strength of the interaction.

Develop and Test Capillary Electrophoresis Methods for Cations

AVF Proj. ID: 9509 / /Cat.: ,
Primary Investigator(s): Andrew L. Waterhouse, Susan E. Ebeler, Roger B. Boultonby

As discussed in the interim report, the cation method has been modified to allow the separation and quantification of ammonium, potassium, calcium and magnesium, as well as arginine, in less than six minutes per sample. Further modifications to the method, which will be tested in the upcoming weeks, may allow the separation of other amino acids as well. As this separation has not proven suitable for the analysis of copper and iron, other methods from the literature are being evaluated for those metals. The comparison of results from this method with results from the atomic absorption spectrometer and flame photometer will be conducted later in the summer as juices from the 1996 season become available. The separation of amino acids in wine has proven to be somewhat more difficult than anticipated. Of the methods tested to date, none provide a means of analyzing all of the important amino acids in a single analysis. The use of low pH phosphate buffer allows a very rapid separation of arginine, the aromatic amino acids, the sulfur containing amino acids and some of the other basic amino acids; there are several methods in the literature which could be used for the analysis of the remaining amino acids. We are currently evaluating the best combination of methods that will allow rapid separation of the majority of amino acids of interest. We will also evaluate the rapid derivitization technique developed by Waterhouse and Butzke under a separate AVF grant for its potential use in capillary electrophoretic separations of the amino acids. Juice samples from the 1996 harvest will be collected and analyzed using the separation methods developed above. Previously collected samples from the 1995 harvest will be analyzed as test samples during the developmental stage. The goal to field test methods developed during the course of this project will be met through an ongoing process. Several California wineries now have capillary electrophoresis instrumentation in-house; we maintain regular contact with these wineries for the testing of new methods of analysis as they are developed. In May Tom Collins taught a University Extension course on the use of capillary electrophoresis in wine analysis; several winery representatives expressed interest in participating in future field trials with the instrument. Collins will continue to pursue the involvement of these wineries in such trials, as well as contacting the instrument manufacturers concerning the use of their instruments for these trials.

Interaction of Flavors with Polyphenols Compounds Found In Wine

AVF Proj. ID: 9522 / /Cat.: ,
Primary Investigator(s): Susan Ebeler, Linda Bissonby

Research to date has focused on validating a gas chromatographic headspace method for measuring the volatility of flavor compounds in solution. Using this procedure with model odorant compounds, we monitored the time course for obtaining equilibrium between the gas and liquid phases and observed that equilibrium times of greater than 30 minutes were needed to obtain accurate measurements of headspace concentration. The method is sensitive allowing the measurement of the concentration of odorants in the gas phase above aqueous solutions containing <2 ppm odorant. The model odorants are generally found in foods and wine at levels above this concentration. Coefficients of variability for replicate analyses were less than 10%indicating good reproducibility. Using this method we have shown that catechin, a major wine polyphenol, significantly depressed the headspace concentration of ethyl benzoate in aqueous solution. These findings provide evidence for an association between catechin and ethyl benzoate which results in decreased ethyl benzoate volatility. However, catechin had no significant effect on the headspace concentration of n-butylpropionate, indicating that there is no interaction between the flavonoid and the unsaturated ester.