Mitchell Davey | Danielle Fox | James Harbertson

Comparison of Freeze-Killed versus Freeze-Dried Leaves for the Production of Frost Tainted Cabernet Sauvignon Wines

Mitchell Davey, Danielle Fox, and James Harbertson*
*Washington State University, 2710 Crimson Way, Richland, WA, 99354-1671 (jfharbertson@wsu.edu)

A commercial freeze-dryer was used to emulate the effects of freeze-killed leaf material on Cabernet Sauvignon wines in 2023. Fresh leaves were collected from a vineyard in Sunnyside, WA one month prior to harvest, and freeze-killed leaves (FK) were collected at harvest. The fresh leaves were subjected to freeze-drying (FD). The FK and FD leaves were added directly to the must prior to fermentation at two rates (0.9 and 3.6 g/kg must) including a control (0 g/kg must). Basic wine chemistry was unaffected by the additions. Untargeted solid-phase microextraction gas chromatography-mass spectrometry was used to identify major aroma compounds present. Tentatively identified compounds were evaluated statistically. Twenty-three compounds were found to vary significantly based on the treatments. Alcohols were significantly reduced by both leaf treatments. Terpenoids and norisoprenoids significantly increased with increased leaf dosages. A previously-suggested frost taint marker, 6-methyl-5-hepten-2-ol, which smells like coriander was only found in the FK-treated wines. Esters significantly increased in both treatments, consistent with dosage. Phenolics were measured in the wines using the Adams-Harbertson assay. Counter to previous results, wines made with both FK and FD leaves had significantly more phenolics and anthocyanins were unaffected. With the exception of the single frost taint marker, the FD treatments emulated the FK-treated wines.

Funding Support: Washington Wine Commission

Charity Maosah | Tom Collins | James Harbertson

Use of Reverse Osmosis, Immobilized β-glucosidase, and Adsorption as Remedy for Smoke-Affected Wine

Charity Maosah*, Tom Collins and James Harbertson
*Washington State University, 1900 Stevens Dr., Apt 615, Richland, WA, 99354-2131 (charity.maosah@wsu.edu)

Every year, wine producing countries are affected by smoke from wildfires. Wine made from grapes that have been exposed to smoke from wildfires can have unpleasant burnt, leather, smoky, and other sensory attributes. Smoke-related volatile phenols that play a role in the smoky characteristics exist as free phenols and sugar-bound glycosides. Free volatile phenols contribute to smoky aromas in wine. After wine fermentation, acid-mediated hydrolysis can release bound phenols, leading to increased smoky aromas. To improve the quality of smoke-affected wines, both free and bound smoke-related phenols must be removed or reduced. There are no effective means to reduce both free and bound smoke phenols in affected wines. Use of reverse osmosis (RO) with solid phase adsorption reduces free-smoke related phenols but not bound-smoke related phenols. This can result in smoke recurrence after some time. This study evaluates the use of immobilized β-glucosidase to release bound-smoke related phenols in wine and permeate which can then be removed by RO and suitable adsorption technique. Smoke-affected wines were treated using RO to create a permeate containing the phenol glycosides. β-glucosidase was immobilized in cross-linked chitosan-silica microspheres. Both original wine and permeate were subjected to hydrolysis by immobilized β-glucosidase at different temperatures for four hours. Released volatile phenols can then be removed by adsorption using activated carbon. Enzyme hydrolysis of phenol-glycosides was dependent on sample type (wine or permeate), temperature, and incubation period. In both wine and permeate, 35°C had highest hydrolysis of phenol-glycosides (21.47% and 33.82%, respectively) with a high hydrolysis rate within the first two hours of incubation. This combination of immobilized enzyme with RO and adsorption can improve the quality of smoke-affected wines and greatly reduce losses incurred by grapegrowers in the case of wildfires.

Funding Support: USDA Washington State Wine Agricultural Research Service

Jedediah Fitzgerald | Zhi Wang | David Garcia | Robert Guzman | Qun Sun

Using Cover Crop to Mitigate the Effects of Winery Wastewater Application on Soil, Grape, and Wine Quality

Jedediah Fitzgerald, Zhi Wang, David Garcia, Robert Guzman, and Qun Sun*
*California State University, Fresno, 2360 E. Barstow Avenue, MS VR89, Fresno, CA, 93740 (qsun@mail.fresnostate.edu)

The San Joaquin Valley is facing drought and saline conditions, both of which have adverse effects on grapevine growth. Approximately 3000 to 5000 L wastewater are generated per metric tonne of crushed grapes annually, posing risks to vineyard soil physical and chemical properties, and ultimately affecting grape and wine quality. This study aims to examine the effects of cover crops on soil properties, determine the effect of cover crops on grape and wine quality, and promote effective water and soil management within the framework of sustainable viticulture.

Eight-year-old Ruby Cabernet vines from a commercial vineyard in Fresno, California were used for this study. The experimental design was a randomized complete block design with eight treatments replicated four times. The treatments were: a control (no-till with residential vegetation), tilled, UC 937 barley, WB patron wheat, Pacheco triticale, Sierra oats, rye grass, and Dairyland magnum salt alfalfa. Each experimental unit consisted of one vine row, a quarter-mile in length. The vineyard was furrow-irrigated with wastewater before planting the cover crop.

Results from the two years of the study revealed significant effects of cover crops on soil chemical compositions. Barley, oat, rye grass, triticale, and wheat sequestered carbon at higher levels (200 to 400 kg/acre) than the control. Oat, rye grass, triticale, and wheat absorbed nitrogen at higher levels (6 to 10 kg/acre) than the control. Barley and rye grass absorbed higher sodium content than the control. Triticale and wheat reduced soil electrical conductivity. Rye grass, triticale, and wheat resulted in lower soil sodium. Yield components exhibited no difference among treatments. However, preliminary wine sensory analysis has shown that the treatments may influence both aromatic properties and color. The project is ongoing and additional data will be provided after completion of the third-season trial.

Funding Support: Agricultural Research Institute (ARI)

Quinn Cahoon | Shunping Ding | Shijian Zhuang | Qun Sun

Effect of Biofungicides on Grape Quality, Composition, Fermentation, and Sensory Characteristics of Wines from California

Quinn Cahoon,* Shunping Ding, Shijian Zhuang, and Qun Sun
*Department of Viticulture and Enology, California State University Fresno, 5241 N Maple Ave, Fresno, CA, 93740 (qcahoon@mail.fresnostate.edu)

Synthetic fungicides have demonstrated adverse environmental effects on finished wine attributes. Alternatives such as biofungicides employ organisms to control pathogens. The purpose of this study is to assess the effects of biofungicides on grape and wine chemistry, fermentation, and wine sensory characteristics. Four separate grape crops treated with biofungicides were vinified: Pinot noir and Chardonnay from the Central Coast, and Carignan and Chardonnay from the San Joaquin Valley. Treatments consisted of three different biofungicides: two bacterial strains (Bacillus subtilis strain QST 713 and Streptomyces lydicus strain WYEC 108) and an extract of Reynoutria sachalinensis. Two controls consisted of synthetic fungicide applications and no fungicide. All treatments were carried out in quadruplicate. Data has been collected on fermentation and basic grape and wine chemical parameters (total soluble solids [TSS], pH, tiratable acidity, and ethanol). Preliminary results for the Central Coast indicate significant differences in TSS compared to untreated controls, while no significant differences were found in grape and wine chemical parameters for the San Joaquin trials. Preliminary sensory trials show differences in color and aromatic intensity between treatments. Further analyses will be conducted on the wine color, phenolic content using high-performance liquid chromatography, and sensory characteristics using descriptive sensory analysis.

Funding Support: CDFA, CSU-ARI

Juliana Pazos | Ryan Doyle | Pierre Davadant | Nataliya Shcherbatyuk | Markus Keller | James Harbertson

Effect of Soil and Foliar Nitrogen Applications on Syrah Fruit and Wine Tannin Concentration and Composition

Juliana Pazos, Ryan Doyle, Pierre Davadant, Nataliya Shcherbatyuk, Markus Keller, and James Harbertson*
*Washington State University, 2710 Crimson Way, Richland, WA, 99354-1671 (jfharbertson@wsu.edu)

Syrah fruit and wines from a Columbia Valley vineyard trial in Washington State were evaluated. Nitrogen was applied at four different rates: to early-season soil at 0, 22.5, 45, and 90 kg N/ha and at veraison foliar at 15 kg N/ha; these treatments were denoted as control, low, medium, and high N and urea. Treatments were applied to three different vine rows in a randomized block design in 2021 to 2023. Fifteen wines were made for each vintage (five treatments, three replicates). Fruit and wine anthocyanins, grape seed, and skin tannins were analyzed for concentration (2021 to 2023) using the Adams-Harbertson assay and composition using phloroglucinolysis (2022-2023). Phenolics were measured daily during fermentation and during aging. At harvest, N-containing compounds including ammonia, yeast available nitrogen (YAN), and protein were measured. YAN and ammonia increased in the high N and urea treatments, with some exceptions. No significant differences were found in seed tannin concentrations for the three vintages, and no clear pattern was observed for skin tannins or anthocyanins. However, wine tannin concentrations were significantly greater in the control than in the high-N application treatment in the 2021 vintage, and both the control and low treatments were significantly greater than the high and urea treatments in the 2022 and 2023 vintages. Protein concentration had the opposite trend, with higher juice protein concentrations in the medium, high, and urea treatments, although the measures varied considerably. The phloroglucinolysis results show no difference in fruit tannin size or composition; however, the high N and urea treatment wines had significantly smaller average polymer size. The results show that the vineyard N applications increased ammonia, YAN, and juice protein, but the increased protein may be responsible for the reduced tannin concentration and size observed in the high N and urea treatments.

Funding Support: USDA-NIFA SCRI, Washington Wine Commission, Washington State Grape and Wine Research Program, Ste. Micelle Wine Estates (in-kind)

Federico Casassa |Sean Kuster | Michael Upton | James Nelson | Bob Coleman

Influence of Sodium and Calcium-based Bentonite Dosage Rates on Alcoholic Fermentation Kinetics in Chardonnay Wines

Sydney Fritsch, Kevin Bargetto, Federico Casassa, and Miguel Pedroza*
*California Polytechnic State University, 1 Grand Avenue, San Luis Obispo, CA, 93407 (miguelp@calpoly.edu)

Bentonite is commonly used in production of white wines to clarify must before alcoholic fermentation and to remove unstable proteins responsible for turbidity. However, it can also remove nitrogen compounds that affect fermentation kinetics and the development of wine aromas. We investigated how sodium-based and sodium-calcium-based bentonite dosage rates affect fermentation kinetics, yeast assimilable nitrogen (YAN), and standard chemical composition in Chardonnay wines from the Edna Valley AVA. Bentonite dosage rates of 0, 0.24, 0.48, and 0.72 g/L were applied to 60 gallons of must with four replicates per treatment at a commercial winery. Our initial findings show that regardless of dosage or bentonite type, wines treated with bentonite experienced a longer lag phase and took approximately one additional day to reach 0 Brix compared to the control wine. In sodium-based bentonite treatments, the 0.72g/L dosage significantly decreased YAN levels by 15 mg/L compared to the control, while with calcium-based bentonite, there were no significant differences observed in YAN concentrations across dosage rates. Future analysis, including gas chromatography-mass spectrometry, color, and phenolic compounds, will provide additional insights into the effects of bentonite treatment on Chardonnay wine quality.

Funding Support: Center of Effort Winery

Megan Meharg | Mackenzie Aragon | Caroline Merrell | Tom Collins

Effect of Yeast Strain Selection on the Concentration of Smoke-Derived Volatile Phenols and Thiophenols in Wine

Megan Meharg, Mackenzie Aragon, Caroline Merrell, and Tom Collins*
*Washington State University, 359 University Dr, Richland, WA, 99354 (tom.collins@wsu.edu)

Wildfire and controlled burn events pose an economic threat to the global wine industry. Smoke-derived volatile phenols (VPs) exist as free and bound glycosidic precursors that are released during fermentation-driven enzymatic hydrolysis, but the role that yeast might play in thiophenol production has not been studied. Free VPs and thiophenols of smoke-affected wines have been shown to cause perception of undesirable attributes such as meaty, smoky, medicinal, and ashy aromas and flavors. Here we studied the effect of commercially available yeast strains on the concentrations of known smoke-derived VP and thiophenol markers. Grapevines (Vitis vinifera cv. Merlot) were experimentally exposed to smoke in a semi-controlled environment. Smoke-exposed and non-exposed grapes were fermented using 12 commercial yeast strains over two vintages. Gas chromatography-mass spectrometry confirmed the increased concentrations of VPs in wines from smoke-affected fruit compared to the control wines, but there were no differences in concentrations of VPs among the tested yeast strains. Investigation of thiophenols using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry revealed differences between control and smoke wines in the relative peak area for thioanisole and thiophenol, but not for thiocresols or thioguaiacol. There were, however, differences among yeast strains for total thiocresols (m-, o-, and p-isomers) and for thioguaiacol. Our study suggests that commercial yeast strains play a role in the release of VPs and the concentration of thiophenols.

Funding Support: USDA, Washington Wine Commission, Jackson Family Wines.

Mary Riley | Justin Siegel | Anita Oberholster | David Mills

Enzymatic Degradation of Undesirable Phenols in Wines Contaminated by Brettanomyces or Smoke Taint

Mary Riley,* Justin Siegel, Anita Oberholster, and David Mills
*University of California, Davis, 1 Shields Ave, Davis, CA, 95616 (myriley@ucdavis.edu)

This is the first year of a multi-year project to discover and design enzymes with exclusive degradation of 4-ethylguaiacol (4EG) and 4-ethylphenol (4EP), the main sensory-active components in wine contaminated with Brettanomyces and responsible for a portion of the ‘taint’ phenols found in smoke-tainted wine (Figure 1). A library of 46 laccase enzymes were produced and tested in different buffered environments for activities on 4EG and 4EP, with multiple laccases degrading both phenols. All laccases were tested further in a model wine environment with 13% ethanol and tartaric acid, at pH 3.5. Multiple laccases quickly and completely degraded 4EG, along with mild activity on 4EP. Sequence analysis of the 4EG- and 4EP-active enzymes revealed two distinct clusters, each with a unique laccase that can degrade 100% of 4EG and 30% of 4EP in industrially relevant times, matrices, and concentrations. 4EP and 4EG docking interactions generated via computational modeling revealed the top-degrading laccases all display open active sites and likely contribute to activity on other, non-target, wine phenols, thereby reducing possible selective degradation of 4EG and 4EP. Currently, novel enzyme structures are being generated computationally to address this deficiency using open-source AI-tools, including RFDiffusion and LigandMPNN, with unprecedented abilities to design proteins and model their structures. The next phase of this project will use the knowledge of how laccases degrade 4EG and 4EP to create additional enzymes capable of fully, and exclusively, degrading the remaining smoke taint marker compounds including guaiacol, 4-methylguaiacol, o-, p-, and m-cresol, and syringol. The larger goal of this effort is to create a suite of novel enzymes that beneficially modulate wine flavor.

Funding Support: American Vineyard Foundation

Jonathan Brumley, | Ipek Aktuna | Charles Edwards

Impact of Yeast Assimilable Nitrogen and Sugar on Sequential Fermentation with Metschnikowia pulcherrima and Saccharomyces cerevisiae

Jonathan Brumley,* Ipek Aktuna, and Charles Edwards
*Washington State University, 1455 NE Brandi Way LL-303, Pullman, WA, 99163 (jonathan.brumley@wsu.edu)

We investigated the application of a low ethanol-producing yeast during co-fermentation with Saccharomyces cerevisiae to lower alcohol concentrations in wine. Specifically, this study investigated the complex relationships between yeast assimilable nitrogen (YAN) and sugar concentrations on Metschnikowia pulcherrima in sequential fermentation. A two × three factorial was designed, with YAN (40 mg N/L or 280 mg N/L) and soluble solids (24, 27, or 30 Brix) as variables, by modification of synthetic grape juice media (SGJM). SGJM was fermented by either S. cerevisiae alone, or by M. pulcherrima and S. cerevisiae, the latter inoculated on day 4. Fermentations inoculated with M. pulcherrima reached similar maximum populations (≈107 CFU/mL), except for those containing 40 mg N/L at 30 Brix, where populations were only 106 CFU/mL. In general, M. pulcherrima exhibited an extended lag phase before log growth in media containing 30 Brix, regardless of initial YAN concentrations. All fermentations with 40 mg N/L progressed slower than fermentations with 280 mg N/L. Low YAN fermentations only reached dryness (completion) at 24 Brix, which occurred six days after high-YAN fermentations at the same sugar level. SGJM fermented with M. pulcherrima and S. cerevisiae had lower glycerol concentrations than single-inoculation fermentations in 27 and 30 Brix treatments. Contrary to previous studies, ethanol concentrations were not affected by the presence of M. pulcherrima, except for the treatment containing 40 mg N/L with 24 Brix, which resulted in 0.7% v/v reduction.

Funding Support: Washington State Grape and Wine Research Program

C. Michael Sonza | Emily Kaneshiro | Tess Snyder | Bjarne Bartlett | James Osborne | Christopher Curtin

Determining the Spoilage Potential of Brettanomyces Strains Isolated from Oregon Vineyards and Cellars

C. Michael Sonza, Emily Kaneshiro, Tess Snyder, Bjarne Bartlett, James Osborne, and Christopher Curtin*
*Department of Food Science and Technology, Oregon State University, 3051 SW Campus Way, Corvallis, OR, 97331 (christopher.curtin@oregonstate.edu)

Oregon wine producers can face economic losses from wine spoilage from Brettanomyces bruxellensis infections. Despite precautions to mitigate infections by B. bruxellensis, such infections still occur. Variation in mitigation efficacy may be due to the emergence of sulfite-tolerant strains of B. bruxellensis in the wine production facility, as well as differences in quantity and strain of B. bruxellensis on the grapes entering the winery. Limited knowledge exists regarding whether known or novel sulfite-tolerant strains are present in OR wine facilities. Furthermore, the extent to which B. bruxellensis exists in vineyards is not well characterized due to the difficulty of isolating this yeast when more-abundant background yeast are present. This study seeks to address the limited understanding of the spoilage potential of B. bruxellensis strains found in OR cellars and vineyards.

We collected 100 Pinot noir cluster samples during the 2023 harvest from five different OR vineyards and will use an additional 105 cluster samples cryopreserved from the 2022 harvest. Collected vineyard samples will be grown in a selective enrichment medium that is currently being optimized to preferentially grow B. bruxellensis and limit background yeast growth. We also collected 52 wine samples and 79 environmental swabs representing seven OR cellars. Any identified B. bruxellensis isolates from the vineyard and cellar will be whole-genome sequenced and evaluated with sequence alignment to determine how related OR strains are to known strains outside of OR, particularly sulfite-tolerant strains. A preliminary enrichment formulation trial using cellobiose as the sole carbon source shows promising inhibition of background yeast, while not impeding growth of most B. bruxellensis isolates. The outcomes of this study will clarify the spoilage capacity of B. bruxellensis in OR wineries and vineyards, supporting wine producers to make informed decisions on how to best protect their wine from infections.

Funding Support: Oregon Wine Board

Rajasekharreddy Bhoomireddy | Ramesh Pilli | Venkateswararao Kadium | Brent Trela | Collin Auwarter | Andrej Svyantek | Harlene Hatterman-Valenti

Genome-Wide Association Study of Anthocyanins and Phenolics in a Cold Climate Winegrape (Vitis spp.) Population

Rajasekharreddy Bhoomireddy,* Ramesh Pilli, Venkateswararao Kadium, Brent Trela, Collin Auwarter, Andrej Svyantek, and Harlene Hatterman-Valenti
*North Dakota State University, 1844 10th St N, Fargo, ND, 58105 (r.bhoomireddy@ndsu.edu)

To ensure superior wine quality and enhanced health benefits, it is desirable to maintain stability of anthocyanins and total phenolics in winegrapes. These compounds not only impart rich color and robust flavor to wine, but also include antioxidants that are beneficial to human health. However, ensuring their stability poses a significant challenge in North Dakota’s harsh and variable climate. Deciphering genetic control of these compounds is crucial to influence the astringency, color, and mouthfeel of the wine. An incomplete diallel population of 1064 individuals was used to examine the genetic basis of key compounds for total phenolic and anthocyanin composition that affect both health benefits and sensory attributes of wine. To understand the genetic variation responsible for these traits, a genome-wide association study (GWAS) was conducted using 24,000 SNP markers. The GWAS revealed six significant SNP associations for anthocyanins on chromosomes 2, 5, 7, and 16 and three significant SNP associations for total phenolics on chromosomes 2, 6, and 11 over a period of two years. These findings will provide insights to understand the genetic factors affecting phenolic and flavonoid levels in winegrapes, offering a valuable resource for viticulture and enology, with potential applications for grapevine breeding for improved wine quality.

Funding Support: Specialty Grant

Bainian Chen | Arran Rumbaugh | Ron Runnebaum | Chen Liang | Annegret Cantu | Hildegarde Heymann

Correlating Volatile Phenol Concentrations in Cabernet Sauvignon Grapes to Unfavorable Sensory Characteristics in Wines

Bainian Chen, Arran Rumbaugh,* Ron Runnebaum, Chen Liang, Annegret Cantu, and Hildegarde Heymann
*USDA-ARS, 595 Hilgard Lane, RMI North, room 3156, Davis, CA, 95616 (arran.rumbaugh@usda.gov)

Within California’s wine industry, wildfire smoke poses a significant threat to vineyards by imparting negative sensory attributes to wines, termed “smoke taint.” Hence, predicting the emergence of such tainted wines is crucial. This study evaluated the efficacy of a proof-of-concept approach to reproducibly expose grapes to smoke conditions. The long-term aim is to establish threshold levels of smoke-derived compounds in grapes that cause negative sensory attributes in the final wines. Around 250 kg of Cabernet Sauvignon grapes was harvested at 25 Brix from the Tyree vineyard at the University of California, Davis. Four experimental groups were exposed to various levels of smoke generated from red oak chips: control-exposure (CE, no smoke exposure), low exposure (LE, 0.5-hr exposure), medium exposure (ME, two-hr exposure), and high exposure (HE, four-hr exposure). Each experiment was conducted in an in-house constructed smoking chamber. Following smoke exposure, duplicate fermentations of each treatment were performed in stainless steel buckets. Volatile phenols (VPs) and their glycoconjugates (bound) were measured in grape and wine samples using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry, respectively. In addition, sensory differences in wines were determined utilizing descriptive analysis (DA). Free and bound VP concentrations in grapes and wines correlated positively with smoke exposure duration and intensity, as expected. Results indicated no significant sensory differences between LE and CE wines. ME samples were described as “medicinal” and significantly lower in fruity aroma than LE and CE samples. HE samples were rated even lower in fruity aromas and higher in “barbeque” and “ashy” aromas than all other treatments. Overall, ME wines had a significant sensory affect compared to CE wines, suggesting a potential range for future threshold experiments. The study was a successful proof-of-concept to develop threshold levels of free and bound VPs in grapes that will lead to unfavorable sensory attributes in the final wines.

Funding Support: USDA-ARS Funded Research

Lauren Picone | Charlotte Decock | Cristina Lazcano

Assessing Long-Term Effects of Regenerative Management Practices on Vineyard Soil Health

Lauren Picone,* Charlotte Decock, and Cristina Lazcano
*Cal Poly, San Luis Obispo, 1802 14th St., Los Osos, CA, 93402 
(laurenmpicone@gmail.com)

Vineyards are often developed on slopes or marginal lands, which results in soils that are particularly susceptible to erosion and degradation. To mitigate these impacts, regenerative management methods—including the use of cover crops, no-till, compost application, and livestock integration— can be used to improve soil health and increase soil organic matter. These practices can improve crop productivity and quality, water infiltration, nutrient availability, and carbon sequestration within soils. Because building soil health is a slow process, there is little knowledge regarding the effects of long-term use of regenerative management practices on vineyard soil health.
To improve soil health and create more resilient agricultural systems, this project aims to assess the effects of long-term management practices on vineyard soil health across an edaphoclimatic gradient in California. Soils were collected from 87 different vineyard blocks across CA, specifically targeting vineyard blocks where one or several regenerative practices have been adopted for five or more years. Following recommendations from the Soil Health Institute, all samples were analyzed for a minimum suite of soil health indicators, which included soil organic carbon concentration, carbon mineralization potential, and aggregate stability. For each vineyard block, growers completed a qualitative survey on the management history and the performance of the vineyard, including yield, crop quality, water, nutrient, and pest management. Our preliminary results show that finer-textured soils had more total soil carbon concentrations than coarse-textured soils in the vine and alley row.  Total soil carbon concentrations in the vine and alley rows increased significantly with the number of years livestock integration had been adopted. Additionally, the years of livestock integration and adoption of no-till increased aggregate stability indices within the vine row. Texture class and location were more important in predicting total carbon and aggregate stability indices than the duration of practice adoption.

Funding Support: Foundation for Food and Agricultural Research, and CDFA

Rushil Patel | Satyanarayana Gouthu | Laurent Deluc

Exploring Auxin Responsive Factor 4’s Influence on Timing of Ripening Initiation Using Molecular Breeding

Rushil Patel, Satyanarayana Gouthu, and Laurent Deluc*
*Oregon State University, 2750 SW Campus Way, Room 4121, Corvallis, OR, 97331 (delucl@oregonstate.edu)

Genetic manipulation is a major tool in determining gene function and its relationship with traits of interest in plants. For any crop, it requires a tractable genetic transformation system and a reliable expression system to turn on or switch off expression of targeted genes. We implemented the microvine system for Agrobacterium tumefaciens-mediated transformation and a plant gene switch system to control the transgene expression by supplying transgenic greenhouse plants (through root-drenching) a chemical inducer, methoxyfenozide. As proof of principle, we conducted gain and loss of function studies on Auxin Responsive Factor 4, a transcription factor that may contribute to the timing of ripening initiation in grape berries. We developed three types of constructs. In construct 1, we aimed to induce over-expression of the native endogenous ARF4. In construct 2, we generated transgenic lines containing a synthetic ARF4 resistant to miRNA-mediated gene silencing (miRARF4). In construct 3, we created ARF4-silencing lines using artificial miRNA technology. To pinpoint plants with the most response to the chemical inducer, eight plants from each construct were grown to maturity and induced over a month with leaf collections at select points. Six collection times were chosen to comprehend both the short-term responses (days 0, 3, 7, and 10) and the reversibility of the system (days 14 and 31). The data extracted using qPCR allowed us to characterize the time period of greatest induced transgene expression. Expression levels of the most responsive plants were eight-fold in the native construct, 100-fold in miRARF4, and 1000-fold increase in the ARF4 silencing line. Ongoing experiments quantitatively compare expression levels in berry samples to verify the inducibility dynamics in the fruit. Once confirmed, the study intends to focus on comparing ARF4’s targetome among the three constructs by using genome-wide transcriptomic analyses.

Funding Support: Oregon Wine Board, Erath/Family Foundation, Oregon Wine Research Institute

Abram Smith | Satyanarayana Gouthu | Charles Dearing | Laurent Deluc

Identifying Protein-Protein Interactions in Red Blotch Infected Vines

Abram Smith, Satyanarayana Gouthu, Charles Dearing, and Laurent Deluc*
*Oregon State University, 2750 SW Campus Way , Corvallis, OR, 97331 (laurent.deluc@oregonstate.edu)

Grapevine red blotch virus (GRBV) poses a significant threat to winegrape production. Host factors critical for red blotch infection remain largely unknown in grapevines, impeding attempts to use CRISPR technology to generate red blotch-resistant grapevines. Our project seeks to identify these host factors. Our two main objectives are to identify the host factors using the yeast two-hybrid (Y2H) system and to validate their interaction with viral proteins in planta. For the Y2H experiment, we will conduct a comprehensive genome-wide screen using GRBV replication-associated proteins (C1, C2, C3, and C1-2) as baits against a comprehensive grapevine cDNA library from GRBV-infected plants as preys. Based on the strength of the interactions and their biological relevance from previous studies, we will select the top two most promising interactors for each bait (C1, C2, C3, C1+2) to conduct bimolecular fluorescence complementary assays from isolated grapevine protoplasts. All four baits have been successfully cloned into yeast, initiating the screening process. A prey library using coding-gene related RNA is currently being constructed. The next step will be to carry out mating of the bait and prey, as well as isolation of mated yeast. Further screenings on a series of selective media will identify high-confidence interactors, followed by Sanger sequencing to identify their biological function. To get ahead of the work on objective 2, we have optimized a protocol to isolate protoplasts from tissue cultured cells of microvines. If we reach our goals, we will lay the groundwork for further studies to produce CRISPR-engineered, GRBV-resistant grapevines.

Funding Support: California Department of Food and Agriculture

Vincenzo Cianciola | Luca Pallotti | Alessandro De Rosa | Peyton Peralez | Desiree Hernandez | Luis Ortiz | William Whalen | Eve Laroche-Pinel | Luca Brillante

A Two-Year study of 12 Sustained and Regulated Deficit Irrigation Schedules for Cabernet Sauvignon in Central California

Vincenzo Cianciola, Luca Pallotti, Alessandro De Rosa, Peyton Peralez, Desiree Hernandez, Luis Ortiz, William Whalen, Eve Laroche-Pinel, and Luca Brillante*
*Department of Viticulture and Enology, California State University Fresno, 2360 E Barstow Ave, Fresno, CA, 93740 (lucabrillante@csufresno.edu)

Amidst escalating drought conditions in the San Joaquin Valley, where grapegrowers face an urgent need for effective water management strategies, this study contributes valuable insights for sustainable viticulture practices. Recognizing the pressing importance of optimizing water use in vineyards, particularly in the context of the West San Joaquin Valley, this research aims to unravel the physiological responses of grapevines under varying intensities and timings of sustained and regulated deficit irrigation.

Focused on a singular commercial Cabernet-Sauvignon × 1103 Paulsen vineyard, this study explored the impact of irrigation treatments administered during 2022 and 2023 seasons. Employing a semi-autonomous irrigation approach, actual water quantities were recorded using flow meters. The study incorporates sustained deficit irrigation (SDI) schedules with varying percentages (40, 60, 80 and 100% of ET_c) and regulated deficit irrigation (RDI) strategies in both preveraison and postveraison periods (e.g., 100/40, 80/60, 60/100).

Stem water potential and gas exchange were measured from June to harvest, grape composition from veraison to harvest and yield components were also assessed at harvest time. Results show that while the 40% SDI significantly underperformed 100% ET_c, it is possible to obtain similar or better results while saving water with RDI and SDI treatments with lower ET_c reductions.

This research determined the primary effects of these irrigation regimes on plant water status, gas exchange, berry composition, and yield components. The findings aim to provide a nuanced understanding that can inform advanced developments in water management strategies, offering practical insights for grapegrowers navigating the challenges of water scarcity in the San Joaquin Valley.

Funding Support: American Vineyard Foundation, California State University – Agriculture Research Institute

Alessandro De Rosa | Luca Pallotti | Eve Laroche-Pinel | William Whalen | Luca Brillante

Enhancing Water Use Efficiency and Berry Ripening in Cabernet Sauvignon: Effects of Irrigation Regimes and Biostimulants

Alessandro De Rosa, Luca Pallotti, Eve Laroche-Pinel, William Whalen, and Luca Brillante*
*Department of Viticulture and Enology, California State University Fresno, 2360 E Barstow Ave, Fresno, CA, 93740 (lucabrillante@csufresno.edu)

Water availability is a key factor for agriculture, as rising temperatures increase plants’ water demand, while water resources become increasingly limited.

Optimizing water use efficiency is essential to enhance vine tolerance to heat stress and ensure high productivity. This study examines the effects of four distinct irrigation regimes: vines irrigated at 50, 75, and 100% of estimated crop evapotranspiration (ET_c), alongside regulated deficit irrigation (RDI), employing 50% of vine ET_c from fruit set to veraison and 100% from veraison to harvest, both independently and in combination with a biostimulant during the 2023 growing season. Measurements were taken for stem water potential, gas exchange, berry ripening, and yield at harvest of Cabernet Sauvignon vines. The biostimulant did not affect vine physiology, as stem water potential, gas exchange, and water use efficiency remained unchanged between treated and untreated vines. There were differences related to irrigation amount, with greater water supply leading to lower stress conditions and increased photosynthetic activity. However, this improvement came at the cost of reduced water use efficiency when compared with the 50% irrigation and RDI treatments, which had the greatest efficiency. The biostimulant increased berry and cluster weight without affecting berry ripening in combination with 50, 75, and 100% irrigation, but not with RDI. The water regime had a significant effect on juice composition, with the 50% treatment hastening berry ripening and the 100% treatment delaying it. Interestingly, RDI had the most pronounced effect on ripening, slowing down sugar accumulation and preserving organic acids.

Even though the effects of the biostimulant were limited, this study underscores that restricting water during periods when vine demand is limited represents a sustainable approach to enhance water use efficiency and delay berry ripening.

Funding Support: N/A

Desiree Hernandez | Alessandro De Rosa | Eve Laroche-Pinel | Luca Brillante

A Comparative Study of Traditional Drip Irrigation and Deep Underground Irrigation to Improve Water Use Efficiency

Desiree Hernandez, Alessandro De Rosa, Eve Laroche-Pinel, and Luca Brillante*
*Department of Viticulture and Enology, California State University Fresno, 2360 E Barstow Ave, Fresno, CA, 93740 (lucabrillante@csufresno.edu)

Water efficiency is crucial for grapevine cultivation, especially in regions like California where water scarcity poses significant challenges. In 2023, a comprehensive project was initiated in Madera, CA, aimed at comparing the efficacy of traditional drip irrigation with a novel, deep-underground irrigation system in sustaining grapevine water status. The experiment comprised eight blocks, each with four vines, with four blocks irrigated conventionally using drip irrigation and the remaining four with a deep underground irrigation setup.

From June to August 2023, three water-related metrics were measured five times: stem water potential (Ψ_stem), stomatal conductance (g_s), and net assimilation rate (AN). Ψ_stem measurements showed a discernible disparity in mid-July, coinciding with peak water stress conditions. During this critical period, vines under traditional drip irrigation exhibited lower average Ψ_stem (-1.24 MPa) than those under deep irrigation (-1.13 MPa). Throughout the observational period, vines subjected to deep irrigation consistently demonstrated superior physiological performance. Specifically, AN remained consistently higher under deep irrigation, with an average difference of 2.6 µmol CO²/m²sec compared to drip irrigation. Similarly, g_s values were consistently elevated under deep irrigation, with an average difference of 65.9 mmol H₂O/m²sec except during the initial stages of the trial.

These findings underscore the efficacy of deep irrigation in mitigating water stress and enhancing grapevine physiological functions compared to traditional drip irrigation methods. The study provides valuable insights into the potential of deep underground irrigation systems to optimize water usage and sustain grapevine health and productivity in water-scarce environments. Ultimately, such innovative irrigation approaches hold promise for improving water efficiency and promoting sustainable viticulture practices in CA and beyond.

Funding Support: N/A

Tingting Ye | Michael Qian | Alexander Levin | Yanping Qian

Effect of Irrigation Initiation Time on Methoxypyrazine and C13-norisoprenoid Composition in Pinot noir Wine

Tingting Ye, Michael Qian,* Alexander Levin, and Yanping Qian
*Department of Food Science and Technology, Oregon State University, 100 Wiegand Hall, Corvallis, OR, 97331 (michael.qian@oregonstate.edu)

This study investigated the influence of initiated irrigation time on the methoxypyrazine and C13-norisoprenoid compositions of Pinot noir wine. The experiment was conducted in three subregions of the Rogue Valley in southern Oregon for two years, and the Pinot noir vines were irrigated at different irrigation initiation times based on normalized vine water status thresholds (ΔSWP). The ΔSWP was defined as the departure of measured midday stem water potential from the calculated non-water-stressed baseline, varied from 0.2 MPa (control) to the most delayed irrigation (1.0 MPa) in five irrigation initiation times, while the irrigation amount was set at 70% ET_c, based on industry standard. Wines produced in each of the two vintages were analyzed. Methoxypyrazines and C13-norisoprenoids in the wine were analyzed by headspace solid-phase microextraction gas chromatography-mass spectrometry and stable-isotope labeled compounds were used as internal standards for quantification. The results showed that the delayed irrigation initiation time decreased the methoxypyrazine levels and increased C13-norisoprenoid levels in the wines, although the trends depended on vintage and subregion. These changes in volatile composition in response to irrigation initiation timing could affect aroma perception and overall wine quality. Determination of the optimal irrigation initiation timing can provide a guideline for grapegrowers to produce high-quality winegrapes and reserve water resources.

Funding Support: Northwest Center for Small Fruits Research Commission, USDA

Kenneth Donsky, | Benjamin Ramsell | Britt Eubanks | Laurent Deluc

Accelerating Molecular Breeding via Development of a Novel Multiplexed Gene-Editing Method for Vitis vinifera

Kenneth Donsky,* Benjamin Ramsell, Britt Eubanks, and Laurent Deluc
*Oregon State University, 1450 NW Division Street, Apartment 32, Corvallis, OR, 97330 (donskyk@oregonstate.edu)

Genome editing can aid breeding programs dedicated to delivering genetically improved material from crops that will not be able to adapt to rapidly evolving climate change. Using the microvine system, we developed a research program to produce high-throughput gene-edited collections of grapevine mutants for easy gene-to-trait interrogations. To achieve this goal, we pursued the following three primary objectives: i) improve plant regeneration from Agrobacterium tumefaciens-mediated transformation events in somatic embryogenic grapevine cells, ii) increase the editing rate using a specific variant of the CRISPR/Cas9 protein, and iii) evaluate the potential of a new nanomaterial, carbon nanodots (CDs), to deliver multiple single guide RNAs for multiplexed editing. The approach to achieve objective 1 involves using the conditional expression of the morphogenic and synthetic gene, miRGRF_GIF4, from Vitis vinifera. For objective 2, we will compare the editing rate of different versions of the CRISPR/Cas9 editing system. In objective 3, we propose to identify the ratio of CD:sgRNA for optimized delivery to embryogenic grapevine cells. Zinc-functionalized CDs have been selected for their ease of synthesis and zinc’s affinity for nucleic acids. If we achieve our goals, we will give the scientific community a new tool for generating KO mutant collections that could expand beyond the grapevine model.

Funding Support: USDA NIFA

Christian Mandelli | Laurent Deluc

Spray-Induced Gene Silencing (SIGS) with Carbon Dots for Enhanced RNA Delivery in Grapevine

Christian Mandelli* and Laurent Deluc
*Oregon State University, 2750 SW Campus Way, Corvallis, OR, 97331 (mandellc@oregonstate.edu)

Spray-induced gene silencing (SIGS) is a recent technology that enhances a plant’s immune system. It involves the ectopic application of double-stranded RNAs (dsRNAs) to trigger a plant’s defense mechanism, known as RNA-interference (RNAi). Unlike traditional methods relying on the generation of transgenic plants, SIGS offers a rapid and targeted disease management solution. By transiently enhancing RNAi, it eliminates the need for resources and time-consuming development of resistant plants. However, SIGS application in tree crops encounters significant challenges, including limited cellular uptake and environmental stability of dsRNAs. To address these limitations, we explored an innovative approach that combines SIGS with branched polyethyleneimine-functionalized carbon dots (CDs) as nanocarriers, which enhanced dsRNA stability and cellular uptake. We developed a cost-effective, microwave-assisted protocol to synthesize 10 nm monodispersed CDs, that achieved >50% protection of dsRNA against RNase III degradation at a 200:1 (w/w) ratio. These CDs effectively improved cellular uptake, demonstrated by the successful delivery of CD-complexed fluorolabeled-dsRNAs (Cy3-RNA) into intact plant tissue-cultured cells. To prove the applicability of this formulation in planta, we sprayed Cy3-RNA with CDs on the abaxial surface of greenhouse grapevine leaves via low-pressure spray. Fluorescence microscopy revealed penetration of Cy3-RNA into stomata and neighboring cells. Additionally, the efficacy of CD-complexed dsRNA in triggering RNAi was validated using a 21-mer dsRNA targeting eGFP in eGFP-expressing microvines. Notably, no cytotoxic effects were observed following CD application. Ongoing research quantitatively compares eGFP expression of microvine leaves sprayed with CD-complexed and naked dsRNA through qPCR, to demonstrate the improvement in nucleic acid delivery associated with CDs. This innovative approach aims to overcome critical barriers for dsRNA delivery, particularly those associated with cell wall-related constraints in plants. Through optimization of RNAi-based defense strategies in grapevine, this research contributes to advancement of sustainable viticulture practices.

Funding Support: Erath Family Foundation, Oregon Wine Board

Bronwyn Riddoch | Myroslava Khomik | Richard Petrone

Testing the Application of a Novel Technology for Assessing Grape Maturity using Spectrometry

Bronwyn Riddoch, Myroslava Khomik, and Richard Petrone*
*University of Waterloo, 200 University Ave W, N2L3G1, Canada
(rpetrone@uwaterloo.ca)

The Niagara region wine industry in Ontario, Canada, is already experiencing the effects of climate change, and more extreme weather events are expected. It is predicted that the changing climate will result in significant consequences for quality wine production. Consistent monitoring of grapes will help maximize their enological potential. However, most monitoring techniques are either destructive to the grapes or time-consuming (e.g., UAV flights). Past research on soft fruits and Chardonnay grapes suggests that the use of the visible light spectra can predict fruit/berry ripeness in a time-efficient and non-destructive manner.

PRecent advancements in compact and inexpensive technology for monitoring individual woody plant health and growth conditions, called TreeTalker, have been adapted to vines (TreeTalker Wine; TTW). This adapted technology allows continuous, real-time monitoring of grape status using spectrometry. Here, we present results from experiments that tested TTW’s ability to monitor grape veraison. We developed empirical relationships between spectral signatures measured by TTWs and berry total soluble solids (TSS) and investigated the possibility of using TTWs to predict berry TSS in situ and continuously on the vine until harvest.

Funding Support: Sources are University of Waterloo, Nature 4.0, and Ontario Grape and Wine Research Inc.

Edgar Godoy-Monterroso | Shunping Ding | Marco Fernandez | Quinn Cahoon | Shijian Zhuang | Qun Sun

Optimizing Biofungicide Use for Control of Grapevine Diseases on the Central Coast of California

Edgar Godoy-Monterroso,* Shunping Ding, Marco Fernandez, Quinn Cahoon, Shijian Zhuang, and Qun Sun
*Department of Viticulture and Enology, California Polytechnic State University-San Luis Obispo, Cal Poly 1 Grand Ave, San Luis Obispo, CA, 93407 (egodoymo@calpoly.edu)

Biofungicide efficacy in grapevine disease management is enhanced by selecting the right active ingredients and modes of action, optimizing application timing and understanding the pathogen’s biology, then integrating them properly into fungicide programs. The efficacy of biopesticides in vineyard production is influenced by local climate and environmental conditions. The objective of this study was to investigate the efficacy of biofungicides applied at different intervals in controlling powdery mildew and Botrytis bunch rot, and their effect on grape quality. The study was conducted in the San Luis Obispo Coast AVA on mature Chardonnay vines. Twelve fungicide programs were designed, with three biofungicides containing different active ingredients and modes of action, application intervals, and integration strategies. A grower-standard program and an untreated control were also included in the experiment. Fungicide applications started at bloom in May 2023 and finished at veraison in August 2023. Fungicide program efficacy was determined based on disease incidence and severity. Yield was measured by weighing all clusters from each plot. Berry chemistry (total soluble solids, pH, and titratable acidity) was assessed following standard laboratory protocols. Biofungicide active ingredient and mode of action significantly affected program efficacy, while the interval of application did not. All programs integrating biofungicides with synthetics had efficacy comparable to the grower standard which, with the exception of one biofungicide stand-alone program, resulted in significantly higher powdery mildew and Botrytis bunch rot disease control than biofungicide stand-alone programs. Yields were also significantly greater in these programs. This suggested that when designing a program with biofungicides in this AVA, integration with synthetics can significantly improve the efficacy of the programs. Berry chemistry was affected significantly by the fungicide program, but results did not necessarily correlate with their efficacy.

Funding Support: CDFA-Specialty Crop Black Grant Program and CSU ARI

Sam Dudley | Isaac K. Uyehara | Andrew McElrone | Megan K. Bartlett

Characterizing Root System Architecture Traits for Drought Tolerance in Grapevine Rootstocks

Sam Dudley,* Isaac K. Uyehara, Andrew McElrone, and Megan K. Bartlett
*University of California, Davis, 750 Anderson Rd., Davis, CA, 95616 (sdudley@ucdavis.edu)

To reduce the effects of climate change on viticulture, breeding more drought-tolerant rootstocks is a promising, but difficult, strategy due to limited information on traits and genes of interest. Our goal is to use an F1 population produced from a drought-tolerant and a drought-sensitive rootstock (110 Richter × 101-14 MGT) to identify molecular markers for root system architecture (RSA) traits in grapevine. Neutron radiography is an imaging technique that can non-destructively visualize entire small root systems. We subjected plants to two-week dry-down and rewatering treatments and used neutron radiography and new semi-automated root classification software to measure RSA. Here, we present preliminary results for a subset of 45 genotypes. We will complete measurements across the entire population and use genotyping by sequencing (GBS) to link RSA traits to molecular markers by next summer.

The 45 genotypes varied significantly (p < 0.001) in nearly all RSA traits, including total root length, mean root angle, aspect (total horizontal/vertical extent), root growth rate, and change in depth during drought. Roots were significantly steeper rooted and thicker in 110 Richter than 101-14 MGT, suggesting these are especially important traits for drought tolerance. Across genotypes, a more vertical aspect was correlated with a steeper root angle (r² = 0.47) and thicker mean root diameter (r² = 0.09), but independent of root system size and fibrosity (root system length/# of tips) (p > 0.05). Altogether, these findings suggest that this population is well-suited to identify genetic associations with RSA, and that selecting for a more vertical aspect, steeper root angles, and thicker roots could enhance drought tolerance in grape rootstocks, though more work is needed to evaluate how these phenotypes perform under field conditions. Genetic associations are pending current experimentation results.

Funding Support: California Department of Food and Agriculture

Luis Ortiz, Luca Pallotti | Eve Laroche-Pinel | William Whalen | Luca Brillante

Enhancing Vine Resilience and Delaying Ripening: Novel Late-Season Practices for Grape Production in Central California

Luis Ortiz, Luca Pallotti, Eve Laroche-Pinel, William Whalen, and Luca Brillante*
*Department of Viticulture and Enology, California State University Fresno, 
2360 E Barstow Ave, Fresno, CA, 93740 (lucabrillante@csufresno.edu)

Global warming is significantly compromising grape production in regions like central California. Late-season practices aimed at reducing the efficiency of the photosynthetic apparatus might be the solution to improve vine resilience and delay ripening. In this trial, an untreated control © was compared with applications of pinolene (P) and diatomaceous earth (D). A fourth treatment involved shoot twisting (T), seeking to achieve the benefits of traditional topping by breaking xylem tissue without overexposing clusters. Leaf area, light interception, midday stem water potential, and gas exchange were measured over the 2023 season. Berry ripening was followed and grape production at harvest was evaluated. Twisting determined the desiccation of the upper portion of the shoot, reducing leaf surface while maintaining a low level of irradiance in the fruiting zone. Stem water potential results showed that D, and especially T, partly alleviated stress conditions, while P led to a worsened vine water status. D had the most pronounced effect on gas exchange, especially transpiration and stomatal conductance, thereby leading to higher water use efficiency trend in the treated vines. D and T fruit exhibited greater berry weights during the first half of the season, likely due to the improved water status, while P berries had reduced weight. All applied treatments significantly affected berry ripening, resulting in increased titratable acidity and lower must pH, without compromising crop yield at harvest.

This trial highlights that innovating traditional, easily mechanizable practices such as topping, and introducing new products like diatomaceous earth can be effective techniques to mitigate summer heat stress and delay berry ripening, particularly in hot, dry regions such as CA’s Central Valley.

Funding Support: N/A