Progress report for GNE24-336
Project Information
The pathogen Plasmopara viticola isresponsible for downy mildew disease in grapevines. This disease significantly reduces yield and can kill vines if unmanaged in the Northeastern United States. This project explores the potential of using biocontrols, like LifeGard, to manage downy mildew. Data from this research can contribute toward growers reducing their reliance on synthetic fungicides by integrating biopesticides into disease management strategies. Through a field experiment, the effects of LifeGard alone and in combination with fungicides on downy mildew incidence, grapevine physiology, and fruit quality were assessed. Preliminary results show that LifeGard, when rotated with fungicides, can effectively control disease and produces outcomes comparable to the use of only conventional fungicides to suppress disease. The first part of the project has been completed while the second part will be completed in the next phase.
Objective 1: To evaluate the efficacy of the biocontrol agent LifeGard, used in an integrated approach with conventional and reduced fungicide applications, for managing downy mildew in vineyards and the resulting physiological response in the plants.
Specific objective:
a. To assess the disease pressure of downy mildew, I will evaluate their incidence and severity on grapevines.
b. To determine the physiological response, I will measure the chlorophyll content in leaves and Brix and pH content in berries.
Objective 2: To investigate the impact of applying biocontrol agent LifeGard on the metabolic profile of susceptible grapevine cultivars
grown in field conditions and explore the relation of these metabolic alterations to improved resistance against downy mildew infections.
a. To determine the presence of resistance-related metabolites, including the potential induction or modulation of defense-related compounds such as phytoalexins, pathogenesis-related (PR) proteins, and other antimicrobial secondary metabolites. Additionally, I will evaluate the effects on the biosynthesis of phenolic compounds, encompassing flavonoids and stilbenes, which play crucial roles in plant defense mechanisms.
b. To determine the metabolites related to improving plant health by evaluating the influence of this biocontrol agent on the production of metabolites associated with enhanced plant vigor and stress tolerance, such as antioxidants, osmolytes, and growth-regulating hormones.
The overall purpose of this project is to find sustainable solutions for grapevine growers in the Northeastern United States by testing the efficacy of a biopesticide that is commercially available to growers. The ultimate goal is to reduce the amount of disease and fungicides used during the growing season. I will determine the efficacy of the biocontrol LifeGard in suppressing the disease incidence and severity of downy mildew Plasmopara viticola on grapevine and determine the plant's physiological and metabolomic response. To work towards the goal of reducing the use of conventional fungicides, LifeGard will be studied when applied in rotation with conventional fungicides as well as in solo sprays.
Downy mildew is one the most important diseases affecting grapes in the Northeastern region of the United States1. The environmental conditions in this area are favorable for the development of the mildews, and each year, they significantly impact wine grape vinyards2–4. Vineyards can see up to 75-100% leaf loss in susceptible vines, leading to substantial yield reductions or complete crop loss 4,5. Growers have identified mildews as the most significant disease threat to their vines3. The economically important susceptible varietals in the Northeast are Chardonnay, Chancellor, and some Vitis vinifera hybrids6.
Growers need effective strategies to manage these highly damaging pathogens, and the cool, humid, and variable weather patterns of the Northeastern US make their management challenging1,7. This has resulted in a reliance on frequent applications of synthetic fungicides 6,7. The overuse of fungicides has raised concerns about their environmental impact, potential development of fungicide resistance in the pathogens, and potential human health risks8. In recent decades, there has been a shift towards exploring alternative strategies to conventional fungicides due to factors such as the emergence of resistance, changing consumer preferences, and government regulations7,9. These limitations associated with fungicide usage have prompted a growing emphasis on developing and implementing more environmentally sustainable approaches to disease control. Leveraging biocontrol agents that harness natural interactions within the plant-microbe ecosystem has emerged as a promising strategy for effective disease management while concurrently promoting sustainable agricultural practices 10,11.
The application of biocontrol agents has demonstrated high potential to manage plant pathogens across a diverse array of crops and production systems12. Bargabus et al. (2002) indicated that the beneficial bacterium Bacillus mycoides was capable of reducing the severity of the fungal pathogen Cercospora beticola in sugar beet through induced systemic resistance (ISR) 13. The mode of action employed by different biocontrol agents to suppress or regulate the target pathogen can vary substantially, contingent upon the specific source organism from which the biocontrol agent is derived. This diversity in mechanisms, ranging from direct antagonism to the elicitation of systemic resistance responses, contributes to the versatility and adaptability of biocontrol strategies in addressing plant disease challenges across diverse agricultural contexts14.
In pursuit of sustainable agricultural practices, the grape industry recognizes the important role of microorganisms in agroecosystems. Biocontrol agents (BCAs) like Bacillus species have gained interest as a sustainable crop protection approach, leveraging their ability to suppress pests and pathogens through various mechanisms15,16. One of the primary mechanisms is antibiosis, producing a diverse array of antimicrobial compounds, including lipopeptides, polyketides, and bacteriocins, that inhibit the growth and development of fungal and bacterial plant pathogens17,18. Bacillus spp. can also produce chitinases to weaken the pathogen's physical defenses19 and stimulate the plant's defense response through a process known as ISR13,14.
This study aims to evaluate the early application of biopesticides in combination with reduced fungicide treatments as a potential strategy for enhancing downy mildew protection in Northeastern vineyards. The wine industry seeks sustainable and cost-effective strategies to enhance grape yields and profitability, but completely abandoning currently used fungicides is impractical. It is crucial to determine the characteristics of the interaction between biocontrol agents and conventional fungicides to gain a better understanding of the mechanisms underlying biocontrol efficacy and the potential synergistic events occurring within the plant. Such insights may facilitate the optimization of biocontrol applications and the development of improved bioproducts, contributing to a reduced reliance on chemical pesticides in agriculture. The rationale behind integrating biopesticides with fungicide trials, the potential benefits of biopesticides, and the efficacy of this integrated approach in mitigating grape losses and enhancing profitability will be elucidated. Concurrently, the study will examine the efficacy of biocontrols and their combination with fungicides, as well as the metabolic responses of grapevines to this integrated pest management strategy, providing insights into the underlying mechanisms and potential synergistic effects.
Research
Materials and methods:
To conduct the first part of the experiment, objectives 1a, 1b and sampling for objective 2 was performed in the field during the summer of 2024. A field experiment was performed to determine the efficacy of the biocontrol LifeGard alone and in combination with a conventional fungicide schedule. The experiment took place at our collaborator’s field sites at the Cornell University pathology vineyards in Geneva, NY. The experiment involved a comparative assessment of four treatments in a randomized complete block design with four replicates per treatment. Each treatment was assigned to a separate row within the plot at the research station. While the overall length of each treatment was determined by the existing row dimensions, all treatments were allocated equal lengths within their assigned rows.
The four treatments are as follows:
- Conventional fungicide application schedule (conventional)
- Rotation of conventional fungicide application schedule AND LifeGard (LifeGard + Fungicides)
- Only Biocontrols: LifeGard and Nat Bio (Bio’s)
- Control
Objective 1a: Assessing downy mildew disease pressure
To assess the disease pressure of downy mildew, the incidence and severity of the disease was evaluated on two plants from the center of each treatment were selected. Chardonnay cultivar was used in this experiment. Each plant was scouted for symptoms and signs of downy mildew. Downy mildew was visually assessed on the lower and upper leaf surface. A total of 30 leaves were selected to assess disease pressure from the two center vines in each treatment. Ratings were made regularly around every 7-10 days in between. An incidence scale of 0 to 100% was used for the disease, representing the number of infected leaves from each plant over time. The mean disease incidence and severity for each treatment, as well as for the entire grapevine, were calculated by averaging the total samples. Initial histograms, boxplots, and ANOVA have been created for these data.
Objective 1b: Leaf and berries measurements
To determine the physiological response of grapevines, chlorophyll content (using the Soil Plant Analysis Development ( SPAD) method) and nitrogen levels in leaves, as well as Brix and pH levels in berries, were measured. A portable chlorophyll meter (GOYOJO, GYJ-C Chlorophyll Meter) was used to assess the relative chlorophyll content of vine leaves. Measurements were taken at the same time as disease assessments for each treatment. A total of 24 leaves were randomly selected per treatment from the two center plants. Data collection occurred at intervals of 7 to 10 days. Measuring leaf chlorophyll and nitrogen content served as an indicator of the plant's photosynthetic capacity and overall vigor.
Representative berry samples were collected from the two center plants in each treatment at harvest. Twenty berries were used to record berry weight. Additional berry samples were collected and analyzed in the lab to measure Brix (soluble solids content) using a handheld digital refractometer, which assessed sugar accumulation. Berry pH was also measured using a calibrated pH meter, as pH is an important indicator of fruit maturity and acidity. Titratable acidity (TA) was also determined from these samples.
By comparing leaf chlorophyll content and berry quality parameters between the integrated biocontrol treatment and the standard fungicide control, the physiological impacts of this alternative management approach on vine performance and fruit composition were evaluated.
Additionally, overall disease pressure ratings were performed on clusters to assess general disease levels.
Objectives 2: Metabolite Sampling
Two vines from the center of the treatment were used to collect samples. For metabolomic analysis, samples were collected at four time points: 24 hours before, 24 hours after, and three, and six days after biocontrol (LifeGard) spraying. Three leaves per each side of the row were selected randomly, making a total of six leaves per treatment. Five punches per leaf were taken. In total 30 leaf punches were taken from the two center plants per treatment, from leaves in the 4th to 8th internode from top to bottom in the branches, placed in 2 mL centrifuge tubes, and flash-frozen in liquid nitrogen. This provided approximately over 100 mg of fresh sample. After collection, samples were stored at -80°C for subsequent metabolic analysis.
Preliminary Results
Preliminary results from field disease rating data show that when the start level of disease is equal for the treatments, spraying the biocontrolLifeGard on the grapevines in rotation with synthetic fungicides can reduce the incidence of disease below 20%. This effect is similar to the use of only synthetic fungicides. These results were observed at the end of the season.
For harvest data, when collecting cluster weight, the "conventional" treatment has a significantly different mean weight compared to the "LifeGard + Fungicides" treatment. No significant difference between "LifeGard + Fungicides" and "Bio’s". In general, "Conventional" showed higher weight on average.
For cluster disease rating, it has shown similar results for Conventional management and LifeGard + Fungicides, which is less than 20% in average, this being around 22% for Bio’s.
Fruit chemistry results indicated that grape berry pH was: 3.6 for LifeGard + Fungicides, 3.57 for Conventional, and 3.65 for Bio’s in average. Control treatment showed a pH of 3.86. In relation to titratable acidity (TA) and Brix, for the different treatments: LifeGard + Fungicides showed 9.5 and 21.1 respectively, Conventional showed 9.9 and 20.5 respectively, and Bio’s showed 10.2 and 20 respectively in average. The Control showed 11.6 and 19.8 for each measurement.
Moving forward regarding objectives 1 and 2 will be followed as follows
For objective 1, final statistical analysis will be performed to determine differences in each treatment and measurement and the effect of the treatments on these characteristics for the plant.
For objective 2, Metabolite Extraction: The protocol provided by the metabolomics core at Penn State will be followed for metabolite extraction as planned. Briefly, plant tissue samples (80–100 mg fresh weight) will be homogenized in 2 mL tubes containing zirconia/silica beads from flash-frozen samples using a tissue lyser and mortar/pestle. An extraction solution (methanol:water:formic acid 80:20:0.05 v/v/v with 1 μM chlorpropamide) will be added, and samples will undergo further homogenization, sonication, and centrifugation to separate the organic phase. After two rounds of extraction, the pooled organic extract will be evaporated to dryness and reconstituted in methanol:water (40:60 v/v). Samples will then be vortexed, sonicated, centrifuged, and transferred to auto-sampler vials for storage at -80°C.
Metabolite LC-MS Analyses
The extracted samples will be sent to the metabolomics core facility at Penn State, where LC-MS analysis will be performed. Specifically, ultra-performance liquid chromatography mass spectrometry (UPLC-MS) will be used for targeted metabolomic profiling.
Data Analysis
The raw data will be processed using MZmine 3 for peak picking, retention time alignment, and isotope/adduct clustering into features, which will then be identified through database searching. Relative quantification of metabolites will be performed based on peak areas, and multivariate statistical analyses will be conducted in MetaboAnalyst to reveal significant differences between experimental groups. Based on the data obtained, further statistical analyses will be carried out using appropriate statistical packages.
Education & Outreach Activities and Participation Summary
Participation Summary:
This project focuses on the New York and Pennsylvania grape-growing community, a cornerstone of the agricultural landscape within the Northeast USA. These growers cultivate a variety of grape cultivars for wine, juice, and table consumption23. One of their primary needs, identified through industry reports, grower surveys, and extension workshops, is to reduce reliance on fungicides24. Concerns encompass the emergence of fungicide resistance, potential environmental consequences of overuse, and rising production costs8. Additionally, there's a growing market demand for sustainable practices in agriculture, driven by consumer preferences and environmental considerations.
While we will share our findings with the research community to contribute to the broader knowledge base on the efficacy of biocontrol agents with reduced fungicide applications and the underlying metabolic mechanisms involved, we will also make a significant effort to ensure that this information is readily accessible and applicable to grapevine growers. This is crucial because the adoption of sustainable practices by growers is essential for addressing the challenges faced by the industry and meeting the market demand for environmentally conscious products12.
To achieve this, I will communicate our results through channels specifically tailored to the grape-growing community. This will include publishing extension articles in publications such as Penn State Extension, which is widely read by field crop farmers in the Northeast. These articles will provide clear and practical guidance on incorporating biocontrol agents and reduced fungicide schedules into vineyard management practices. Additionally, I will present our findings at conferences and events attended by growers, extension educators, and industry representatives focused on sustainable viticulture practices, such as the Pennsylvania Sustainable Agriculture (PASA) conference, which has many growers from NY and New England attend. I’ll also present my results at the American Phytopathological Society (APS). These presentations will not only disseminate our research but also facilitate discussions and gather feedback from industry stakeholders. For the extension articles and presentations, I will translate my research into Spanish to reach a broader audience of growers.
By actively engaging with wine growers and extension professionals through these channels, I aim to facilitate the translation of our research findings into practical recommendations and strategies that can be easily implemented in vineyards. This approach can also be applied by collaborators at Cornell University in New York. The Gold lab in Geneva, New York, works with a large number of private growers, and through them, I will be able to reach a wide audience.
Furthermore, this research will facilitate collaboration with extension educators and industry representatives to integrate our findings into educational modules and resources on sustainable viticulture practices. Penn State Extension has a widely recognized webpage that communicates and disseminates new findings to the grapevine industry and the general public4. By integrating our research into these resources, we can provide growers with comprehensive and accessible information to guide their adoption of sustainable practices.
Overall, this project recognizes the importance of not only conducting rigorous scientific research but also effectively communicating and translating the findings to the target audience – the grape growers. By actively engaging with the industry and leveraging various communication channels, we can ensure that our research contributes to the advancement of sustainable viticulture practices, addressing the needs and concerns of growers while promoting environmentally responsible grape production.