Antifungal Activity of Grapevine-derived Extracts against Botrytis cinerea

Progress report for GNE21-251

Project Type: Graduate Student
Funds awarded in 2021: $14,490.00
Projected End Date: 09/30/2023
Grant Recipient: University of New Hampshire
Region: Northeast
State: New Hampshire
Graduate Student:
Faculty Advisor:
Dr. Subhash Minocha
University of New Hampshire
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Project Information

Project Objectives:

The overall goal of this research is to investigate the putative antifungal activity of field-collected grapevine leaves and cell suspension cultures obtained from cold-hardy grapevine varieties and demonstrate the potential of grapevine-derived products as an alternative to synthetic fungicides. The specific objectives are to:

  1. Investigate the in vitro antifungal activity of field-collected grapevine leaf extracts against Botrytis cinerea.
  2. Investigate the in vitro antifungal activity of cell suspension cultures against Botrytis cinerea.
  3. Test the effectiveness of field-collected grapevine leaf extracts in controlling Botrytis cinerea infection in grapevine leaves and berries.
  4. Test the effectiveness of cell suspension cultures in controlling Botrytis cinerea infection in grapevine leaves and berries.
Introduction:

This project aims at developing a new alternative for disease management to vineyards in New England. Botrytis bunch rot, caused by the fungus Botrytis cinerea, is one of the most destructive grapevine diseases worldwide (33; 55; 58). Botrytis is a significant problem in grapevines in the Northeastern U.S. because of the warm, humid, and summer rains during the growing season that favor disease development (22).

The genus Botrytis consists of ~35 necrotrophic species, some having a vast range (B. cinerea and B. pseudocinerea), impacting ~1400 different plant species (28). Economically important crops are affected by this fungus and are the main contributor to food waste and loss and threats to global food security. This project focuses on B. cinerea infection on grapevines. Botrytis can grow on any succulent plant tissue (young and green), ripened fruit, or dead tissues (15). Botrytis can cause reduced fruit and wine quality, which is a significant concern for wineries (60), and affect the table grape sales in grocery stores. These scenarios can also be experienced in the New England wineries, which can decrease revenue to the growers (47).

Currently, viticulturists rely on sanitation, canopy management, and fungicides to manage Botrytis. However, there is increasing consumer pressure to reduce the use of synthetic chemicals due to concerns over potential adverse effects on non-pest species, on soil microbiome, and on human health (5), as well as the risk of pathogen resistance (18). Several techniques such as leaf removal, maintaining canopy architecture, and microclimate have been developed to adopt more environmentally and economically sustainable practices to manage gray mold (23; 32). Nonetheless, Botrytis continues to be a main challenge to grape growers. One possible alternative practice that could be used is the rotation of plant secondary metabolites with synthetic fungicides to suppress the disease (36). I propose a strategy to use extracts of grapevine debris (leaves) or cell suspension cultures to control B. cinerea infection. Cell suspensions (liquid cultures of undifferentiated cells) of grapevines also have antifungal compounds and have the advantage of direct extraction without sacrificing the whole plant (12; 44) and can be mass-produced in bioreactors (13). Grapevine canes have been successfully analyzed and quantified as a source of bioactive stilbenes (9; 23; 53; 16). The grapevine debris was also used for its phenolics in the pharma and nutraceutical industries (19). Therefore, it is expected that grapevine debris and cell suspension cultures can be used as a source of antifungal compounds to be used in pest management, thus reducing synthetic fungicide usage. This research directly addresses SARE’s mission to provide growers with innovations that improve environmental stewardship, resilience, and profitability. This project addresses four of the identified themes in sustainable agriculture by decreasing reliance on fungicides (reduce ecological and health risks, improve worker safety), improve the long-term health of agricultural systems (protection of natural resources), and reduce losses to disease (increase productivity).  

Research

Materials and methods:

Objective 1: Investigate the in vitro antifungal activity of field-collected grown grapevine leaf extracts against Botrytis cinerea.

The antifungal effect of grapevine leaves depends on their secondary metabolite contents (26). Several studies investigated the antifungal effect of grapevine leaves against different types of fungal pathogens (2; 25). In this objective, the antifungal activity of leaf extracts of field-grown cold-hardy table grapevines will be tested in vitro on B. cinerea. Additionally, leaf phenolic composition will be determined to identify metabolites responsible for the antifungal activity.

Field-collected leaves – The senescent leaves will be collected from the vineyard, and their extracts will be used to test their antifungal properties. The leaves will be collected, brought to the lab, and about 5 g of leaves will be boiled in 250 mL of water. The resulting extract will be filtered and then lyophilized and used to test the antifungal activity in vitro (16; 46). Additional leaves will be collected in replicates and flash-frozen in liquid nitrogen immediately upon harvest and stored at -80 o C until further analysis using high-performance liquid chromatography-diode array detector (HPLC-DAD). This HPLC-DAD analysis will be used to determine the leaf phenolic compounds.

B. cinerea inoculum: To prepare inoculum, fresh B. cinerea will be isolated and cultured on potato dextrose agar (PDA) plates and incubated for 5 days at room temperature in the dark. The plates will then be exposed to 14 h darkness/12 h light at 21o C for 6 days to induce sporulation. Conidia will be harvested from the cultures by rubbing the plates with a glass rod with 10 mL of sterile deionized water. Then the resulting suspension will be filtered through sterile cheesecloth (10). Spore concentrations will be adjusted to the 5x103 concentration using a hemocytometer.

Experimental design: Petri dishes having two compartments (I plates) with PDA-containing leaf extracts will be used for this assay.  One-half of the I plate will be inoculated by spraying a spore suspension of  5x103 conidia per mL to runoff (10; 14; 37). The other half (non-inoculated) will be sprayed with sterile water, which serves as a negative control. The Petri plates will be placed in the tissue culture room (25±2°C under 16-hours photoperiod and relative humidity at 95-99%). Two, three, five, and seven days post-inoculation, the plates will be evaluated for disease incidence. Disease incidence = (the number of spores/the total area of the plate) x 100%. The test will be conducted with 2 mL (50mg/L) of leaf extract in each plate (10 plates with equal amounts of control and treatment) and repeated for two table grape varieties. The design of the experiment will follow a complete randomized design block. The disease lesions will be analyzed for statistical significance using a One-Way ANOVA in GraphPad (Prism software, CA). The HPLC data for both grape varieties will be recorded and analyzed. The model statement will include treatment as the independent variable and disease incidence as the dependent variable. Statistical significance will be assessed at p < 0.05, and a Tukey Honest Significant Difference (HSD) Post-hoc test will be used to separate the means. 

Objective 2: Investigate the in vitro antifungal activity of cell suspension cultures against Botrytis cinerea.

Cell suspension cultures: Initially, solid cultures of undifferentiated cells (calli) will be established from the axillary buds of Canadice and Mars grapevine plants. The cultures will be initiated and maintained on a solid Murashige and Skoog media and grown at 25+ 1 o C, with 16 h light/8 h dark photoperiod illuminated with fluorescent light bulbs. The calli will be routinely sub-cultured in 4-week intervals to maintain the cell lines (42; 39). The calli cultures will be used to establish liquid cultures of undifferentiated cells (cell suspension cultures) using liquid Murashige and Skoog media.

Experimental design: The cell suspension cultures will be dissolved in dimethyl sulphoxide (DMSO) (39) and then mixed with the PDA medium and used for testing antifungal activity similar to the leaf extracts assay described above. One-half of the I plate will be inoculated by spraying a spore suspension of  5x103 conidia per mL to runoff (10; 14). The non-inoculated part will be sprayed with sterile water, which serves as a negative control. The Petri plates will be placed in the tissue culture room (25±2°C under 16-hours photoperiod and relative humidity at 95-99%). Two, three, five, and seven days post-inoculation, the plates will be evaluated for disease incidence. Disease incidence = (the number of spores/the total area of the plate) x 100%. The test will be conducted with 5 mL of suspension culture in each plate (10 plates with equal amounts of control and treatment) will be repeated for two table grape varieties. The design of the experiment will follow a complete randomized design block. The data will be analyzed for statistical significance using a One-Way ANOVA in GraphPad (Prism software, CA). The model statement will include treatment as the independent variable and disease incidence as the dependent variable. Statistical significance will be assessed at p < 0.05, and a Tukey Honest Significant Difference (HSD) Post-hoc test will be used to separate the means. 

Objective 3: Test the effectiveness of field-collected grapevine leaf extracts in controlling Botrytis cinerea infection in grapevine leaves and berries.

Experimental design for leaf infection: Leaf extract prepared according to Objective 1 will be sprayed on the greenhouse-grown grapevine plants in different concentrations (25, and 50 mg/L) until runoff. Sterile distilled water will be used as control. The design of the experiment will follow the randomized complete block pattern with 8 plants per treatment (24 plants total). Then plants will be challenged by spraying a 5x103 B. cinerea conidia per mL to runoff using the inoculum described in objective 1 (10; 14; 37). After inoculation, the plants will be placed in a humid chamber (18°C and 100% relative humidity for 48 hours and then returned to the greenhouse. Two, three, five, and seven days post-inoculation, the plants will be evaluated for disease incidence and disease severity. Disease incidence = (the number of lesions/the total number of leaves) x 100%. At 14 days post-inoculation, overall plant disease severity will be measured using a five-point scale rating (0=no disease, 1=20%, 3=60%, 4=80%, and 5=100% total plant collapse). Disease severity and disease incidence will be analyzed for statistical significance using a One-Way Analysis of Variance (ANOVA) in GraphPad (Prism software, CA). The model statement will include crude extract concentration as the independent variable, block as the random variable, and disease severity, and disease incidence as dependent variables. Statistical significance will be assessed at p < 0.05, and a Tukey Honest Significant Difference (HSD) Post-hoc test will be used to separate the means. 

Experimental design for berry infection: The experiment will be conducted with cold-hardy table grape cv “Mars” and cv “Canadice” treated by field-collected grapevine leaf extracts. Table grape berries will be sprayed with 10 mL of leaf extract solutions (50 mg/L). After air-dried at room temperature for 30 minutes, each berry will be inoculated with 5x103 conidia per mL to runoff of B. cinerea. This experiment will be repeated twice with four replicates, consisting of 50 berries per treatment (61). All the berries will be placed in plastic containers and kept at 22 o C in the dark for 4 to 7 days. Disease incidence will be calculated by counting the percentage of infected berries, and rot lesion diameter will be measured. Using One-Way Analysis of Variance (ANOVA) in GraphPad (Prism software, CA) and the statistical significance will be assessed at p < 0.05, and a Tukey Honest Significant Difference (HSD) Post-hoc test will be used to separate the means. 

Objective 4: Test the effectiveness of cell suspension cultures in controlling Botrytis cinerea infection in grapevine leaves and berries.

Experimental design for leaf infection: The cell suspension extracts prepared according to Objective 2 will be sprayed on the greenhouse-grown grapevine plants in different concentrations (5, and 10 mg/mL) until runoff and control (distilled water) be maintained. The design of the experiment will follow the randomized complete block design with 8 plants per treatment (24 plants total). Then plants will be challenged by spraying a 5x103 conidia per mL to runoff using the method described in objective 1 (10; 14; 37). After inoculation, the plants will be placed in a humid chamber (18°C and 100% relative humidity for 48 hours and then returned to the greenhouse. Two, three, five-, and seven days post-inoculation, the plants will be evaluated for disease incidence and disease severity. Disease incidence = (the number of lesions/the total number of leaves) x 100%. At 14 days post inoculum, overall plant disease severity will be measured using a five-point scale rating (0=no disease, 1=20%, 3=60%, 4=80%, and 5=100% total plant collapse). Disease severity and disease incidence will be analyzed for statistical significance using a One-Way Analysis of Variance (ANOVA) in GraphPad (Prism software, CA). The model statement will include cell suspension culture extracts concentration as the independent variable, block as the random variable, and disease severity and disease incidence as dependent variables. Statistical significance will be assessed at p < 0.05, and a Tukey Honest Significant Difference (HSD) Post-hoc test will be used to separate the means. 

Experimental design for berry infection: The experiment will be conducted with cold-hardy table grape cv “Mars” and cv “Canadice” treated by cell suspension culture extracts. Table grape berries will be sprayed with 10 mg/mL of cell suspension culture extracts. After air-dried at room temperature for 30 minutes, each berry will be inoculated with 5x103 conidia per mL to runoff of B. cinerea. This experiment will be repeated twice with four replicates, consisting of 50 berries per treatment (61). All the berries will be placed in plastic containers and kept at 22 o C in the dark for 4 to 7 days. Disease incidence will be calculated by counting the percentage of infected berries, and rot lesion diameter will be measured. Using One-Way Analysis of Variance (ANOVA) in GraphPad (Prism software, CA) and the statistical significance will be assessed at p < 0.05, and a Tukey Honest Significant Difference (HSD) Post-hoc test will be used to separate the means. 

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

Throughout this proposal, I will synthesize results and present them to stakeholders. Dr. Becky Sideman from UNH Extension will help me maintaining contact with grape growers around NH (see attached letter of support). In the summer of 2022, I will present results from the controlled greenhouse trials at one of the twilight meetings organized by the NH Fruits Growers Association and UNH Extension. Furthermore, I will present during one of the Under the Vine meetings (annual grape and kiwi berry twilight meeting held at the NH Agricultural Experiment Station’s Woodman Farm), showcasing our research and answering questions about the greenhouse trials to the public. During this meeting, I will survey grape growers with the following questions: 1) How big of a problem is botrytis on your vineyard? 2) What did you learn today? 3) Do you currently control disease using approaches other than using fungicides? 4) What is the likelihood of you implementing new tools as a part of your IPM strategy?

The grape growers are likely to attend the yearly NH Winery Association-sponsored meetings in January and February each year. I will present the results of the experiments conducted in the Jan-Feb 2023 and 2024 meetings. At the NH Fruit Growers annual meeting in March 2023 and the 2023 twilight meetings, I will present our data from our first season of greenhouse trials. At these meetings, growers will be surveyed with the following questions: 1) What did you learn today? 2) In the future, would you consider using a grapevine-derived product to help manage disease in your vineyards? 3) Do you feel that this research is valuable to you as a New Hampshire grapevine grower? After these meetings, I will be able to send out follow-up surveys and contact farmers who showed interest in implementing these practices. These events draw in grape growers from around NH and ensure that our research is communicated to most of NH’s viticulturists.

 

Post the end of this project, I will also present the results of this research to plant science researchers at the 2023 American Pathological Society symposium. This will allow us to reach other state audiences. I will write a report on this research and share it with the NH vegetable and fruit news. I anticipate publishing data from this research in a peer-reviewed journal, such as the Journal of Plant Physiology and Pathology or the Journal of Plant Diseases and Biomarkers. Publishing our data will increase our outreach nationally and internationally. Thus, through extension meetings, professional conferences, and publications, I hope to enhance the grape grower’s adoption of new tools, reduce losses, decrease reliance on fungicides, increase revenue, and create a more sustainable agricultural system. 

Project Outcomes

Assessment of Project Approach and Areas of Further Study:

The grapevines of two cold-hardy varieties proposed in my research were propagated and started growing in the greenhouse. These grapevines will be utilized for callus production using their meristematic tissues.

 

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.