Use of tartaric acid in the prevention and treatment of fungal diseases of grape

Abstract

This invention discloses the application of tartaric acid in the control of grape fungal diseases, belonging to the field of plant-derived antifungal agents. The invention involves uniformly spraying a tartaric acid solution onto the surface of grape berries and leaves, effectively preventing the occurrence of white rot, black rot, and gray mold. This result demonstrates that plant-derived tartaric acid possesses inhibitory activity against white rot fungi, black rot fungi, and gray mold, and can be used as an agricultural disease inhibitor or fungicide to control these diseases. It effectively reduces the use of chemical pesticides, exhibits good biosafety, and has significant application value and market prospects in grape disease control.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived antibacterial agents, and relates to the application of tartaric acid in the prevention and control of fungal diseases in grapes. Background Technology

[0002] Grape diseases are a significant factor affecting grape production, greatly impacting vine growth, yield, and quality. White rot and black rot are the main fungal diseases affecting grapes. White rot primarily damages the grape berries, but can also affect young shoots and leaves, causing berries to rot and fall off the grape bunches. Black rot mainly affects young tissues such as young leaves, shoots, and young berries, causing leaf deformities and drop, shoot death, and rendering the fruit unmarketable. Furthermore, gray mold not only infects flower bunches and young berries but also easily breaks out during fruit ripening and post-harvest storage and transportation. It spreads through wounds or direct infection, causing the fruit to rot under humid conditions by developing a gray mold layer. The pathogen has a latent infection characteristic, making control difficult. Especially in rainy regions and years, these diseases cause significant economic losses to the grape industry.

[0003] Grape diseases are primarily controlled through chemical pesticides. However, the long-term and repeated use of single chemical fungicides easily leads to drug resistance in pathogens, resulting in ineffective control. This not only increases grape cultivation and management costs and affects grape product quality but also poses potential harm to the environment, soil microorganisms, and human health. Therefore, there is an urgent need to develop more efficient and safer green control technologies. Plant-derived antifungal agents or fungicides have less environmental pollution, lower costs, are safer for humans and animals, and are less likely to induce drug resistance in pathogens. They can reduce the amount of chemical pesticides used and save energy, making them an important means of gradually replacing chemical control. This is of great significance for solving the problem of grape disease control, reducing the use of chemical agents, promoting food safety, and ensuring the sustainable development of the grape industry.

[0004] Tartaric acid is an important organic acid widely distributed in plants, including horticultural crops, especially in grapes. Due to its non-toxicity and lack of environmental pollution, it is often used as an acidulant in fruit juices, wines, candies, breads, etc., to enhance flavor. It can also act as an antioxidant and fermentation agent to regulate the acidity of food. However, there are currently no publicly reported studies on its use as a direct active ingredient for controlling grape fungal diseases. Summary of the Invention

[0005] The purpose of this invention is to provide an application of tartaric acid in the prevention and control of grape fungal diseases. Experiments have shown that tartaric acid has an inhibitory effect on grape white rot fungus, black rot fungus, and gray mold fungus, and can effectively prevent and control grape white rot, black rot, and gray mold. It can be used as a reference agent for green prevention and control of these three grape diseases.

[0006] The technical solution provided by this invention is as follows:

[0007] In a first aspect, the present invention provides the application of tartaric acid in the prevention and control of fungal diseases in grapes.

[0008] In a preferred embodiment of the present invention, the tartaric acid is used to prevent and control grape white rot, black rot, or gray mold. The grape white rot is caused by *Phytophthora infestans* (…). Coniella vitis The blackhead disease is caused by the fungus *Trichophyton mentagrophytes* (…). Elsinoe ampelina The gray mold is caused by the fungus *Botrytis cinerea* (…). Botrytis cinerea Caused by ).

[0009] In a preferred embodiment of the present invention, the tartaric acid is prepared into an aqueous solution and sprayed onto the target plant.

[0010] In a preferred embodiment of the present invention, the concentration of the tartaric acid aqueous solution is 2.5~5 mg / mL.

[0011] In a preferred embodiment of the present invention, the pH of the tartaric acid aqueous solution is 3-4. Different pH values ​​for tartaric acid aqueous solutions can be achieved by adjusting the concentration of tartaric acid itself, resulting in a final concentration of 2.5-5 mg / mL for the tartaric acid aqueous solution.

[0012] More preferably, the pH of the tartaric acid aqueous solution is 3.

[0013] In a preferred embodiment of the present invention, the spraying is to spray an aqueous solution of tartaric acid onto the surface of the grapes or leaves.

[0014] In a second aspect, the present invention provides a medicament for preventing and controlling grape fungal diseases, the active ingredient of which is tartaric acid as described above, and the medicament is used to inhibit grape white rot fungus, black rot fungus or gray mold.

[0015] In a preferred embodiment of the present invention, the agent includes pesticide-acceptable excipients.

[0016] More preferably, the agent is a powder, microemulsion, suspension, or aqueous solution.

[0017] In a preferred embodiment of the present invention, the reagent is an aqueous solution of tartaric acid.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) This invention provides an application of tartaric acid in the prevention and control of fungal diseases in grapes. Tartaric acid is widely found in horticultural crops and has the characteristics of low cost, safety and no pollution. It is a green and environmentally friendly disease control agent. Experiments have shown that tartaric acid has the highest inhibition rate of 96.4% against white rot fungi and the highest inhibition rate of 60.0% against black rot fungi.

[0020] (2) This invention can reduce the occurrence of grape white rot and black rot. The indoor control efficiency of tartaric acid against grape white rot is 25.3% to 55.6%, and the field control efficiency is 57.1% to 78.6%; the field control efficiency of tartaric acid against black rot is 97.7%; and the control efficiency of tartaric acid against postharvest grape gray mold is 41.3% to 76.4%. It is of great significance for reducing the use of chemical pesticides and ensuring the safe production of grapes. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention, and the illustrative embodiments of the invention and their descriptions are used to explain the invention.

[0022] Figure 1 This figure shows the effect of tartaric acid on the germination of conidia of grape white rot fungus. Data in the figure are mean ± standard error; different letters indicate significant differences (P < 0.05, One-way ANOVA analysis).

[0023] Figure 2 This section describes the indoor control effect of tartaric acid on grape white rot. A shows the control effect graph, and B and C show the disease index statistics. Data in the graphs are presented as mean ± standard error, with different letters indicating significant differences (P < 0.05, One-way ANOVA analysis).

[0024] Figure 3 This shows the field control effect of tartaric acid on grape white rot. A. Control effect diagram; B. Rot rate statistics. Data in the figures are mean ± standard error, and different letters indicate significant differences (P<0.05, One-way ANOVA analysis).

[0025] Figure 4 This study examines the effects of tartaric acid on *Phytophthora rotundifolia* and its control efficacy against the disease. A. Effect of tartaric acid on conidial germination of *Phytophthora rotundifolia*; B. Control efficacy diagram; C. Disease index statistics. Data in the figures are expressed as mean ± standard error. Different letters indicate significant differences (P < 0.05, One-way ANOVA analysis).

[0026] Figure 5 This diagram shows the control effect of tartaric acid on postharvest gray mold in grapes. A shows the control effect of tartaric acid on grape berries against gray mold; B shows the disease index statistics. Data in the diagrams are mean ± standard error, and different letters indicate significant differences (P < 0.05, One-way ANOVA analysis). Detailed Implementation

[0027] To better explain the present invention, the main contents of the invention are further illustrated below with reference to specific embodiments, but the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the technical solutions described in this invention are conventional techniques in the art. Unless otherwise specified, the materials and reagents used in this invention are commercially available. Furthermore, other terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.

[0028] The experimental materials and instruments used in the embodiments of this invention are as follows:

[0029] Pathogen: Grape white rot fungus Coniella vitis QNYT13637, kindly provided by Professor Zhou Shanyue of the College of Plant Medicine, Qingdao Agricultural University, is documented in: Zhou, S., & Li, B. (2020). Genome sequence resource of Coniella vitis , a fungal pathogen causing grape white rot disease. Molecular Plant-Microbe Interactions, 33(6), 787-789.;Grape black rot fungus is Elsinoe ampelina YL-1, isolated from diseased leaves of the 'Red Globe' grape variety, is described in the literature: Li, Z., Dang, H., Yuan, X., He, J., Hu, Z., & Wang, X. (2018). Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina , thecausal organism of grapevine anthracnose. Journal of Phytopathology, 166(6),420-428.

[0030] Grape gray mold RG-1, isolated from diseased 'Red Globe' grape berries, is described in the literature: Hou, X., Zhang, G., Han, R., Wan, R., Li, Z., & Wang, X. (2022). Ultrastructural observations of Botrytis cinereaand physical changes in resistant and sustainable grapevines. Phytopathology, 112(2), 387-395.

[0031] Grapes: Leaves of the Eurasian grape variety 'Seedless White', fruits of 'Beauty Finger', 'Golden Finger', and 'Red Globe' were all collected from the Grape Germplasm Resources and Breeding Experimental Base of Northwest A&F University in Yangling, Shaanxi Province.

[0032] Culture medium: Potato glucose solid medium (PDA), consisting of 200 g / L potato, 20 g / L glucose, 15 g / L agar powder, and the remainder water, at natural pH. Sterilize at 121℃ for 30 min. PDA medium without agar powder is called PDB liquid medium.

[0033] Experimental instruments: biochemical incubator, light incubator, optical microscope, sterilizer, and clean bench.

[0034] Example 1: Antimicrobial activity of tartaric acid against white rot fungi in grapes

[0035] Grape white rot fungus was inoculated onto PDA medium and cultured at 25°C for one week. After conidia were produced, they were added to centrifuge tubes containing 1 mL of PDB medium and to PDB medium prepared with tartaric acid at different pH values ​​(2, 3, 4, and 5). A control was prepared without tartaric acid. The spore concentration was adjusted to 1 × 10⁻⁶ using a hemocytometer under a microscope. 5 / mL. After incubation at 25℃ for 24 h, the germination rate of conidia of white-rot fungi was counted under a microscope.

[0036] The treatment and control were each replicated three times. The germination of 300 conidia was recorded in each replicate. The germination rate (%) was calculated as: (Number of germinated conidia / Total number of conidia) × 100%.

[0037] The inhibition rate is calculated based on the germination rate. Inhibition rate (%) = (control germination rate - treatment germination rate) / control germination rate × 100%.

[0038] Figure 1 The results showed that tartaric acid inhibited the germination of conidia of white rot fungi by 17.3%–96.4%, and tartaric acid (pH=2) had the strongest inhibitory effect on grape white rot fungi conidia.

[0039] Example 2: Indoor control of grape white rot using tartaric acid

[0040] Prepare a suspension of grape white rot fungus conidia (1×10⁻⁶) using sterile water. 5Prepare tartaric acid solutions of 5 mg / mL (pH=3) and 2.5 mg / mL (pH=4) with sterile water. Collect 'Beauty Finger' and 'Golden Finger' grapes, disinfect with 70% alcohol for 30 seconds, and wash three times with sterile water. Spray with tartaric acid solutions (pH=3 and 4) until the solution no longer drips from the fruit surface, using sterile water as a control. Then, puncture and inoculate with 10 µL of grape white rot fungus conidia suspension. Place the inoculated fruit in a plastic box lined with moist filter paper, seal with plastic wrap to maintain moisture, and then place in a 25℃ light incubator. Take photos and count the area of ​​lesions 3 days after inoculation.

[0041] ImageJ software was used to measure the area of ​​fruit lesions and the total fruit surface area, and the disease index was calculated.

[0042] Disease index (%) = [Area of ​​fruit lesions / Total fruit surface area] × 100%.

[0043] Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0044] Figure 2 The results showed that 3 days after inoculation, tartaric acid at concentrations of 5 mg / mL (pH=3) and 2.5 mg / mL (pH=4) had control efficiencies of 55.6% and 38.5% on 'Beauty Finger' grapes, respectively, and 46.7% and 25.3% on 'Golden Finger' grapes, respectively. Among them, 5 mg / mL tartaric acid (pH=3) showed the best control effect.

[0045] Example 3: Field control of grape white rot using tartaric acid

[0046] Prepare a suspension of grape white rot fungus conidia (1×10⁻⁶) using sterile water. 5 Prepare tartaric acid solutions of 5 mg / mL (pH=3) and 2.5 mg / mL (pH=4) with sterile water. Using mature 'Golden Finger' grape bunches from the field as material, spray with tartaric acid (pH=3 and 4) solutions until the solution no longer drips from the fruit surface. Use sterile water as a control. Then inoculate with a suspension of grape white rot fungi conidia. Seal the inoculated bunches in plastic bags to maintain moisture. Take photos and count the rate of bunch rot at 6 and 10 days after inoculation.

[0047] Fruit rot rate (%) = [number of diseased or rotten fruits per bunch / total number of fruits per bunch] × 100%.

[0048] Prevention and control effect (%) = (rot rate of control group - rot rate of treatment group) / rot rate of control group × 100%.

[0049] Figure 3The results showed that 6 days after inoculation, the control efficiencies of 5 mg / mL (pH=3) and 2.5 mg / mL (pH=4) tartaric acid on 'Golden Finger' grapes were 78.6% and 61.3%, respectively; 10 days after inoculation, the control efficiencies were 74.8% and 57.1%, respectively, indicating that 5 mg / mL tartaric acid (pH=3) had the best control effect.

[0050] Example 4: Antibacterial activity of tartaric acid against Staphylococcus aureus

[0051] Grape black rot fungus was inoculated onto PDA medium and cultured at 25°C for 25 days. Sporulation was then induced by treatment at 21°C for 24 hours. After conidia were produced, they were added to centrifuge tubes containing 1 mL of PDA liquid medium. The pH of the PDA liquid medium was adjusted to 2, 3, 4, and 5 with tartaric acid at concentrations of 50 mg / mL, 7.2 mg / mL, 1.2 mg / mL, and 0.3 mg / mL, respectively, with a control group not containing tartaric acid. The spore concentration was adjusted to 2 × 10⁻⁶ using a hemocytometer under a microscope. 6 / mL. After incubation at 25℃ for 24 h, the germination rate of *Aureobasidium nigra* conidia was counted under a microscope.

[0052] The treatment and control were each replicated three times. The germination of 300 conidia was recorded in each replicate. The germination rate (%) was calculated as: (Number of germinated conidia / Total number of conidia) × 100%.

[0053] The inhibition rate is calculated based on the germination rate. Inhibition rate (%) = (control germination rate - treatment germination rate) / control germination rate × 100%.

[0054] Figure 4 The results from Figure A show that tartaric acid (pH=2) has the highest inhibition rate of 60.0% on the germination of conidia of *Pseudomonas aeruginosa*.

[0055] Example 5: The prevention and treatment of grape black rot with tartaric acid

[0056] Prepare a suspension of Staphylococcus aureus conidia (2×10⁻⁶) using sterile water. 6 Prepare a 5 mg / mL (pH=3) tartaric acid solution using sterile water. Collect leaves of 'Seedless White' grapes, disinfect with 70% alcohol for 30 seconds, and wash three times with sterile water. Spray with 5 mg / mL (pH=3) tartaric acid solution until the solution no longer drips from the leaf surface. Use sterile water as a control. Then spray with a suspension of grape black rot conidia until the spore suspension no longer drips. Place the inoculated leaves in a plastic box lined with moist filter paper, seal with plastic wrap to maintain moisture, and then place in a 25℃ light incubator. Take photos and count the area of ​​lesions 5 days after inoculation.

[0057] ImageJ software was used to measure the leaf lesion area and total leaf area and to calculate the disease index.

[0058] Disease index (%) = [Leaf lesion area / Total leaf area] × 100%.

[0059] Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0060] Figure 4 Figures B and C show that treatment of grape leaves with 5 mg / mL tartaric acid (pH=3) significantly reduced the symptoms of grape black rot, with a control efficiency of 97.7%.

[0061] Example 6: The control of grape gray mold by tartaric acid

[0062] Prepare a suspension of Botrytis cinerea conidia (2×10⁻⁶) with sterile water. 6 Prepare tartaric acid solutions (5 mg / mL, pH=3 and 2.5 mg / mL, pH=4) with sterile water. Collect 'Red Globe' grapes, disinfect with 70% alcohol for 30 seconds, and wash three times with sterile water. Spray with tartaric acid (pH=3 and 4) solutions until the solution no longer drips from the fruit surface, using sterile water as a control. Then, puncture and inoculate with a 10 µL suspension of grape botrytis conidia. Place the inoculated fruit in a plastic box lined with moistened filter paper, seal with plastic wrap to maintain moisture, and then place in a 25℃ light incubator. Take photos and count the area of ​​lesions 3 days after inoculation.

[0063] ImageJ software was used to measure the area of ​​fruit lesions and the total fruit surface area, and the disease index was calculated.

[0064] Disease index (%) = [Area of ​​fruit lesions / Total fruit surface area] × 100%.

[0065] Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0066] Figure 5 The results showed that 2 days after inoculation, the control efficiencies of 5 mg / mL (pH=3) and 2.5 mg / mL (pH=4) tartaric acid on 'Red Globe' grape berries were 76.4% and 58.8%, respectively; 3 days after inoculation, the control efficiencies were 70.9% and 41.3%, respectively, indicating that 5 mg / mL tartaric acid (pH=3) had the best control effect.

[0067] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of tartaric acid in the control of grape fungal diseases, characterized in that, The tartaric acid is used to prevent and treat [the condition caused by] Coniella vitis This causes white rot in grapes.

2. The application according to claim 1, characterized in that, The tartaric acid was prepared into an aqueous solution and sprayed onto the target plants.

3. The application according to claim 2, characterized in that, The concentration of tartaric acid aqueous solution is 2.5~5 mg / mL.

4. The application according to claim 2, characterized in that, The spraying involves applying an aqueous solution of tartaric acid to the surface of the grapes or leaves.

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