Application of sodium tartrate in controlling grape botrytis blight, aspergillus infection and OTA production

Abstract

This invention belongs to the field of green preservation technology for fruits and vegetables, specifically involving the application of sodium tartrate in controlling grape gray mold, Aspergillus infection, and OTA production. The steps are as follows: grapes are soaked in a sodium tartrate solution. This soaking process enables the application of sodium tartrate in controlling grape gray mold, Aspergillus infection, and OTA production. This invention is the first to utilize sodium tartrate to efficiently control pathogen infection on grapes, achieving significant technical effects in controlling grape gray mold and Aspergillus niger. While controlling diseases, sodium tartrate synergistically inhibits OTA production, reducing OTA production by over 100% compared to the control group, achieving unexpected technical results. Furthermore, this invention uses food-grade sodium tartrate, making the preservative safe, easy to operate, and with broad market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of green preservation technology for fruits and vegetables, specifically involving the application of sodium tartrate in controlling major grape diseases and ochratoxin A (OTA) contamination. Background Technology

[0002] Grapes are one of the world's major fruit production areas. my country's grape industry primarily focuses on table grapes, with varieties such as 'Kyoho', 'Xiahei', and 'Sunshine Rose' being particularly popular. However, grapes have thin skins, are juicy, and have a high sugar content, making them highly susceptible to mechanical damage during harvesting and transportation, and vulnerable to Botrytis cinerea (a type of fungus). Botrytis cinerea ), Aspergillus carbonisata ( Aspergillus carbonarius ), Westerdick Aspergillus ( Aspergillus westerdijkiae Infection by pathogens such as ) A. carbonarius and A. westerdijkiae It can also produce carcinogens such as OTA on the fruit. Currently, traditional methods for controlling grape diseases mainly include physical methods such as low-temperature refrigeration and treatment with chemical fungicides (such as sulfur dioxide and fludioxonil). Although chemical methods are low-cost and effective, they have problems such as pesticide residues, environmental harm, and inducing drug resistance in pathogens. Biological control is also a current research hotspot, mainly using antagonistic microorganisms for biological control. The core is to screen and isolate beneficial microorganisms with antagonistic activity against postharvest pathogens, and achieve disease control through artificial inoculation. Commonly used strains cover three major categories: bacteria, yeasts, and filamentous fungi, and have advantages such as safety and environmental friendliness. However, postharvest rot of fruits and vegetables is often caused by multiple diseases from multiple pathogens. The number of targeted biocontrol bacteria is small, and the activity of biocontrol bacteria is easily affected by the environment, resulting in poor control effects. Biocontrol bacteria that can control both diseases and toxin production are even rarer. Therefore, developing green, safe, and efficient new preservation methods is particularly important for improving the shelf quality of grapes.

[0003] Sodium tartrate is a GRAS (Generally Recognized as Safe) substance, belonging to the carboxylate food additive category. Currently, sodium tartrate has a wide range of applications in the food industry, primarily for regulating food acidity, acting as an antioxidant, inhibiting squid autolysis, lowering the gelation temperature of methylcellulose, and enhancing the gel strength of surimi. However, its application in the preservation of fruits and vegetables such as grapes has not been explored.

[0004] Based on this, the present invention proposes an application of sodium tartrate to control grape gray mold, Aspergillus niger infection and OTA production, which can achieve control of disease and fungal toxin pollution; furthermore, no research has been found in the prior art on methods for controlling fruit and vegetable preservation with sodium tartrate. Summary of the Invention

[0005] To overcome the problems existing in the current grape preservation technology, this invention provides the application of sodium tartrate in grape preservation. Based on sodium tartrate, the synergistic control of major grape diseases and ochratoxin A (OTA) contamination is achieved, and significant technical effects are obtained, providing a theoretical basis for its application in postharvest storage and preservation of grapes.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application of sodium tartrate in controlling grape gray mold, Aspergillus infection, and OTA production involves the following steps: (1) Activation of pathogens: First, the pathogens are activated using PDA medium; the pathogens include B. cinerea , A. carbonarius and A. westerdijkiae ; (2) Preparation of spore suspension: The activated spores were then placed in the spore suspension. B. cinerea , A. carbonarius and A. westerdijkiae The culture medium was inoculated into PDB medium for the first activation culture to obtain the first activated culture solution; the first activated culture solution was then transferred to fresh PDB medium for a second activation culture to obtain... B. cinerea , A. carbonarius and A. westerdijkiae The corresponding activated colonies were then added to sterile water, and the spore concentration was determined using a hemocytometer to obtain a suspension of pathogenic spores. (3) Preparation of sodium tartrate solution: Sterilize deionized water to obtain sterile water; add sodium tartrate (food grade) to sterile water to obtain sodium tartrate solution; (4) Grape treatment: After the grapes are disinfected with sodium hypochlorite or kept in their natural state (i.e. without disinfection), they are soaked in sodium tartrate solution. Soaking can realize the application of sodium tartrate to control grape gray mold, Aspergillus fungal infection and OTA production. (4.1) Verification of disease control: After soaking, grapes were divided into groups, and holes were punched on the surface of the grapes using a sterile puncher. Then, they were inoculated separately. B. cinerea , A. carbonarius and A. westerdijkiae Spore suspension, thus verifying the effectiveness of sodium tartrate in controlling grape diseases; (4.2) OTA extraction and detection: mining A. carbonarius and A. westerdijkiae The OTA content in the infected, rotten parts of the fruit and the surrounding healthy pulp tissue was determined by HPLC-FLD method, thereby verifying the control of OTA content in fruit samples by sodium tartrate.

[0007] The specific steps are as follows: excavation A. carbonarius and A. westerdijkiae The infected, rotten parts of the fruit and the surrounding healthy pulp were flash-frozen in liquid nitrogen and stored at -80 °C. A 20 g sample of frozen fruit was placed in a mortar, 20 mL of dichloromethane was added, and the mixture was ground into a homogenate. This homogenate was then transferred to a 250 mL wide-mouth bottle, and 60 mL of dichloromethane was added to extract the sample. The mixture was placed in a shaker at 25 °C and shaken slowly in the dark for 24 h to obtain a mixture. The mixture was poured into a separatory funnel and allowed to separate into layers. The lower layer was collected and evaporated using a rotary evaporator at 40 °C for 15–20 min. The remaining liquid in the receiving bottle was collected into a 5 mL centrifuge tube, and 1 mL of chromatographic methanol was added to the centrifuge tube to reconstitute the mixture. The mixture was then centrifuged at 10000 × g at 4 °C for 15 min. The supernatant was filtered through a 0.22 µm organic filter membrane and stored in a chromatographic sample vial for later use.

[0008] The content of OTA in fruit samples was determined by HPLC-FLD using a Shimadzu LC-2060 high-performance liquid chromatograph with a fluorescence detector. A Shim-pack GIST C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was a mixture of acetonitrile and 1% acetic acid (60:40 v / v). Detection conditions were: flow rate 1.0 mL / min, injection volume 10 μL, column temperature 30 ℃, excitation wavelength 333 nm, and emission wavelength 460 nm. Quantification was performed using the external standard method.

[0009] Preferably, the PDA culture medium in step (1) consists of the following components: 9.6 g of potato dextrose agar per 500 mL, with the remainder being distilled water, sterilized at 121 °C for 20 min; and activated culture conditions of 22-25 °C for 48-72 h.

[0010] Preferably, the conditions for the first and second activation cultures in step (1) are: 22~25 ℃, 150~180 rpm, 48~72 h.

[0011] Preferably, the sterile water in step (1) is deionized water that has been sterilized at 121 °C for 20 min.

[0012] Preferably, in step (2), the spore concentration is calculated using the following formula: C × 5 × Dilution factor × 10 4 In the formula: C is the number of spores counted by the hemocytometer; B. cinerea , A. carbonarius and A. westerdijkiae The concentration of the spore suspensions was 1 × 10⁻⁶. 5spores / mL.

[0013] Preferably, the sterilization conditions in step (3) are: sterilization at 121 °C for 20 min, and sodium tartrate solution concentration of 10 g / kg.

[0014] Preferably, in step (4), the immersion disinfection is carried out by immersion in sodium hypochlorite with a mass fraction of 1~2% for 1~2 minutes.

[0015] Preferably, the hole diameter and depth of the hole punched in step (4.1) are 3 mm.

[0016] Preferably, the healthy pulp tissue surrounding the lesion in step (4.2) refers to the pulp within 5 mm of the lesion, centered on the lesion.

[0017] The beneficial effects of this invention are: This invention is the first to effectively control sodium tartrate. B. cinerea , A. carbonarius and A. westerdijkiae The infection on grapes was effectively controlled; experimental results showed that sodium tartrate treatment significantly reduced the infection rate. A. carbonarius and A. westerdijkiae The onset of the disease was delayed by 24 hours. When the incidence rate in the control group was 100%, the incidence rate in the treatment group was still 0. Regarding lesion control, sodium tartrate was effective against lesions 72 hours after treatment. B. cinerea The diameter of lesions caused by infected grapes with gray mold was reduced by more than 30% compared to the control group; after 3 days of treatment, sodium tartrate showed a significant effect on... A. carbonarius The diameter of lesions infected with Aspergillus niger in grapes was reduced by more than 150% compared to the control group; after 3 days of treatment, sodium tartrate showed a significant effect on... A. westerdijkiae The diameter of lesions infected with Aspergillus niger ear rot was reduced by more than 200% compared to the control group; On the other hand, sodium tartrate treatment not only achieved control of major grape diseases, but also synergistic control of ochratoxin A (OTA) contamination; experimental results showed that inoculation... A. carbonarius On day 1, the OTA content in the 5 mm pulp tissue surrounding the lesions of fruits treated with sodium tartrate (10 g / kg) was 2.54 ng / g, while the OTA content in the 5 mm pulp tissue surrounding the lesions of the control group was 5.29 ng / g, representing a reduction of over 100% compared to the control group; inoculation A. westerdijkiae On the fruit, at 1 day, the OTA content in the 5 mm pulp tissue around the lesion of the fruit treated with sodium tartrate (10 g / kg) was 4.29 ng / g, while the OTA content in the 5 mm pulp tissue around the lesion of the control group was 14.90 ng / g, which was more than 200% lower than that of the control group, achieving unexpected technical results. Attached Figure Description

[0018] Figure 1 Sodium tartrate for inoculation B. cinerea The effect of the diameter of lesions on grape fruit is shown in the figure.

[0019] Figure 2 Sodium tartrate for inoculation A. carbonarius The effect of disease incidence (A) and lesion diameter (B) on grape fruit.

[0020] Figure 3 Sodium tartrate for inoculation A. westerdijkiae The effect of disease incidence (A) and lesion diameter (B) on grape fruit.

[0021] Figure 4 Sodium tartrate inhibition A. carbonarius The effect of producing OTA in grape lesions (A) and healthy tissue (B).

[0022] Figure 5 Sodium tartrate inhibition A. westerdijkiae The effect of producing OTA in grape lesions (A) and healthy tissue (B). Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings; reagents or materials not specifically described are all conventional materials and can be obtained through commercial channels.

[0027] (1) Culture medium: The PDA culture medium consists of 9.6 g potato dextrose agar and distilled water in 500 mL. Sterilize at 121 °C for 20 min. The only difference between PDB medium and PDA medium is that it does not contain agar; that is, potato dextrose agar is replaced with potato dextrose.

[0028] (2) Preparation of spore suspension: B. cinerea ( Botrytis cinerea ), A. carbonarius ( Aspergillus carbonarius )and A. westerdijkiae ( Aspergillus westerdijkiae The culture medium was first activated at 25 ℃ and 160 rpm for 72 h to obtain the first activated culture medium. The first activated culture medium was then inoculated into fresh PDA medium at a 1% inoculation rate and subjected to a second activation culture under the same conditions to obtain the desired result. B. cinerea , A. carbonarius and A. westerdijkiae The corresponding activated colonies were obtained; activated colonies were added to sterile water, and the spore concentration was determined using a hemocytometer to obtain a pathogenic spore suspension, with the concentration controlled at 1 × 10⁻⁶. 5 spores / mL; The formula for calculating spore concentration is as follows: C × 5 × Dilution factor × 10 4 In the formula: C is the number of spores counted by the hemocytometer.

[0029] In this example, using 'Sunshine Rose' grapes as the experimental material, the study investigated the postharvest preservation effect of food-grade sodium tartrate solution treatment on grapes. Example 1: Inhibitory effect of sodium tartrate on grape gray mold (1) Preparation before the experiment: Before the experiment, activate the PDA medium. B. cinereaCalculate the required number of grapes, the amounts of sodium tartrate and sodium hypochlorite, and prepare the necessary egg cartons, containers, and measuring instruments. One day in advance, sterilize the sterile water, gauze, 5 mL centrifuge tubes, and 5 mL pipette tips.

[0030] (2) Grape pretreatment and disinfection: Lay absorbent paper on the experimental table, cut off the grapes and retain a 5 mm stem. Select grapes that are uniform in size, color, and shape and have no mechanical damage. Prepare a 2% sodium hypochlorite solution and soak the grapes in batches for 2 min. After removing them, place them on absorbent paper to air dry naturally for 40 min.

[0031] (3) Sodium tartrate soaking treatment: A 10 g / kg sodium tartrate solution was prepared using sterile water. The dried grapes were soaked in the sodium tartrate solution for 60 min, and then dried again for 40 min. A control group was set up, in which the grapes were soaked in sterile water.

[0032] (4) Pathogen inoculation and culture: Clean the laminar flow hood, place the pipette tip, pipette, physiological saline, etc. inside, and sterilize with ultraviolet light for 30 min. Make holes (3 mm wide and 3 mm deep) at the equatorial region of each grape, remove the juice from the holes with a pipette, and continue to air dry at room temperature. Prepare a solution with a concentration of 1 × 10⁻⁶. 5 Inoculate each well with a spore suspension of 3 μL per spores / mL. After allowing to air dry, place the grape berries in a sterilized egg carton and incubate at 20 °C. This incubation process is highly susceptible to infection of grapes, leading to gray mold. Observe and measure the incidence rate and lesion diameter every 12 h after treatment.

[0033] Control effect: The onset of gray mold disease began at 48 hours, and the diameter of the lesions was recorded at 12-hour intervals until the 96th hour.

[0034] Inoculate at the wound site of the fruit B. cinerea The spore suspension was dried at room temperature and then stored for 48 hours, denoted as 48 h.

[0035] like Figure 1 As shown, at 48 h, the diameter of lesions on fruits treated with sodium tartrate (10 g / kg) was 0.60 cm, while that in the control group was 0.65 cm, with no significant difference between the two.

[0036] At 60 h, the diameter of lesions on fruits treated with sodium tartrate was 0.99 cm, while that in the control group was 1.27 cm, with the control group showing a 22.13% higher diameter than the treated group. At 72 h, the diameter of lesions on fruits treated with sodium tartrate was 1.23 cm, while that in the control group was 1.62 cm, with the control group showing a 24.10% increase compared to the treated group. At 84 h, the diameter of lesions on fruits treated with sodium tartrate was 1.67 cm, while that in the control group was 2.04 cm, with the control group showing a 18.12% higher diameter than the treated group. At 96 h, the diameter of lesions on fruits treated with sodium tartrate was 1.78 cm, while that in the control group was 2.17 cm. The control group was 17.78% larger than the treated group.

[0037] Example 2: Sodium tartrate on Aspergillus fungi on grapes ( A. carbonarius Inhibition effect of infection (1) Preparation before the experiment: Activate the PDA medium 5-7 days before the experiment. A. carbonarius Calculate the required number of grapes, the amounts of sodium tartrate and sodium hypochlorite, and prepare the necessary egg cartons, containers, and measuring instruments. One day in advance, sterilize the sterile water, gauze, 5 mL centrifuge tubes, and 5 mL pipette tips.

[0038] (2) Grape pretreatment and disinfection: Absorbent paper was laid on the experimental table, and grapes were cut off, retaining a 5 mm stem. Fruits of uniform size, color, and shape, without mechanical damage, were selected. A 2% sodium hypochlorite solution was prepared, and the grapes were soaked in batches for 2 minutes. After removal, they were placed on absorbent paper to air dry naturally for 40 minutes. A control group was also established, in which grapes were soaked in sterile water.

[0039] (3) Sodium tartrate soaking treatment: Prepare a 10 g / kg sodium tartrate solution using sterile water. Soak the dried grapes in the sodium tartrate solution for 60 min. After soaking, dry them again for 40 min.

[0040] (4) Pathogen inoculation and culture: Clean the laminar flow hood, place the pipette tip, pipette, physiological saline, etc. inside, and sterilize with ultraviolet light for 30 min. Make holes (3 mm wide and 3 mm deep) at the equatorial region of each grape, remove the juice from the holes with a pipette, and continue to air dry at room temperature. Prepare a solution with a concentration of 1 × 10⁻⁶. 5 Inoculate 3 μL of a spore suspension (spores / mL) into each well. After allowing the grapes to air dry, place them in a sterilized egg carton and incubate at 20 °C. This incubation period makes the grapes susceptible to Aspergillus niger. Observe and measure the incidence rate every 12 h after treatment, and observe and measure the diameter of lesions every 24 h.

[0041] Control effect: A. carbonarius The incidence rate was observed starting 12 hours after vaccination, with results recorded at 12-hour intervals; the lesion diameter was recorded at the time of vaccination. A. carbonarius Starting from day 1, record the results daily until day 5.

[0042] Inoculate at the wound site of the fruit A. carbonariusThe spore suspension was dried at room temperature and then stored for 12 h, denoted as 12 h. Inoculate at the wound site of the fruit A. carbonarius The spore suspension was dried at room temperature and stored for 1 day, which is recorded as 1 day.

[0043] like Figure 2 As shown in Figure A, the disease incidence rate of fruits treated with sodium tartrate (10 g / kg) was 0 at 12 h, 24 h, and 36 h. At 24 h, the control group fruits began to develop the disease, and the disease incidence rates at 24 h and 36 h were 79.2% and 100%, respectively.

[0044] like Figure 2 As shown in Figure B, at 1 day, the diameter of lesions on fruits treated with sodium tartrate was 0 cm, while that in the control group was 0.24 cm, meaning the control group was 100.00% larger than the treated group. At 2 days, the diameter of lesions on fruits treated with sodium tartrate was 0.40 cm, while that in the control group was 0.90 cm, with the control group showing a 55.20% increase compared to the treated group. At 3 days, the diameter of lesions on fruits treated with sodium tartrate was 0.60 cm, while that in the control group was 1.51 cm, with the control group showing a 60.66% higher diameter than the treated group. At 4 days, the diameter of lesions on fruits treated with sodium tartrate was 1.00 cm, while that in the control group was 2.01 cm, with the control group showing a 50.05% increase compared to the treated group. At 5 days, the diameter of lesions on fruits treated with sodium tartrate was 1.48 cm, while that in the control group was 2.45 cm. The control group was 39.54% larger than the treated group.

[0045] Example 3: Sodium tartrate on Aspergillus fungi on grapes ( A. westerdijkiae Inhibition effect of infection (1) Preparation before the experiment: Activate the PDA medium 5-7 days before the experiment. A. westerdijkiae Calculate the required number of grapes, the amounts of sodium tartrate and sodium hypochlorite, and prepare the necessary egg cartons, containers, and measuring instruments. One day in advance, sterilize the sterile water, gauze, 5 mL centrifuge tubes, and 5 mL pipette tips.

[0046] (2) Grape pretreatment and disinfection: Lay absorbent paper on the experimental table, cut off the grapes and retain a 5 mm stem. Select grapes that are uniform in size, color, and shape and have no mechanical damage. Prepare a 2% sodium hypochlorite solution and soak the grapes in batches for 2 min. After removing them, place them on absorbent paper to air dry naturally for 40 min.

[0047] (3) Sodium tartrate soaking treatment: A 10 g / kg sodium tartrate solution was prepared using sterile water. The dried grapes were soaked in the sodium tartrate solution for 60 min. After soaking, they were dried again for 40 min. A control group was set up, in which the grapes were soaked in sterile water.

[0048] (4) Pathogen inoculation and culture: Clean the laminar flow hood, place the pipette tip, pipette, physiological saline, etc. inside, and sterilize with ultraviolet light for 30 min. Make holes (3 mm wide and 3 mm deep) at the equatorial region of each grape, remove the juice from the holes with a pipette, and continue to air dry at room temperature. Prepare a solution with a concentration of 1 × 10⁻⁶. 5 Inoculate 3 μL of a spore suspension (spores / mL) into each well. After allowing the grapes to air dry, place them in a sterilized egg carton and incubate at 20 °C. This incubation period makes the grapes susceptible to Aspergillus rot. Observe and measure the incidence rate every 12 h after treatment, and observe and measure the diameter of lesions every 24 h.

[0049] Control effect: A. westerdijkiae The incidence rate was observed starting 12 hours after vaccination, with results recorded at 12-hour intervals; the lesion diameter was recorded at the time of vaccination. A. westerdijkiae Starting from day 1, record the results daily until day 5.

[0050] Inoculate at the wound site of the fruit A. westerdijkiae The spore suspension was dried at room temperature and then stored for 12 hours, denoted as 12h. Inoculate at the wound site of the fruit A. westerdijkiae The spore suspension was dried at room temperature and stored for 1 day, which is recorded as 1 day.

[0051] like Figure 3 As shown in Figure A, at 24 h and 36 h, the incidence rate of fruits treated with sodium tartrate (10 g / kg) was 0, while the incidence rates of fruits in the control group were 91.6% and 100%, respectively.

[0052] like Figure 3 As shown in Figure B, at 1 day, the diameter of lesions on fruits treated with sodium tartrate was 0 cm, while that in the control group was 0.28 cm, meaning the control group was 100.00% larger than the treated group. At 2 days, the diameter of lesions on fruits treated with sodium tartrate was 0.40 cm, while that in the control group was 0.88 cm, with the control group showing a 54.48% higher diameter than the treated group. At 3 days, the diameter of lesions on fruits treated with sodium tartrate was 0.46 cm, while that in the control group was 1.53 cm, with the control group showing a 70.16% higher lesion diameter than the treated group. At 4 days, the diameter of lesions on fruits treated with sodium tartrate was 0.77 cm, while that in the control group was 2.03 cm, with the control group showing a 61.91% higher diameter than the treated group. At 5 days, the diameter of lesions on fruits treated with sodium tartrate was 1.19 cm, while that in the control group was 2.44 cm. The control group was 51.24% larger than the treated group.

[0053] Example 4: Sodium tartrate on A. carbonarius and A. westerdijkiae Inhibitory effect of OTA production in grape berries (1) Preparation before the experiment: On the 5th day after treatment, dig up A. carbonarius or A. westerdijkiae The infected grape rotten tissue was collected, and the pulp within 5 mm around the rotten tissue was removed, flash-frozen with liquid nitrogen, and then stored in a -80 ℃ freezer.

[0054] (2) Extraction of OTA from grape berries: Take 20 g of frozen fruit sample and place it in a mortar. Add 20 mL of dichloromethane and grind it into a homogenate in an ice bath. Transfer the homogenate to a 250 mL wide-mouth bottle and add 60 mL of dichloromethane to extract the sample. Place the sample in a shaker at 25 ℃ and shake slowly for 24 h. Under light-protected conditions, pour the mixture into a separatory funnel and allow it to stand for separation. Collect the lower layer liquid and evaporate it by rotary evaporation for 15-20 min. Collect the remaining liquid in the receiving bottle into a 5 mL centrifuge tube. Add 1 mL of chromatographic grade methanol to the centrifuge tube to reconstitute the liquid. Then centrifuge at 10000 × g and 4 ℃ for 15 min. Take the supernatant and filter it through a 0.22 µm organic filter membrane. Store the supernatant in a chromatographic sample bottle for later use.

[0055] (3) Detection of OTA content in grape berries: The OTA content in grape berries was determined by HPLC-FLD method using a Shimadzu LC-2060 high-performance liquid chromatograph with a fluorescence detector. The detection conditions were: Shim-pack GIST C18 column (250 mm × 4.6 mm, 5 μm), mobile phase: acetonitrile:1% acetic acid (60:40, v / v), flow rate: 1.0 mL / min, injection volume: 10 μL, column temperature: 30 ℃, excitation wavelength: 333 nm, emission wavelength: 460 nm. Quantification was performed using the external standard method.

[0056] Control effect: A. carbonarius After infecting grape berries, the extraction time for OTA was set to 5 days; A. westerdijkiae After infecting the grape berries, the extraction time for OTA was set to 5 days.

[0057] Inoculate at the wound site of the fruit A. carbonarius or A. westerdijkiae The spore suspension was dried at room temperature and stored for 1 day, which is recorded as 1 day.

[0058] like Figure 4 As shown in A, vaccinationA. carbonarius On day 1, the OTA content in the lesions of fruits treated with sodium tartrate (10 g / kg) was 443.99 ng / g, while the OTA content in the lesions of the control group was 508.84 ng / g, which was 12.74% higher than that of the treatment group, showing a significant difference between the two.

[0059] like Figure 4 As shown in B, vaccination A. carbonarius On day 1, the OTA content in the 5 mm pulp tissue around the lesions of the fruit treated with sodium tartrate (10 g / kg) was 2.54 ng / g, while the OTA content in the 5 mm pulp tissue around the lesions of the control group was 5.29 ng / g, which was 51.97% higher than that of the treatment group, showing a significant difference between the two.

[0060] like Figure 5 As shown in A, vaccination A. westerdijkiae On day 1, the OTA content in the lesions of fruits treated with sodium tartrate (10 g / kg) was 596.66 ng / g, while the OTA content in the lesions of the control group was 989.20 ng / g, which was 39.68% higher than that of the treatment group, showing a significant difference between the two.

[0061] like Figure 5 As shown in B, vaccination A. westerdijkiae On day 1, the OTA content in the 5 mm pulp tissue around the lesions of the fruit treated with sodium tartrate (10 g / kg) was 4.29 ng / g, while the OTA content in the 5 mm pulp tissue around the lesions of the control group was 14.90 ng / g, which was 71.25% higher than that of the treatment group, showing a significant difference between the two.

[0062] In summary, this invention utilizes sodium tartrate to effectively control grape gray mold (… B. cinerea ) and Aspergillus fungi ( A. carbonarius and A. westerdijkiae It can significantly inhibit the infection of ) and also significantly inhibit A. carbonarius and A. westerdijkiae OTA generation on grape berries.

[0063] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Therefore, although this specification has described the present invention in detail with reference to the various embodiments above, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. An application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production, characterized in that, The steps are as follows: (1) Activation of pathogens: First, the pathogens were activated using PDA medium to obtain activated pathogens; the pathogens included B. cinerea , A. carbonarius and A. westerdijkiae ; (2) Preparation of spore suspension: The activated spores were then placed in the spore suspension. B. cinerea , A. carbonarius and A. westerdijkiae The culture medium was inoculated into PDB medium for the first activation culture to obtain the first activated culture solution; the first activated culture solution was then transferred to fresh PDB medium for a second activation culture to obtain... B. cinerea , A. carbonarius and A. westerdijkiae The corresponding activated colonies were then added to sterile water, and the spore concentration was determined using a hemocytometer to obtain a suspension of pathogenic spores. (3) Preparation of sodium tartrate solution: Sterilize deionized water to obtain sterile water; add sodium tartrate to sterile water to obtain sodium tartrate solution; (4) Grape treatment: After disinfection with sodium hypochlorite or kept in its natural state, grapes are soaked in sodium tartrate solution. Soaking can realize the application of sodium tartrate to control grape gray mold, Aspergillus fungal infection and OTA production. (4.1) Verification of disease control: After soaking, grapes were divided into groups, and holes were punched on the surface of the grapes using a sterile puncher. Then, they were inoculated separately. B. cinerea , A. carbonarius and A. westerdijkiae Spore suspension, thus verifying the effectiveness of sodium tartrate in controlling grape diseases; (4.2) OTA extraction and detection: mining A. carbonarius and A. westerdijkiae The OTA content in the infected, rotten parts of the fruit and the surrounding healthy pulp tissue was determined by HPLC-FLD method, thereby verifying the control of OTA content in fruit samples by sodium tartrate.

2. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, The PDA culture medium in step (1) consists of the following components: 9.6 g potato dextrose agar per 500 mL, with the remainder being distilled water. The medium is sterilized at 121 °C for 20 min. The activation culture conditions are: 22-25 °C for 48-72 h.

3. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, The conditions for the first activation culture in step (2) are: 22~25 ℃, 150~180 rpm, 48~72 h; the conditions for the second activation culture are: 22~25 ℃, 48~72 h.

4. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, In step (2), the formula for calculating spore concentration is as follows: C × 5 × Dilution factor × 10 4 In the formula: C is the number of spores counted by the hemocytometer; B. cinerea , A. carbonarius and A. westerdijkiae The concentration of the spore suspensions was 1×10⁻⁶. 5 spores / mL.

5. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, The sodium tartrate soaking time in step (2) is 60 min.

6. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, The sterilization conditions in step (3) are: sterilization at 121 °C for 20 min, and sodium tartrate solution concentration of 10 g / kg.

7. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, In step (4), the immersion disinfection is carried out by immersing in sodium hypochlorite with a mass fraction of 1~2% for 1~2 minutes.

8. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, The hole diameter and depth of the hole described in step (4.1) are 3 mm.

9. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, The specific operation of step (4.2) is as follows: excavation A. carbonarius and A. westerdijkiae The infected, rotten parts of the fruit and the surrounding healthy pulp were flash-frozen in liquid nitrogen and stored at -80 ℃. Then, 20 g of the frozen fruit sample was placed in a mortar, 20 mL of dichloromethane was added, and the mixture was ground into a homogenate. The homogenate was then transferred to a 250 mL wide-mouth bottle, and 60 mL of dichloromethane was added to extract the sample. The sample was then placed in a shaker at 25 ℃ and shaken slowly in the dark for 24 h to obtain a mixture. The mixture was poured into a separatory funnel and allowed to stand for separation. The lower layer of liquid was collected and evaporated by rotary evaporation at 40 ℃ for 15-20 min. The remaining liquid in the receiving bottle was collected into a 5 mL centrifuge tube, 1 mL of chromatographic methanol was added to the centrifuge tube to reconstitute the liquid, and then the mixture was centrifuged at 10000 × g at 4 ℃ for 15 min. The supernatant was then filtered through a 0.22 µm organic filter membrane and stored in a chromatographic sample bottle for later use. The content of OTA in fruit samples was determined by HPLC-FLD using a Shimadzu LC-2060 high-performance liquid chromatograph with a fluorescence detector; a Shim-pack GIST C18 column with dimensions of 250 mm × 4.6 mm and 5 μm was used; the mobile phase was a mixture of acetonitrile and 1% acetic acid (volume ratio 60:40); the detection conditions were: flow rate 1.0 mL / min, injection volume 10 μL, column temperature 30 ℃, excitation wavelength 333 nm, and emission wavelength 460 nm.

10. The application of sodium tartrate for controlling grape gray mold, Aspergillus infection, and OTA production according to claim 1, characterized in that, In step (4.2), the healthy pulp tissue surrounding the lesion refers to the pulp within 5 mm of the lesion's periphery.

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