Method for preventing and treating postharvest diseases of grapes and storing and preserving grapes through cyclic adenylate induced culture of sporidiobolus pink Y16
Through cAMP induced culture of Pseudo-Spore-Spore Y16, the problem of prevention and treatment of post-harvest acid rot was solved, safe and efficient storage and preservation were achieved, environmental and health hazards of chemical fungicides were avoided, and the quality of grapes was maintained.
Patent Information
- Application Number
- CN202510515387.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art has environmental pollution and health hazards caused by chemical fungicides in preventing and treating post-harvest acid rot of grapes. Physical methods consume high energy and affect the quality of fruits and vegetables. The biological control method has low efficacy and is unstable.
Cyclic adenylate (cAMP) was used to induce culture of Pseudo-Pink Loops Y16. By injecting yeast suspension into the wound on the surface of grapes and inoculating Aspergillus niger spores, the post-harvest diseases of grapes were controlled and stored and preserved.
Significantly reduce the occurrence of post-harvest acid rot of grapes, reduce the natural rot rate, maintain the quality of grapes, and avoid the residual harm of chemical fungicides. It has the advantages of safety, greenness and environmental protection.
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Figure CN120283829A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological control of post-harvest diseases of fruits, and particularly relates to a method for controlling post-harvest diseases of grapes by cyclic adenosine monophosphate-induced cultivation of Saccharomyces pseudopinkana Y16 and storing and preserving grapes. Background Art
[0002] Grapes are bright-looking and juicy fruits that are rich in vitamins, fruit acids, and anthocyanins. Regular consumption of grapes can not only improve the body's antioxidant and anti-aging abilities, but also promote digestion, nourish blood, and enhance immunity. Grapes can be eaten fresh or used to make wine and raisins. They are known as the top of the world's four major fruits.
[0003] Grapes have thin and juicy skins, which are very susceptible to mechanical damage during harvesting, storage and transportation, providing conditions for infection by pathogens. Therefore, grapes are prone to infectious diseases during post-harvest storage, causing them to rot and deteriorate, causing huge economic losses to the industry. Among them, sour rot caused by Aspergillus carbonarius is a common fungal disease of post-harvest grapes. This pathogen is also the main producer of the fungal toxin ochratoxin A (OTA), which can seriously endanger the health of consumers. Therefore, it is very necessary to adopt safe, green and efficient methods to prevent and control post-harvest sour rot of grapes.
[0004] At present, the prevention and control methods of post-harvest diseases of fruits and vegetables mainly include: physical method, chemical method and biological control method. The physical method mainly uses low-temperature storage, controlled atmosphere storage and heat treatment. Although it can effectively inhibit the growth of pathogens and extend the shelf life of fruits and vegetables, it often has the disadvantages of high equipment requirements and high energy consumption. Some physical methods will also affect the sensory quality of fruits and vegetables. The chemical method mainly uses chemical fungicides, such as carbendazim, sec-butylamine, etc., which have high prevention and control efficiency, but are easy to cause pathogens to develop drug resistance, pollute the environment, and endanger the health of consumers. The biological control method mainly uses antagonistic microorganisms or their metabolites to achieve the purpose of preventing and controlling post-harvest diseases of fruits and vegetables. This method has the advantages of safety and environmental friendliness. However, compared with chemical fungicides, the use of antagonistic microorganisms alone has the disadvantages of low prevention and control efficacy and unstable antibacterial activity.
[0005] Cyclic adenosine monophosphate (cAMP) is the "second messenger" of life information transmission. It is formed by ATP (adenosine triphosphate) under the catalysis of AC (adenylate cyclase) and can regulate the growth, metabolism, signal transduction and gene expression of microorganisms. However, the application of cAMP as an elicitor to induce the cultivation of antagonistic yeast in the field of biological control, especially the control of post-harvest grape diseases, has not been reported so far. Summary of the invention
[0006] In view of the deficiencies of the prior art, the present invention creatively uses cAMP to induce the cultivation of Sporidiobolus pararoseus Y16, and applies it to the prevention and control of postharvest diseases of grapes and the storage and preservation of grapes, achieving remarkable effects; it can effectively control the occurrence of postharvest sour rot of grapes, reduce the loss of edible value and commercial value caused by diseases, and has good application value.
[0007] To achieve the above objectives, the present invention provides the following solutions:
[0008] The Sporidiobolus pararoseus Y16 provided by the present invention is a publicly disclosed strain, from the China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 2.5351. cAMP is a safe and non-toxic organic substance, presenting as white crystalline powder. Research has proven that compared with the uninduced Sporidiobolus pararoseus Y16, the cAMP-induced cultivation of Sporidiobolus pararoseus Y16 can significantly reduce the occurrence of postharvest sour rot of grapes, reduce the natural decay of grapes after harvest, and achieve the purpose of grape storage and preservation.
[0009] The method for preventing and controlling postharvest diseases of grapes and storing and preserving grapes by inducing the cultivation of Sporidiobolus pararoseus Y16 with cyclic adenosine monophosphate is carried out according to the following steps:
[0010] (1) First, inoculate Sporidiobolus pararoseus Y16 into NYDB medium for the first cultivation to obtain a culture solution; then suck the culture solution and transfer it again into NYDB medium for the second cultivation to obtain a yeast culture solution; then transfer the yeast culture solution into NYDB medium containing cyclic adenosine monophosphate for induced cultivation. After cultivation, the culture solution is centrifuged to obtain a bacterial sludge, which is centrifuged and washed with sterile physiological saline. The washed bacterial sludge is resuspended with sterile physiological saline again to obtain a Sporidiobolus pararoseus Y16 bacterial suspension; the final concentration of cyclic adenosine monophosphate in the NYDB medium is 0.05 - 0.5 mM;
[0011] (2) Select grapes with uniform size, no obvious mechanical damage and diseases, and consistent maturity. Soak and treat them with a disinfectant solution, and then rinse them with running water. Put the washed grapes into a disinfected plastic basket to dry; punch holes on the surface of the dried grapes to form wounds of a certain size and depth, inject an equal volume of the Sporidiobolus pararoseus Y16 bacterial suspension prepared in step (1) into each wound, let it stand at room temperature for a period of time, then inject an Aspergillus carbonarius spore suspension into each wound, dry it, seal it with a supporting disinfected plastic lid, and store it in a constant temperature and humidity incubator to achieve the control of postharvest sour rot of grapes;
[0012] Alternatively, select grapes that are disease-free, free from mechanical damage, have a uniform color, and are of similar size. Without any disinfection treatment, keep the grapes in their natural state and evenly spray the Sporidiobolus pararoseus Y16 bacterial suspension prepared in step (1) on the surface of the grapes, then the use for preventing post-harvest diseases of grapes and storage and preservation can be achieved.
[0013] Preferably, the NYDB medium described in step (1) (per 1 L) is: yeast extract 5 g, glucose 10 g, beef extract 8 g, made up to 1000 mL with distilled water, natural pH, sterilized at 115 °C for 20 min.
[0014] Preferably, the conditions for the first culture and the second culture in step (1) are both: temperature 28 °C, rotation speed 180 - 200 rpm, time 20 - 24 h; the temperature for the induction culture is 28 °C, rotation speed 180 rpm, and time 24 h.
[0015] Preferably, the inoculation amount inoculated into the NYDB medium in step (1) is 1% - 2%; the centrifugation conditions are: 20 °C, 8000 rpm, 10 min.
[0016] Preferably, the final concentration of cyclic adenosine monophosphate in the NYDB medium in step (1) is 0.05 mM, and the concentration of the Sporidiobolus pararoseus Y16 bacterial suspension is 1×10 8 cells / mL.
[0017] Preferably, the disinfectant solution in step (2) is an aqueous solution of sodium hypochlorite with a concentration of 0.5%, and the soaking time is 5 - 10 min.
[0018] Preferably, the operation for disinfecting the plastic baskets and plastic lids in step (2) is: clean and dry them, then immerse them in an aqueous solution of sodium hypochlorite with a concentration of 1% for 2 - 3 h, and air-dry at room temperature.
[0019] Preferably, the Sporidiobolus pararoseus Y16 bacterial suspension and the Aspergillus carbonarius spore suspension injected into each wound in step (2) are both 10 μL; the standing time is 2 h.
[0020] Preferably, the temperature for culturing in the constant temperature and humidity incubator in step (2) is 20 °C, and the relative humidity is 95%;
[0021] Preferably, when evenly spraying the Sporidiobolus pararoseus Y16 bacterial suspension prepared in step (1) on the surface of the grapes in step (2), the specific dosage is 0.5 - 1.5 mL of the bacterial suspension sprayed on each grape.
[0022] Compared with the prior art, the advantages of the present invention are:
[0023] (1) The Sporidiobolus salmonicolor Y16 used in the present invention is isolated and purified in this laboratory. It has been verified as a non-toxic yeast through animal acute toxicity experiments and is safe and non-toxic to humans. At the same time, the cAMP used in the present invention is a non-toxic and harmless organic compound that can be safely applied to food. Moreover, there is currently no relevant research on using cAMP-induced culture of antagonistic yeast for controlling post-harvest diseases of grapes. The present invention is original.
[0024] (2) The present invention uses Sporidiobolus salmonicolor Y16 induced by cAMP to treat grapes. Compared with the treatment with non-induced Sporidiobolus salmonicolor Y16, the induced treatment can significantly reduce the occurrence of post-harvest sour rot of grapes and lower the natural decay rate of post-harvest grapes, achieving remarkable results.
[0025] (3) The Sporidiobolus salmonicolor Y16 induced by cAMP used in the present invention to treat grapes has no significant adverse effects on the main quality indicators of post-harvest grapes, such as weight loss rate, soluble solids, titratable acid, ascorbic acid, and hardness, compared with non-induced antagonistic yeast.
[0026] (4) The Sporidiobolus salmonicolor Y16 induced by cAMP used in the present invention can replace chemical fungicides to control post-harvest diseases of grapes and is used for the storage and preservation of post-harvest grapes. It can reduce the harm of chemical fungicide residues to the health of consumers and the environment, has the advantages of safety, green environmental protection, and broad application prospects. Description of the Drawings
[0027] Figure 1 Effects of Sporidiobolus salmonicolor Y16 induced by different concentrations of cAMP on the decay rate and decay diameter of post-harvest sour rot of grapes; Note: A is the decay rate of grapes stored for 4 days and 5 days, B is the decay diameter of grapes stored for 4 days and 5 days; among them, CK is the control group, that is, grapes treated with sterile physiological saline; 0, 0.01, 0.05, 0.1, 0.5, 1 are suspensions of Sporidiobolus salmonicolor Y16 induced by cAMP with final concentrations of 0 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM (1×10 8 cells / mL) - treated grapes; the concentration of Aspergillus carbonarius spores is 1×10 5 spores / mL; different lowercase letters represent significant differences (P < 0.05).
[0028] Figure 2Effect of cAMP at 0.05 mM on the decay rate and decay diameter of postharvest sour rot of grapes induced by Sporidiobolus pararoseus Y16 cultured for different times; Note: A is the decay rate of grapes stored for 4 d and 5 d, B is the decay diameter of grapes stored for 4 d and 5 d; where CK is the control group, i.e., grapes treated with sterile saline; 0, 12, 24, 36, 48 are grapes treated with the suspension of Sporidiobolus pararoseus Y16 (1×10 8 cells / mL) induced by cAMP at a final concentration of 0.05 mM for 0 h, 12 h, 24 h, 36 h, 48 h respectively; the concentration of Aspergillus carbonarius spores is 1×10 5 spores / mL; different lowercase letters represent significant differences (P < 0.05).
[0029] Figure 3 Control effect of Sporidiobolus pararoseus Y16 induced by cAMP on natural decay of grapes; Note: CK is the control group, i.e., grapes treated with sterile saline; Y is grapes treated with the suspension of Sporidiobolus pararoseus Y16 (1×10 8 cells / mL) without cAMP induction; Y+C is grapes treated with the suspension of Sporidiobolus pararoseus Y16 (1×10 8 cells / mL) induced by cAMP at a final concentration of 0.05 mM for 24 h; different lowercase letters represent significant differences (P < 0.05). Detailed implementation mode
[0030] The present invention is more detailedly interpreted by means of the following implementation examples. The following examples are only illustrative and the present invention is not limited by these examples.
[0031] The culture procedure of Sporidiobolus pararoseus Y16 is as follows:
[0032] First activation: Pipette 1 mL of Sporidiobolus pararoseus Y16 from the glycerol tube stored at -80°C with a sterile pipette tip and inoculate it into 50 mL of NYDB medium (inoculation amount is 2%), and culture it at 28°C and 180 rpm for 24 h to obtain a culture solution;
[0033] Second liquid activation: Pipette 1 mL of the culture solution with a sterile pipette tip and inoculate it into 50 mL of NYDB medium (inoculation amount is 2%), and culture it at 28°C and 180 rpm for 24 h to obtain a culture solution;
[0034] Induction culture: Take the activated culture solution and inoculate it into a new NYDB medium (containing cAMP at a final concentration of 0.05 - 0.5 mM) at the same inoculation amount of 2%, and culture it at 28°C and 180 rpm for a certain time to obtain a culture solution.
[0035] Preparation of suspension: The above-mentioned culture medium was centrifuged at 20 °C and 8000 rpm for 10 min, and washed 3 times with sterile physiological saline to remove the culture medium matrix, obtaining bacterial sludge. Then it was resuspended with sterile physiological saline and adjusted to the required yeast concentration (1×10 8 cells / mL).
[0036] The activation step of Aspergillus carbonarius before use was as follows: Aspergillus carbonarius was evenly coated on PDA medium with a sterile spreading rod and cultured in the dark at 25 °C for 5 days. The spores were scraped into sterile physiological saline, and then filtered through eight layers of sterilized gauze to obtain a mold spore suspension, which was adjusted to a final concentration of 1×10 5 spores / mL with sterile physiological saline.
[0037] Example 1:
[0038] Sporidiobolus pararoseus Y16 induced and cultured with different concentrations of cAMP was used for the control of postharvest sour rot of grapes;
[0039] I. Test scheme
[0040] Grapes of uniform size, without diseases and mechanical damage, and consistent maturity were selected, soaked in 0.5% sodium hypochlorite for 10 minutes, then rinsed with running water, and the washed grapes were placed in a sterilized plastic box and dried at room temperature. Wounds with a size of 3 mm × 3 mm (diameter × depth) were made at the equatorial part of the grapes, and the following treatments were carried out respectively:
[0041] 1) Inject 10 μL of sterile physiological saline;
[0042] 2) Inject 10 μL of a suspension of Sporidiobolus pararoseus Y16 (1×10 8 cells / mL) induced and cultured with 0 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM cAMP for 24 h;
[0043] 3) After standing at room temperature for 2 h, inject 10 μL of an Aspergillus carbonarius spore suspension (1×10 5 spores / mL) into the wound. After drying, it was placed in a constant temperature and humidity incubator at 20 °C and a relative humidity of 95% for storage for 4 d and 5 d, and then the grape decay rate was counted and the decay diameter was measured. There were 3 parallels for each treatment, and 24 grapes for each parallel.
[0044] Decay rate (%) = number of decayed / total number × 100%;
[0045] II. Test results
[0046] From Figure 1As can be seen from Figure A, on the 4th day of storage, the decay rate of the control group had reached 100%. Among them, the decay rate of grapes treated with Sporidiobolus pararoseus Y16 cultured with 0.05 mM cAMP was only 13.77%, which was significantly lower than that of the control group and other treatment groups (P < 0.05). On the 5th day, except for the control group, the decay rates of all treatment groups increased significantly. However, the decay rate of grapes treated with Sporidiobolus pararoseus Y16 cultured with 0.05 mM cAMP was 56.25%, still significantly lower than that of the control group and the non-induced antagonistic yeast treatment group (P < 0.05), and it was the lowest among all treatment groups.
[0047] As can be seen from Figure 1 Figure B, on the 4th and 5th days of the storage period, the decay diameters of grapes treated with Sporidiobolus pararoseus Y16 cultured with 0.05 mM cAMP were 8.03 mm and 9.44 mm respectively, which were significantly lower than those of the control group and the non-induced antagonistic yeast treatment group at the same time point (P < 0.05), and it was the lowest among all treatment groups.
[0048] Therefore, Sporidiobolus pararoseus Y16 cultured with 0.05 mM cAMP can more effectively control postharvest sour rot of grapes, achieving unexpected technical effects. Subsequently, the effect of different induction times on the biocontrol efficacy of antagonistic yeast was explored on the basis of this concentration.
[0049] Example 2:
[0050] Sporidiobolus pararoseus Y16 cultured with 0.05 mM cAMP for different times was used to control postharvest sour rot of grapes;
[0051] I. Test scheme
[0052] Grapes of uniform size, without diseases and mechanical damage, and consistent maturity were selected, soaked in 0.5% sodium hypochlorite for 10 minutes, then rinsed with running water, and the washed grapes were placed in a sterilized plastic box and dried at room temperature. At the equatorial part of the grapes, wounds with a size of 3 mm × 3 mm (diameter × depth) were made with a sterile punch, and the following treatments were carried out respectively:
[0053] 1) Inject 10 μL of sterile normal saline;
[0054] 2) Inject 10 μL of cell suspension of Sporidiobolus pararoseus Y16 (1 × 10 8 cells / mL) cultured with 0.05 mM cAMP for 0 h, 12 h, 24 h, 36 h, and 48 h;
[0055] 3) After standing at room temperature for 2 h, inject 10 μL of Aspergillus carbonarius spore suspension (1 × 10 5(spores / mL). After air-drying, they were placed in a constant temperature and humidity incubator at 20 °C and a relative humidity of 95% for 4 days and 5 days, and then the grape rot rate was counted and the rot diameter was measured. There were 3 replicates for each treatment, and 24 grapes for each replicate.
[0056] Rot rate = number of rotted ones / total number × 100%;
[0057] II. Test results
[0058] From Figure 2 Figure A in it can be seen that at the 4th day of storage, the rot rates of grapes treated with Sporidiobolus pararoseus Y16 induced by 0.05 mM cAMP for different induction times were all significantly lower than those of the control group (P < 0.05). Among them, the rot rate of the group treated with Sporidiobolus pararoseus Y16 induced for 24 h was only 6%, which was significantly lower than that of the control group and the group treated with antagonistic yeast induced for 0 h (P < 0.05), and was the lowest among all treatment groups. At the 5th day, the rot rate of the yeast treatment group induced for 24 h was 25%, which was significantly lower than that of the control group and the group treated with antagonistic yeast induced for 0 h (P < 0.05), and was the lowest among all treatment groups. From Figure 2 Figure B in it can be seen that at the 4th and 5th days of storage, the rot diameters of different induction time antagonistic yeast treatment groups were all significantly lower than those of the control group; moreover, the rot diameters of grapes treated with Sporidiobolus pararoseus Y16 induced for 24 h were 9.06 mm and 9.17 mm respectively, which were significantly lower than those of the control group and the group treated with antagonistic yeast induced for 0 h (P < 0.05), and were the lowest among all treatment groups.
[0059] Comprehensively analyzing the effects of different treatments on the rot rate and rot diameter of grapes, it can be seen that Sporidiobolus pararoseus Y16 (1×10 8 cells / mL) induced by 0.05 mM cAMP for 24 h can more effectively control postharvest sour rot of grapes.
[0060] Example 3:
[0061] Use of Sporidiobolus pararoseus Y16 induced by cAMP for grape storage and preservation
[0062] I. Test scheme
[0063] Select grapes with uniform size, no diseases and mechanical damage, and consistent maturity, without any treatment, keep the natural condition of the grapes, and directly spray evenly on the grape surface: (1) sterile normal saline; (2) Sporidiobolus pararoseus Y16 bacterial suspension (1×10 8 cells / mL); (3) Sporidiobolus pararoseus Y16 bacterial suspension (1×10 8cells / mL). After air-drying naturally at room temperature, it was sealed in a sterilized plastic box and stored in a constant temperature and humidity incubator (20 °C, relative humidity 95%) for 6 d, 12 d, 18 d, and 24 d. Then, the natural decay rate of grapes was counted, and indexes such as weight loss rate, soluble solids, titratable acid, ascorbic acid, and hardness were measured. There were 3 parallels for each treatment, and 24 grapes for each parallel.
[0064] Natural decay rate (%) = Number of decayed fruits / Total number of fruits × 100%
[0065] The specific measurement methods for quality indexes are as follows:
[0066] 1. Weight loss rate: Weight loss rate (%) = (Mass before treatment - Mass after storage) / Mass before treatment × 100%
[0067] 2. Soluble solids content: The soluble solids (TSS) content (g / 100 g) was measured using a hand-held refractometer.
[0068] 3. Titratable acid content: It was determined by the sodium hydroxide titration method. Weigh 5 g of grape samples and grind them in a mortar, transfer them to a graduated cylinder, add distilled water to make up the volume to 50 mL, let it stand for 30 min, then transfer it to a 50 mL centrifuge tube, centrifuge for 10 min (4 °C, 8000 rpm). Take 10 mL of the supernatant, add 2 drops of 1% phenolphthalein indicator, and titrate with 0.1 M NaOH until the solution just shows a pink color and does not fade within 30 s as the end point. Use distilled water instead of the supernatant as a control, and the result is expressed as a mass fraction (%).
[0069] Titratable acid content (%) = (V × c × (V1 - V0) × 0.075) / (Vs × m) × 100%
[0070] Where V is the total volume of the sample extract (mL), c is the concentration of NaOH (mol / L), V1 is the volume of the NaOH solution consumed for titrating the sample solution (mL), V0 is the volume of the sodium hydroxide solution consumed for titrating distilled water (mL), Vs is the volume of the sample solution taken for titration (mL), m is the sample mass (g), and 0.075 is the tartaric acid conversion coefficient (g / mmol).
[0071] 4. Ascorbic acid content: It was determined by the 2,6-dichlorophenol indophenol titration method. Weigh 5 g of grape samples and grind them in a mortar, then transfer them to a graduated cylinder. Add oxalic acid solution with a concentration of 20 g / L to make the volume up to 50 mL. After standing for 10 min, transfer it to a 50 mL centrifuge tube and centrifuge for 10 min (at 4 °C, 8000 rpm). Take 10 mL of the supernatant and add it to a conical flask, and titrate it with the calibrated 2,6-dichlorophenol indophenol solution. The titration end point is when the solution shows a faint pink color and does not fade within 15 s. At the same time, use 10 mL of 20 g / L oxalic acid solution as a blank control. Calculate the ascorbic acid content in grapes according to the consumption of the dye, expressed as the mass of ascorbic acid contained in every 100 g of the sample, that is, mg / 100 g.
[0072] Titration degree T = (c×V) / (V1 - V0)
[0073] Where c is the mass concentration of the ascorbic acid standard solution (mg / mL), V is the volume of the ascorbic acid standard solution absorbed (mL), V1 is the volume of the 2,6-dichlorophenol indophenol solution consumed when titrating the standard liquid (mL), and V0 is the volume of the 2,6-dichlorophenol indophenol solution consumed when titrating the blank (20 g / L oxalic acid) (mL).
[0074] Ascorbic acid content (mg / 100 g) = [V×(V1 - V0)×T] / (Vs×m)×100
[0075] Where V is the total volume of the sample extraction solution (mL), V1 is the volume of the dye consumed in the sample titration (mL), V0 is the volume of the dye consumed when titrating the blank (mL), Vs is the volume of the sample solution taken during titration (mL), and m is the sample mass (g).
[0076] 5. Hardness: Use a TA-XT2i physical property analyzer to measure the hardness of grapes. Select a P2 probe, the test speed is 1 mm / s, and the test depth is 5 mm. Select the equator of the grape for testing. The maximum resistance received when the probe inserts into the grape is recorded as the hardness (N).
[0077] II. Test results
[0078] It can be seen from Figure 3 that under the storage condition of 20 °C, the natural decay rate of grapes in the treatment group of Sporidiobolus pararoseus Y16 induced by cAMP was at the lowest level during the whole storage period. Among them, the decay rates at the 12th d and 18th d were 43% and 48.67% respectively, which were significantly lower than those of the control group (66%, 71.67%) and the treatment group of uninduced Sporidiobolus pararoseus Y16 (55.33%, 66.33%).
[0079] The grape quality indexes measured according to the above steps are shown in Table 1. During the entire storage period, there were no significant differences in the indexes such as the weight loss rate, soluble solids, titratable acids, ascorbic acid, and hardness of the grapes between the treatment group of Sporidiobolus pararoseus Y16 cultured with cAMP and the control group and the treatment group of uninduced Sporidiobolus pararoseus Y16. It can be seen that culturing with cAMP can improve the prevention and control effect of Sporidiobolus pararoseus Y16 on the natural decay of grapes and has no significant adverse effect on the grape quality.
[0080] Table 1 Effects of Sporidiobolus pararoseus Y16 cultured with cAMP on the storage quality of grapes
[0081]
[0082]
[0083] Note: CK is the control group, that is, grapes treated with sterile normal saline; Y is the grapes treated with uninduced Sporidiobolus pararoseus Y16 (1×10 8 cells / mL), and Y+C is the grapes treated with Sporidiobolus pararoseus Y16 (1×10 8 cells / mL) cultured with 0.05 mM cAMP; Different lowercase letters represent significant differences (P<0.05).
[0084] Explanation: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; Therefore, although this specification has described the present invention in detail with reference to the above respective embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their 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. A method for preventing and controlling post - harvest diseases and storing and preserving grapes by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate, characterized in that, The specific steps are as follows: (1) First, inoculate Sporidiobolus pararoseus Y16 into NYDB medium for the first culture to obtain a culture solution; then suck the culture solution and transfer it again into NYDB medium for the second culture to obtain a yeast culture solution; then transfer the yeast culture solution into NYDB medium containing cyclic adenosine monophosphate for induced culture. The cultured culture solution is centrifuged to obtain a bacterial sludge, which is centrifuged and washed with sterile normal saline. The washed bacterial sludge is resuspended with sterile normal saline again to obtain a Sporidiobolus pararoseus Y16 bacterial suspension; the final concentration of cyclic adenosine monophosphate in the NYDB medium is 0.05 - 0.5 mM; (2) Select grapes with uniform size, no obvious mechanical damage and diseases, and consistent maturity. Soak them in a disinfectant solution, then rinse with running water. Put the washed grapes into a disinfected plastic basket to dry. Punch holes on the surface of the dried grapes to form wounds of a certain size and depth. Inject an equal volume of the Sporidiobolus pararoseus Y16 bacterial suspension prepared in step (1) into each wound. After standing at room temperature for a period of time, inject an Aspergillus carbonarius spore suspension into each wound again. After drying, seal it with a supporting disinfected plastic lid and store it in a constant temperature and humidity incubator to control postharvest sour rot of grapes; Or select grapes without diseases, mechanical damage, with uniform color and similar size. Without any disinfection treatment, keep the natural condition of the grapes. Uniformly spray the Sporidiobolus pararoseus Y16 bacterial suspension prepared in step (1) on the surface of the grapes to achieve the prevention and control of postharvest diseases of grapes and the purpose of storage and preservation; The Sporidiobolus pararoseus Y16 is specifically Sporidiobolus pararoseus Y16, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is: CGMCC No. 2.5351.
2. The method for preventing and controlling postharvest diseases of grapes and storing and preserving them by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, characterized in that, The components of the NYDB medium described in step (1) are as follows, based on 1 L: 5 g of yeast extract, 10 g of glucose, 8 g of beef extract, made up to 1000 mL with distilled water, natural pH, sterilized at 115 °C for 20 min.
3. The method for preventing and controlling postharvest diseases of grapes and storing and preserving them by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, characterized in that, The conditions for the first culture and the second culture described in step (1) are both: temperature 28 °C, rotation speed 180 - 200 rpm, time 20 - 24 h; the temperature for induced culture is 28 °C, rotation speed 180 rpm, and time is 24 h.
4. The method for preventing and controlling postharvest diseases of grapes and storing and preserving freshness by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, wherein The inoculation amount inoculated into the NYDB medium in step (1) is 1% - 2%; the centrifugation conditions are: 20 °C, 8000 rpm, 10 min.
5. The method for preventing and controlling postharvest diseases of grapes and storing and preserving freshness by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, wherein In step (1), the final concentration of cyclic adenosine monophosphate in the NYDB medium is 0.05 mM, and the concentration of the suspension of **Sporidiobolus pararoseus** Y16 is 1×10 8 cells / mL.
6. The method for preventing and controlling postharvest diseases of grapes and storing and preserving them by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, characterized in that, The disinfectant solution in step (2) is a 0.5% sodium hypochlorite aqueous solution, and the soaking treatment time is 5 - 10 min.
7. The method for preventing and controlling postharvest diseases of grapes and storing and preserving fresh grapes by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, wherein, The disinfection operation of the plastic basket and the plastic lid in step (2) is: clean and dry, then immerse in a 1% sodium hypochlorite aqueous solution for 2 - 3 h, and dry at room temperature.
8. The method for preventing and controlling postharvest diseases of grapes and storing and preserving freshness by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, characterized in that The Sporidiobolus pararoseus Y16 bacterial suspension and the Aspergillus carbonarius spore suspension injected into each wound in step (2) are both 10 μL; the standing time for a period of time is 2 h.
9. The method for preventing and controlling postharvest diseases of grapes and storing and preserving freshness by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, wherein, The temperature for culturing in the constant temperature and humidity incubator in step (2) is 20 °C, and the relative humidity is 95%.
10. The method for preventing and controlling postharvest diseases of grapes and storing and preserving them by culturing Sporidiobolus salmonicolor Y16 induced by cyclic adenosine monophosphate according to claim 1, characterized in that, In step (2), the prepared Sporidiobolus salmonicolor Y16 bacterial suspension in step (1) is evenly sprayed on the surface of grapes, and the specific dosage is 0.5 - 1.5 mL of the bacterial suspension sprayed on each grape.