阅读说明：本技术 γ-氨基丁酸诱导培养拟粉红锁掷孢酵母Y16对葡萄采后病害防治的方法 (Method for preventing and treating postharvest diseases of grapes by gamma-aminobutyric acid induced culture of pseudopink Sporobolomyces Y16 ) 是由 张红印 许美秋 赵利娜 肖金玮 张晓云 杨其亚 王凯利 于 2021-07-13 设计创作，主要内容包括：本发明属于生物防治技术领域,具体涉及γ-氨基丁酸诱导培养拟粉红锁掷孢酵母Y16对葡萄采后病害防治的方法；步骤为：将酵母活化培养后,接种到添加有γ-氨基丁酸的NYDB培养基中进行诱导培养,离心得到菌体,用无菌水稀释为1×10～(8)cells/mL的菌悬液；在葡萄表面打孔后注入菌悬液,2h后注入等体积的塔宾曲霉孢子悬浮液,实现对塔宾曲霉的有效控制；或者直接将菌悬液均匀喷洒在葡萄表面,自然晾干,即可实现葡萄采后病害的防治和贮藏保鲜的用途；本发明可以有效控制葡萄采后由塔宾曲霉引起的病害和自然腐烂,同时对葡萄采后主要品质指标无不良显著影响,操作方便且安全环保,具有显著的经济效益和社会效益。(The invention belongs to the technical field of biological control, and particularly relates to a method for controlling postharvest diseases of grapes by using gamma-aminobutyric acid to induce and culture pseudopink Sporobolomyces Y16; the method comprises the following steps: activating yeast, culturing, inoculating to NYDB medium containing gamma-aminobutyric acid, inducing, culturing, centrifuging to obtain thallus, diluting with sterile water to 1 × 10 8 cell/mL of bacterial suspension; injecting bacterial suspension after punching holes on the surface of the grape, and injecting aspergillus tubingensis spore suspension with the same volume after 2 hours to realize effective control on aspergillus tubingensis; or directly and uniformly spraying the bacterial suspension on the surface of the grape, and naturally airing, so that the purposes of preventing and treating the postharvest diseases of the grape and storing and refreshing the grape can be realized; the invention can effectively control the disease and natural decay caused by the aspergillus tubingensis after the grapes are picked, and simultaneously, the grapes are pickedThe main quality index is not affected obviously, the operation is convenient, the safety and the environmental protection are realized, and the economic benefit and the social benefit are obvious.)

1. The method for preventing and controlling postharvest diseases of grapes by using gamma-aminobutyric acid to induce and culture pseudopink Sporobolomyces Y16 is characterized by comprising the following specific steps of:

activating and culturing Sporobolomyces pinkishinouye Y16 in NYDB culture medium to obtainActivating the culture solution; then NYDB is taken for culture, and gamma-aminobutyric acid is added to obtain a mixed culture solution; inoculating the activated culture solution into mixed culture solution, inducing culture, centrifuging to obtain thallus precipitate, washing thallus precipitate with sterile water for several times, and diluting to obtain 1 × 10⁸cells/mL yeast suspension; perforating the grape surface with aseptic perforator, injecting yeast suspension into each hole, inoculating Aspergillus tubingensis spore suspension with the same volume as the yeast suspension into each hole after a period of time, wherein the concentration of the Aspergillus tubingensis spore suspension is 1 × 10⁵spores/mL, and realizes effective prevention and control of grape diseases caused by Aspergillus tubingensis;

or the grapes are not treated, the grapes are placed in a plastic basket in a natural state, yeast suspension prepared by dilution after induced culture is uniformly sprayed on the surfaces of the grapes, the grapes are placed in a freshness protection bag after being naturally air-dried, then the grapes are placed in the plastic basket, and the grapes are sealed by a freshness protection film and then stored at room temperature, so that the prevention and control of the diseases after the grapes are picked, and the storage and freshness protection of the grapes can be realized;

the pink-lock Sporobolomyces Y16 is concretely pink-lock Sporomyces (Sporidiobolus pararosaeus) Y16, is preserved in China general microbiological culture Collection center, and has the preservation number: CGMCC No. 2.5351.

2. The method for preventing and treating postharvest diseases of grapes by using the gamma-aminobutyric acid for induction culture of the pinkish locksmithworm yeast Y16 according to claim 1, wherein the mass concentration of the gamma-aminobutyric acid in the mixed culture solution is 0.05% -1.6%.

3. The method for preventing and treating postharvest diseases of grapes by using gamma-aminobutyric acid to induce and culture pseudopink Sporobolomyces Y16 according to claim 1, wherein the NYDB culture medium comprises the following components in 1L: 5g of yeast extract, 10g of glucose, 8g of beef extract and distilled water are added to a constant volume of 1000mL, the pH is natural, and the mixture is sterilized at 115 ℃ for 20 min.

4. The method for preventing and treating postharvest diseases of grapes by using gamma-aminobutyric acid to induce and culture pseudopink Sporobolomyces Y16, according to claim 1, wherein the inoculation volume ratio of the activated culture inoculated in the mixed culture solution is 1-2: 100.

5. The method for preventing and treating postharvest diseases of grapes by using gamma-aminobutyric acid to culture the pinkish locky spore yeast Y16 in an induction way, according to claim 1, wherein the conditions of the activation culture and the induction culture are as follows: the temperature is 28 ℃, the rotating speed is 180-200 rpm, and the time is 20-24 h.

6. The method for preventing and treating postharvest diseases of grapes by using gamma-aminobutyric acid to culture pseudopink Sporobolomyces Y16 in an induction manner, according to claim 1, wherein the centrifugation conditions are as follows: the centrifugal force is 8000g, and the time is 10-15 min.

7. The method for controlling postharvest diseases of grapes through gamma-aminobutyric acid induced culture of pseudopink Sporobolomyces Y16, according to claim 1, wherein the diameter of the punched holes is 3mm, and the depth of the punched holes is 3 mm; the period of time is 2 hours.

8. The method for preventing and treating postharvest diseases of grapes by using gamma-aminobutyric acid for induced culture of pinkish rosette yeast Y16, according to claim 1, wherein the volumes of the yeast suspension and the Aspergillus tubingensis spore suspension injected into the hole are 10 μ L.

Technical Field

The invention belongs to the technical field of biological prevention and control of postharvest diseases of fruits, and particularly relates to a method for preventing and controlling postharvest diseases of grapes by using gamma-aminobutyric acid to induce and culture pseudopink Sporobolomyces Y16.

Background

Grape (Vitis vinifera L.) is one of the most consumed fruits in the world. They have high nutritional values, such as a wide variety of amino acids, sugars, organic acids and vitamins, and are well received by consumers. The grape planting area is wide and the variety is complete in China, the grape planting method is mainly used for brewing grape wine, making fruit wine, jam, preserved fruit and the like, and has great economic development value and wide market prospect. However, after picking, during storage and sale, fruit granules are easy to rot due to mechanical extrusion and pathogen infection, so that the fruit granules lose commodity value and directly cause huge economic loss to related practitioners. The postharvest diseases of grapes mainly include physiological diseases such as pulp browning, fruit cracking and freezing damage caused by fruit aging, environmental temperature, sulfur dioxide damage and the like, and invasive diseases such as gray mold, green rot, root mold, black spot and black powdery mildew caused by fungal infection of penicillium, aspergillus and rhizopus. Merlea (2015) et al report that Aspergillus tubingensis, one of the Aspergillus niger group species, is one of the major pathogens after grape harvest. Medina (2005) et al found that Aspergillus tubingensis has a strong pathogenic ability to grapes, and that ochratoxin A (OTA) is produced by grape infestation. Ochratoxin a is a ubiquitous mycotoxin and has nephrotoxicity, hepatotoxicity, teratogenic toxicity and immunotoxicity in animals. More seriously, ochratoxin A exists in grapes and grape products, such as wine, not only harms human health, but also brings great problems to the industries of grapes and grape products, and causes huge economic loss. Ochratoxin a contamination has been identified as a worldwide problem and the european union committee has set maximum allowable limits for ochratoxin a in certain food products.

At present, the control method aiming at the postharvest disease of the fruit mainly comprises a physical method, a chemical method and a biological control method. At present, the most common control method is chemical bactericide, which has the advantages of low price, high efficiency, convenience and the like, however, long-term use of the chemical bactericide can cause pathogenic bacteria to generate drug resistance, harm the environment and human health, and greatly limit the application of the chemical bactericide. The physical methods mainly comprise refrigeration, heat treatment (hot water treatment and hot air treatment), radiation and the like, can inhibit the activity and toxin accumulation of pathogenic bacteria to different degrees, but have great limitation in practical application due to the problems of high equipment cost, great industrialization difficulty, bad change of physicochemical properties and sensory quality of fruits caused by improper treatment and the like. The biological control method is to control the postharvest diseases of fruits by using antagonistic microorganisms (bacteria, mold and yeast), and compared with physical methods and chemical methods, the biological control method is safe and harmless, has no side effect and has good control effect. Compared with other antagonistic microorganisms, the antagonistic yeast has the advantages of rapid proliferation, no generation of antibiotics and mycotoxins, genetic stability, high safety and the like. The partially antagonistic yeast has good prevention and treatment effects on postharvest diseases of fruits, can degrade toxins generated by pathogenic bacteria, and has great significance for large-scale practical application of biological prevention and treatment. However, compared with chemical bactericides, the antagonistic microbe has lower control efficiency, and how to improve the biocontrol efficiency is an urgent problem to be solved.

Gamma-aminobutyric acid (GABA), white or near-white crystalline powder, is a natural non-protein amino acid and is widely distributed in animals, plants and microorganisms. At present, the production process of the gamma-aminobutyric acid mainly adopts a plant enrichment method and a microbiological method, has the advantages of mild conditions, high safety, good controllability and the like, and is widely applied to the food industry. However, no relevant report is found in the field of biological control, especially in the aspect of biological control of postharvest diseases of grapes.

Disclosure of Invention

The invention aims to improve the biocontrol efficiency of the Sporobolomyces roseus Y16(Sporidiobolus pararosaeus Y16) on postharvest diseases of grapes by utilizing gamma-aminobutyric acid to induce and culture the Sporobolomyces roseus Y16, reduce the loss caused by the postharvest diseases and have potential application value.

In order to achieve the above object, the present invention provides the following solutions:

the pseudopink locked Sporobolomyces Y16 is screened by the laboratory, has stronger antagonistic effect and is safe and harmless to human bodies. The gamma-aminobutyric acid is a natural non-protein amino acid, is widely applied and is non-toxic and harmless. The research proves that the gamma-aminobutyric acid induced culture of the pinkish locky Sporobolomyces Y16 can obviously improve the biocontrol effect on the postharvest diseases of the grapes, reduce the natural rotting rate of the grapes after the grapes are harvested, have no adverse effect on the quality of the grapes, achieve the aim of storing and preserving fruits and achieve the unexpected outstanding effect.

The preservation information of the Pink-like Sporobolomyces Y16 is as follows: the preservation name is: sporobolomyces pinkistroostii Y16(Sporidiobolus pararosaus Y16); the preservation number is: CGMCC No. 2.5351; the preservation unit: china general microbiological culture Collection center; and (4) storage address: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing; the preservation date is as follows: 7/17/2017.

The invention also provides a method for preventing and treating postharvest diseases of grapes by using the gamma-aminobutyric acid to induce and culture the pseudopink Sporobolomyces Y16, which comprises the following specific steps:

carrying out activated culture on the pinkish Sporobolomyces Y16 in an NYDB culture medium to obtain an activated culture solution; then NYDB is taken for culture, and gamma-aminobutyric acid is added to obtain a mixed culture solution; inoculating the activated culture solution into mixed culture solution, inducing culture, centrifuging to obtain thallus precipitate, washing thallus precipitate with sterile water for several times, and diluting to obtain 1 × 10⁸cells/mL yeast suspension; perforating the grape surface with aseptic perforator, injecting yeast suspension into each hole, inoculating Aspergillus tubingensis spore suspension with the same volume as the yeast suspension into each hole after a period of time, wherein the concentration of the Aspergillus tubingensis spore suspension is 1 × 10⁵spores/mL, and realizes effective prevention and control of diseases caused by aspergillus tubingensis;

or the grapes are not treated, the grapes are placed in a plastic basket in a natural state, yeast suspension prepared by dilution after induced culture is uniformly sprayed on the surfaces of the grapes, the grapes are placed in a freshness protection bag after being naturally air-dried, then the grapes are placed in the plastic basket, and the grapes are sealed by a freshness protection film and then stored at room temperature, so that the purposes of preventing and treating the diseases after the grapes are picked, storing and keeping freshness can be achieved.

Wherein the mass concentration of the gamma-aminobutyric acid in the mixed culture solution is 0.05-1.6%.

Wherein the NYDB culture medium (calculated by 1L) is as follows: 5g of yeast extract, 10g of glucose, 8g of beef extract and distilled water are added to a constant volume of 1000mL, the pH is natural, and the mixture is sterilized at 115 ℃ for 20 min.

Wherein the inoculation volume ratio of the activated culture inoculated in the mixed culture solution is 1-2: 100.

Wherein the conditions of the activation culture and the induction culture are as follows: the temperature is 28 ℃, the rotating speed is 180-200 rpm, and the time is 20-24 h.

Wherein the centrifugation conditions are as follows: the centrifugal force is 8000g, and the time is 10-15 min.

Wherein the diameter of the punched hole is 3mm, and the depth of the punched hole is 3 mm; the period of time is 2 hours.

Wherein the volumes of the yeast suspension and the Aspergillus tubingensis spore suspension injected into the holes are both 10 mu L.

The invention has the advantages that:

(1) the pseudopink locked Sporobolomyces Y16 used in the invention is screened by the laboratory, has strong antagonistic effect and is harmless to human body.

(2) The invention uses gamma-aminobutyric acid to induce and culture the pseudopink-lock Sporobolomyces Y16, compared with the traditional glucose culture medium, the antagonistic activity of the pseudopink-lock Sporomyces Y16 can be obviously improved, thereby reducing the natural rotting rate of the picked grapes, further achieving the purpose of storing and preserving the grapes and obtaining obvious effects.

(3) Compared with a control group, the gamma-aminobutyric acid induced and cultured pseudopink locked Sporobolomyces Y16 used in the invention has no any adverse significant influence on main quality indexes of harvested grapes, such as weight loss rate, hardness, soluble solid, titratable acid, soluble protein and ascorbic acid, and has the effect of slowing down the reduction of the quality indexes, such as the weight loss rate of grapes treated by yeast is significantly reduced.

(4) The rosette sporotrichum tosporum Y16 induced and cultured by gamma-aminobutyric acid can replace a chemical bactericide to prevent and treat postharvest diseases of grapes, avoid potential harm of the chemical bactericide to human bodies and the environment, reduce economic and energy burdens caused by physical sterilization and storage, and have remarkable economic and social benefits.

Drawings

FIG. 1 is a graph showing the effect of Sporobolomyces rosepinorum Y16 after induction culture of mixed culture solution containing gamma-aminobutyric acid in different mass concentrations on the rate of disease rot caused by Aspergillus tubingensis after grape harvest; note: CK represents sterile water treatment, and is a control group; y represents Pinolopsis pininosa Y16 (yeast concentration is 1X 10)⁸cells/mL); YA, YB, YC, YD, YE and YF are respectively gamma-aminobutyric acid induction cultured pseudopink Sporobolomyces Y16 (yeast concentration is 1 × 10) with mass concentration of 0.05%, 0.1%, 0.2%, 0.4%, 0.8% and 1.6%⁸cells/mL); different lower case letters represent significant differences between treatments (P)<0.05)。

FIG. 2 is a graph showing the effect of Sporobolomyces rosepinorum Y16 after induction culture of mixed culture solution containing gamma-aminobutyric acid in different mass concentrations on the diameter of disease rot caused by Aspergillus tubingensis after grape harvest. Note: CK represents sterile water treatment, and is a control group; y represents Pinolopsis pininosa Y16 (concentration 1X 10) cultured without induction of mixed culture solution⁸cells/mL); YA, YB, YC, YD, YE and YF are respectively gamma-aminobutyric acid induction cultured pseudopink Sporobolomyces Y16 (concentration is 1 × 10) with mass concentration of 0.05%, 0.1%, 0.2%, 0.4%, 0.8% and 1.6%⁸cells/mL); different lower case letters represent significant differences between treatments (P)<0.05)。

The specific implementation mode is as follows:

the invention will be explained in more detail by means of the following examples; the following examples are illustrative only, and the present invention is not limited by these examples.

Example 1:

sporidiobolus pararoseus Y16(Sporidiobolus pararosaus Y16) is a safe and nontoxic antagonistic yeast screened from Zhenjiang sentence-tolerant vineyard in the laboratory. Is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number as follows: CGMCC No.2.5351 (based on the applied patent of the yeast, CN201710753559.5 strain of yeast for controlling penicilliosis of apple after harvest and application thereof).

The culture procedure is as follows: (1) liquid activation: inoculating Pink-like Sporobolomyces Y16 in NYDB culture medium, and culturing at 180rpm and 28 deg.C for 24 hr to obtain activated culture solution; (2) and (3) induction culture: the culture can be carried out using the step a or the step b; a. centrifuging the activated culture solution (8000 Xg, 10min), collecting thallus, washing with sterile water repeatedly to remove culture medium, adding appropriate amount of sterile distilled water, counting with blood cell counting plate, and adjusting yeast suspension concentration to 5X 10⁸cells/mL. Respectively filling 50mL of NYDB culture medium into 250mL triangular flasks, adding gamma-aminobutyric acid to ensure that the mass concentration of the gamma-aminobutyric acid in the NYDB is 0.05%, 0.1%, 0.2%, 0.4%, 0.8% and 1.6% in sequence, adding 1mL of yeast suspension with the concentration, and culturing at 180rpm and 28 ℃ for 24 hours; b. respectively filling 50mL of NYDB culture medium into 250mL of triangular flasks, adding gamma-aminobutyric acid to ensure that the mass concentration of the gamma-aminobutyric acid in the NYDB is 0.05%, 0.1%, 0.2%, 0.4%, 0.8% and 1.6% in sequence, adding 1mL of activated culture solution, and culturing at 180rpm and 28 ℃ for 24 hours; (3) after induction culture, respectively centrifuging (8000 Xg, 10min) the yeast culture medium with different concentrations of gamma-aminobutyric acid for induction treatment to collect thallus, washing with sterile distilled water for 3 times to remove the culture medium, re-suspending with sterile distilled water, and adjusting the yeast cell concentration to 1X 10 with a blood counting chamber⁸cells/mL。

Example 2:

the control effect of the gamma-aminobutyric acid induced pink locked Sporobolomyces Y16 on diseases caused by Aspergillus tubingensis after grape harvest.

Test protocol

The test variety is grape (vitas vinifera L.), and the selected grape has good commercial maturity, no damage on the surface, no infection and consistent specification. The surface was rinsed with tap water, then disinfected with 0.2% (v/v) sodium hypochlorite (NaClO) for 2min, rinsed with tap water to remove residual sodium hypochlorite, and placed in a plastic basket to dry for use.

The mould activation steps before use are as follows: (1) 1mL of the mold spore suspension was inoculated into sterilized PDB medium and cultured for 24 hours on a constant temperature shaker (120rpm, 25 ℃). (2) Taking a proper amount of mould to scribe on a PDA culture medium, culturing in a constant temperature and humidity box for 7d, taking mould spores on a flat plate, washing twice in sterile physiological saline, and adjusting to the required concentration by adopting a binocular microscope.

1 wound (diameter 3 mm. times. depth 3mm) was made at the equator of each grape using a sterile punch, and 10. mu.L of (1) yeast suspension cultured in NYDB medium was injected into each wound; (2) yeast suspension subjected to induction culture by using a gamma-aminobutyric acid supplementary culture medium; (3) sterile water was used as a control. After standing at room temperature for 2 hours, 10. mu.L of Aspergillus tubingensis spore suspension (1X 10) was injected into the wound⁵spores/mL); airing at room temperature, placing in a constant-temperature constant-humidity incubator (20 ℃, RH 95 percent) for storage, observing the rotting condition of the pomes after 5d, and recording the rotting rate and the rotting diameter; each treatment was performed in 3 replicates of 18 grapes each, and the experiment was repeated twice.

The formula for the incidence of disease is as follows:

incidence (%) of disease ═ total number of fruits/fruit affected × 100%

(II) test results

According to the test steps, the results of counting the rotting rate and the lesion diameter of the fruits are as follows:

as shown in fig. 1, treatment with uninduced pinolopsis Y16 and treatment with gamma-aminobutyric acid-induced cultured pinolopsis Y16 both reduced the incidence of postharvest disease in grapes, but gamma-aminobutyric acid-induced pinolopsis Y16 was more effective in inhibiting aspergillus tubingensis than pinolopsis Y16 alone; the induction effect of 0.4 percent of gamma-aminobutyric acid on the pink-locking Sporobolomyces Y16 is particularly remarkable. After storage for 5 days, the decay rate of the control group reached 100%, the decay rate of the pink-lock Sporobolomyces Y16 alone (not induced) also exceeded 50%, while the incidence of the 0.4% gamma-aminobutyric acid-induced pink-lock Sporomyces Y16 treated group was only 16.67%.

As shown in FIG. 2, the diameter of the grape lesion of the sterile water-treated control group was 11.98mm, whereas the diameter of the grape lesion of the 0.4% GABA-induced Sporobolomyces pinkishinouye Y16-treated group was only 7.08 mm.

Example 3:

influence of gamma-aminobutyric acid induced culture of pseudopink locked Sporobolomyces Y16 on natural decay and storage quality of picked grapes

Test protocol

Selecting grapes which are free of mechanical damage, uniform in size and normal in commercial maturity for testing, keeping the grapes in a natural state after picking, dividing the grapes into three groups, and directly spraying (1) yeast suspension (Y) cultured by an NYDB culture medium on the surface of the grapes; (2) yeast suspension (Y + GABA) induced by gamma-aminobutyric acid supplemented medium; (3) sterile water (as control, CK). After the grapes are dried at room temperature, the grapes are independently packaged by a freshness protection package, then the grapes are placed in a plastic basket, sealed by a freshness protection film and placed in a constant temperature and humidity incubator (20 ℃ and RH 95%) for storage, and after 9 days, the related indexes of the natural morbidity and the natural storage quality of the fruits are measured. Each treatment was performed in 3 replicates, each with 18 grapes, and the experiment was repeated three times. The inhibitory effect of gamma-aminobutyric acid induced Pink-like Sporobolomyces rosenbergii Y16 on the natural rot of grapes was expressed as morbidity.

(1) The formula for the incidence of disease is as follows:

incidence (%) of disease ═ total number of fruits/fruit affected × 100%

(2) The storage quality was measured as follows:

1. weight loss rate: weight loss rate (mass before treatment-mass after storage)/mass before treatment × 100%

2. Soluble Solids (TSS): the measurement was carried out by a hand-held sugar meter, and the measurement result was expressed as g/100g of sample. The experiment was repeated three times.

3. Titratable Acid (TA): homogenizing 20g pulp tissue with 30mL distilled water, leaching in 80 deg.C water bath for 30min, diluting to 100mL, and filtering to obtain filtrate. 20mL of the filtrate was taken, 2-5 drops of phenolphthalein indicator were added dropwise, and the titration was performed with a standard solution (0.1M NaOH) until the solution became pink and did not fade within 0.5min, which was taken as the end point of the titration, and the results were expressed as percentage of tartaric acid. The experiment was repeated three times.

4. Soluble protein: weighing 2g of grape tissue sample, adding 5mL of distilled water or buffer solution, grinding into homogenate, centrifuging at 4 ℃ and 12000 rpm for 20min, and collecting supernatant, namely the crude extract of soluble protein. Sucking 1mL of sample, extracting supernatant (diluting properly according to protein content), placing into a test tube with a plug, adding 5mL of Coomassie brilliant blue G-250 solution, mixing thoroughly, standing for 2min, performing color comparison at 595nm, and determining absorbance value according to the same method for preparing standard curve. This was repeated three times. The results are expressed in terms of the mass of soluble protein contained per gram of grape pulp, i.e. mg/g.

5. Ascorbic acid (Vc): and (3) mixing a 20g fruit sample with a small amount of 20g/L oxalic acid solution, grinding in an ice bath, homogenizing, transferring to a 50mL volumetric flask, metering to a scale with oxalic acid, and extracting for 10 min. Centrifuging at 10000 rpm and 4 deg.C for 10min, and collecting supernatant for titration. 20mL of the supernatant was pipetted into a 100mL Erlenmeyer flask and titrated with a calibrated 2, 6-dichlorophenol indophenol solution until reddish color appeared and fading did not occur for 15 seconds, and the dye usage was recorded, using 20mL of oxalic acid as a blank. This was repeated three times. The results are expressed in terms of the mass of ascorbic acid contained in 100g of sample (fresh weight), i.e., mg/100 g.

6. Hardness (N): the test is carried out by adopting a TA-XT2 Plus texture analyzer, a P2 probe is selected, parameters are set to be in a pressure measurement mode, the speed is 1mm/s, and the test depth is 3 mm. Three points equidistant at the equator were selected for testing. The maximum resistance (in N) to which the probe is inserted into a grape is defined as the stiffness of the grape. The experiment was repeated three times.

(II) test results

TABLE 1 influence of gamma-aminobutyric acid induced Sporobolomyces pinkishinouye Y16 on the Natural rot Rate and storage quality of grapes

Note: CK is a control group treated with sterile water, Y represents Sporobolomyces pinkishinouye Y16 treatment, GABA + Y represents gamma-aminobutyric acid (0.4%) induction Sporobolomyces pinkishinouye Y16 treatment group. Different lower case letters represent significance of difference (P < 0.05).

As shown in Table 1, after being stored at 20 ℃ for 9 days, the incidence rate of natural diseases of grapes treated by the pink locked Sporobolomyces Y16 induced by gamma-aminobutyric acid (0.4%) is remarkably reduced to only 5.56%, the effect is very remarkable, and an unexpected effect is generated; the incidence of natural disease in grapes treated with Pink-like Sporobolomyces roseus Y16 was 12.5%, while the incidence of natural disease in grapes sprayed with sterile water was 30.56%. There were no significant adverse changes in storage quality, such as weight loss rate, hardness, soluble solids, titratable acids, soluble proteins, and ascorbic acid, compared to the control. Therefore, it is considered that induction of the pink-locking Sporobolomyces Y16 by gamma-aminobutyric acid (0.4%) can reduce the rotting rate of grapes without significantly adversely affecting the storage quality of grapes.

Preservation of Sporobolomyces pinkishinouye Y16:

optionally, NYDA culture medium can be stored at 4 deg.C in refrigerator, or yeast can be stored at-80 deg.C in 50% glycerol tube for a long period.

Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations that do not depart from the spirit and scope of the invention are intended to be included within the scope of the appended claims.