Composite antibiological inoculant for preventing and treating fruit and vegetable diseases as well as preparation method and application of composite antibiological inoculant

Abstract

The invention provides a composite biocontrol inoculant for preventing and treating fruit and vegetable diseases as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The liquid compound microbial agent is prepared from the following raw materials: Meyerozyma caribba, Metschnikowia zizyphicola Y-3, Pichia rassica, and an additive, and is characterized in that the liquid compound microbial agent is prepared from the following raw materials: Meyerozyma caribba, Metschnikowia zizyphicola Y-3, Pichia rassica, and an additive. The liquid fungicide has the advantages of simple preparation method, easiness in storage, convenience in transportation, short starting time and the like, can achieve the purposes of quickly starting a reactor and improving the treatment efficiency, and has great development potential and application prospect in practice.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a composite biocontrol agent for preventing and treating fruit and vegetable diseases, and a preparation method and application thereof. Background Art

[0002] Postharvest damage to fruits and vegetables often results in significant economic losses due to various factors. In recent years, biological antagonists have been widely used in postharvest pest control. Yeasts have become a key component of postharvest pest control research due to their effective antagonistic properties, lack of toxin production, and compatibility with chemical fungicides.

[0003] Under natural conditions, antagonistic yeasts often lose their biocontrol effectiveness or even vitality due to adverse conditions such as oxidative stress, extreme temperatures, and pH imbalances. Prolonging their survival time to ensure their effectiveness during post-harvest storage, transportation, and marketing of fruits and vegetables is a pressing issue. Therefore, developing commercially viable yeast agents is crucial. Summary of the Invention

[0004] The embodiments of the present disclosure aim to solve at least one of the technical problems existing in the prior art, and disclose a composite biocontrol agent for preventing and controlling fruit and vegetable diseases, as well as a preparation method and application.

[0005] In one aspect of the present disclosure, a composite biocontrol agent for preventing and controlling fruit and vegetable diseases is provided, wherein the composite biocontrol agent comprises: Meyerozyma caribbica, Metschnikowiazizyphicola Y-3, Laras

[0006] Pichia rarassimilans and additives; wherein,

[0007] The Meyerozyma caribbica was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on June 12, 2023, with the deposit number being CGMCC No. 27604.

[0008] The Metschnikowia zizyphicola Y-3 was deposited in the China Center for Type Culture Collection on October 17, 2016, with a deposit number of CCTCC NO: M 2016564.

[0009] The Pichia rarassimilans was deposited in the General Microbiology Center of the China Culture Collection Administration on December 2, 2024, and its deposit number is CGMCC No.32875.

[0010] Optionally, the mass ratio of the Meyerozyma caribbica, the Metschnikowia zizyphicola Y-3, and the Pichia rarassimilans is 1:2:3.

[0011] Optionally, the additives include thickeners, emulsifiers and antioxidants.

[0012] Optionally, the thickener is any one of gum arabic with a concentration of 0.3%, gum arabic with a concentration of 0.6%, sodium alginate with a concentration of 0.3%, sodium alginate with a concentration of 0.6%, agar with a concentration of 0.3%, and agar with a concentration of 0.6%; and / or,

[0013] The emulsifier is any one of Tween 20 with a concentration of 0.2%, Tween 20 with a concentration of 0.4%, Tween 80 with a concentration of 0.2%, and Tween 80 with a concentration of 0.4%; and / or

[0014] The antioxidants include ascorbic acid with a concentration of 0.03%, ascorbic acid with a concentration of 0.06%, tea polyphenol with a concentration of 0.03%, tea polyphenol with a concentration of 0.06%, 2,6-di-tert-butyl-p-cresol with a concentration of 0.03%, and 2,6-di-tert-butyl-p-cresol with a concentration of 0.06%.

[0015] Optionally, the thickener is 0.3% gum arabic; and / or,

[0016] The emulsifier is Tween 80 with a concentration of 0.4%; and / or,

[0017] The antioxidant is ascorbic acid with a concentration of 0.06%.

[0018] The present disclosure provides a method for preparing the composite biocontrol agent described above, the method comprising:

[0019] Meyerozyma caribbica, Metschnikowiazizyphicola Y-3, and Pichia rarassimilans are used as a mixed seed liquid, the mixed seed liquid is purified and inoculated into a liquid culture medium, and a mixed bacterial suspension is formed through culturing and preparation;

[0020] Additives are added to the mixed bacterial suspension to prepare a composite biocontrol agent.

[0021] Optionally, the volume ratio of the mixed bacterial suspension to the additive is 1:1.

[0022] Optionally, the liquid culture medium comprises 10-20 g / L of a carbon source, 10-20 g / L of a nitrogen source, and 0.2-1.8 g / L of an inorganic salt.

[0023] Optionally, the carbon source is sucrose, and the concentration of the sucrose is 15 g / L; and / or, the nitrogen source is yeast extract powder, and the concentration of the yeast extract powder is 15 g / L; and / or, the inorganic salts are dipotassium hydrogen phosphate and sodium chloride, and the concentration of the dipotassium hydrogen phosphate is 1 g / L, and the concentration of the sodium chloride is 1 g / L; and / or,

[0024] The inoculation amount of the mixed seed liquid is 0.5-4%, the pH value during the culture process is 5-9, the culture temperature is 20-36° C., and the culture time is 24-72 hours.

[0025] Another aspect of the present disclosure provides an application of a composite biocontrol agent, wherein the composite biocontrol agent described above is used to control at least one of gray mold, penicillium, black spot, and aspergillosis of fruits and vegetables.

[0026] The present disclosure proposes a composite biocontrol agent for preventing and controlling fruit and vegetable diseases, as well as its preparation method and application. The liquid composite microbial agent has the advantages of simple preparation method, easy storage, convenient transportation, and short startup time. It can achieve the purpose of quickly starting the reactor and improving treatment efficiency, and has great development potential and application prospects in practice.

[0027] Preservation Instructions

[0028] Depository: General Microbiology Center, China Culture Collection Administration;

[0029] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0030] Deposit date: June 12, 2023;

[0031] Deposit number: CGMCC No.27604;

[0032] Classification and nomenclature of biological materials: Meyer yeast Kalibik;

[0033] Latin name of the biomaterial: Meyerozyma caribbica.

[0034] Depository: China Center for Type Culture Collection;

[0035] Deposit address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0036] Deposit date: October 17, 2016;

[0037] Deposit number: CCTCC NO: M 2016564;

[0038] Classification and nomenclature of biological materials: Metschnikowia;

[0039] The Latin name of the biomaterial: Metschnikowia zizyphicola Y-3.

[0040] Depository: General Microbiology Center, China Culture Collection Administration;

[0041] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0042] Deposit date: December 2, 2024;

[0043] Deposit number: CGMCC No.32875;

[0044] Classification and nomenclature of biological materials: Pichia laris;

[0045] The Latin name of the biomaterial: Pichia rarassimilans. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the drawings, the same or similar reference numerals represent the same or similar elements.

[0047] Figure 1 This is a flowchart of a method for preparing a composite biocontrol agent according to a specific embodiment of the present disclosure;

[0048] Figure 2 Schematic diagram of the effect of carbon source and optimal carbon source concentration on mixed bacterial culture in Example 1 of the present disclosure; wherein, Figure 2 (a) is the result of mixed culture when using different carbon sources. Figure 2 (b) shows the results of mixed culture with different concentrations of sucrose;

[0049] Figure 3 Schematic diagram of the effect of nitrogen source and optimal nitrogen source concentration on mixed bacterial culture in Example 2 of the present disclosure; wherein, Figure 3 (a) is the result of mixed culture when different nitrogen sources are used. Figure 3 (b) shows the results of mixed culture when different concentrations of yeast extract powder were used;

[0050] Figure 4 Schematic diagram of the effects of inorganic salts and two optimal inorganic salt concentrations on mixed bacterial culture in Example 3 of the present disclosure; wherein, Figure 4 (a) is the result of mixed bacterial culture when different inorganic salts are used. Figure 4 (b) shows the results of mixed bacterial culture when using different concentrations of dipotassium hydrogen phosphate; Figure 4 (c) shows the results of mixed bacterial culture when different concentrations of sodium chloride and potassium hydrogen phosphate were used;

[0051] Figure 5 Schematic diagram of the effects of pH value, inoculation amount, culture temperature and culture time on mixed culture in Example 5 of the present disclosure; wherein, Figure 5 (a) is the result of mixed culture at different pH values. Figure 5 (b) shows the results of mixed culture with different inoculation amounts; Figure 5 (c) shows the results of mixed culture at different temperatures; Figure 5 (d) shows the results of mixed culture with different culture times;

[0052] Figure 6 This is a schematic diagram of the effects of different additives on the bacterial agent in Example 6 of the present disclosure; Figure 6 (a) shows the effect of different thickeners on bacterial agents; Figure 6 (b) shows the effects of different emulsifiers on bacterial agents; Figure 6 (c) shows the effects of different antioxidants on bacterial agents;

[0053] Figure 7 Schematic diagram of the antibacterial effect of different concentrations of the composite biocontrol agent on strawberry gray mold and blue mold in Example 7 of the present disclosure;

[0054] Figure 8 Schematic diagram of the antibacterial effect of composite biocontrol agents of different concentrations on pear gray mold and black spot disease in Example 8 of the present disclosure;

[0055] Figure 9 Schematic diagram of the antibacterial effect of composite biocontrol agents of different concentrations on gray mold and black spot of winter jujube according to Example 9 of the present disclosure;

[0056] Figure 10 Schematic diagram of the antibacterial effect of the composite biocontrol agent of different concentrations on gray mold and blue mold of kiwifruit according to Example 10 of the present disclosure;

[0057] Figure 11 Schematic diagram of the antibacterial effect of the composite biocontrol agent of different concentrations on blueberry aspergillus and gray mold in Example 11 of the present disclosure;

[0058] Figure 12Schematic diagram of the antibacterial effect of the composite biocontrol agent of different concentrations on cucumber aspergillus and gray mold in Example 12 of the present disclosure;

[0059] Figure 13 Schematic diagram of the antibacterial effect of different concentrations of the composite biocontrol agent on apple blue mold in Example 13 of the present disclosure;

[0060] Figure 14 Schematic diagram of the antibacterial effect of different concentrations of the composite biocontrol agent on orange Penicillium odorifera in Example 14 of the present disclosure;

[0061] Figure 15 Schematic diagram of the antibacterial effect of different concentrations of the composite biocontrol agent on tomato gray mold and blue mold according to Example 15 of the present disclosure. DETAILED DESCRIPTION

[0062] In order to enable those skilled in the art to better understand the technical solutions of the present disclosure, the present disclosure is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0063] In one aspect of the present disclosure, a composite biocontrol agent for preventing and controlling fruit and vegetable diseases is provided, the composite biocontrol agent comprising: Meyerozyma caribbica, Metschnikowia zizyphicola Y-3, Pichia rarassimilans, and an additive; wherein Meyerozyma caribbica Y-3 was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms on June 12, 2023, with a deposit number of CGMCC No. 27604; the Metschnikowia zizyphicola Y-3 was deposited in the China Center for Type Culture Collection on October 17, 2016, with a deposit number of CCTCC NO: M 2016564; the Pichia rarassimilans was deposited in the China Center for Type Culture Collection on October 17, 2016, with a deposit number of CCTCC NO: M 2016564; the Pichia rarassimilans was deposited in the China Center for Type Culture Collection on October 17, 2016, with a deposit number of CCTCC NO: M 2016564. rarassimilans) was deposited in the General Microbiology Center of China Culture Collection Administration on December 2, 2024, and its deposit number is CGMCC No.32875.

[0064] The liquid composite microbial agent of this embodiment is formed by a composite of multiple antagonistic bacteria, which can control the occurrence and spread of plant diseases, reduce the rot of fruits and vegetables caused by pathogens, and reduce the loss of agricultural product yield and quality. It has the advantages of easy storage, convenient transportation, short startup time, and long survival time. It can achieve the purpose of quickly starting the reactor and improving treatment efficiency. In practice, it has great development potential and application prospects and meets the needs of commercial applications.

[0065] In some preferred embodiments, the mass ratio of Meyerozyma caribbica, Metschnikowia zizyphicola Y-3, and Pichia rarassimilans is 1:2:3.

[0066] In other preferred embodiments, the additives include thickeners, emulsifiers, and antioxidants.

[0067] As a further preferred embodiment, the thickener is any one of gum arabic at a concentration of 0.3%, gum arabic at a concentration of 0.6%, sodium alginate at a concentration of 0.3%, sodium alginate at a concentration of 0.6%, agar at a concentration of 0.3%, and agar at a concentration of 0.6%. For example, the thickener may preferably be gum arabic at a concentration of 0.3%;

[0068] As a further preferred embodiment, the emulsifier is any one of 0.2% Tween 20, 0.4% Tween 20, 0.2% Tween 80, and 0.4% Tween 80. For example, the emulsifier may preferably be 0.4% Tween 80.

[0069] As a further preferred embodiment, the antioxidants include ascorbic acid at a concentration of 0.03%, ascorbic acid at a concentration of 0.06%, tea polyphenols at a concentration of 0.03%, tea polyphenols at a concentration of 0.06%, 2,6-di-tert-butyl-p-cresol at a concentration of 0.03%, and 2,6-di-tert-butyl-p-cresol at a concentration of 0.06%. For example, the antioxidant may preferably be ascorbic acid at a concentration of 0.06%.

[0070] like Figure 1 As shown, another aspect of the present disclosure provides a method S100 for preparing the composite biocontrol agent described above, the method comprising the following specific steps S110-S120:

[0071] S110. Meyerozyma caribbica, Metschnikowia zizyphicola Y-3, and Pichia rarassimilans were used as a mixed seed solution, which was purified and inoculated into NYDB liquid medium. The mixture was cultured at 20-36°C and 180 rpm to prepare the seed solution. The solution was diluted with sterile water to 1×10 8 cfu / mL of mixed bacterial suspension for later use.

[0072] In some preferred embodiments, the inoculation amount of the mixed seed solution is 0.5-4%, the pH of the culture process is 5-9, the culture temperature is 20-36° C., and the culture time is 24-72 h.

[0073] As a further preferred solution, the inoculation amount of the mixed seed solution may be preferably 1%, the pH of the culture process may be preferably 7, the culture temperature may be 24° C., and the culture time may be 60 h.

[0074] In other preferred embodiments, the NYDB liquid culture medium comprises 10-20 g / L of a carbon source, 10-20 g / L of a nitrogen source, and 0.2-1.8 g / L of an inorganic salt.

[0075] As a further preferred embodiment, the carbon source is sucrose, glucose, lactose, molasses, soluble starch, etc. For example, sucrose is preferred, and the concentration of sucrose is preferably 15 g / L.

[0076] As a further preferred embodiment, the nitrogen source is yeast extract powder, yeast extract, beef extract, tryptone, ammonium sulfate, ammonium chloride, urea, etc. For example, yeast extract powder is preferred, and the concentration of the yeast extract powder is preferably 15 g / L.

[0077] As a further preferred embodiment, the inorganic salt is dipotassium hydrogen phosphate, sodium chloride, potassium dihydrogen sulfate, magnesium sulfate, zinc sulfate, sodium chloride, potassium sulfate, etc. For example, dipotassium hydrogen phosphate and sodium chloride are preferred, and the concentration of dipotassium hydrogen phosphate is 1 g / L, and the concentration of sodium chloride is 1 g / L.

[0078] S120, adding additives to the mixed bacterial suspension to form a composite biocontrol agent.

[0079] In some preferred embodiments, the volume ratio of the mixed bacterial suspension to the additive is 1: 1. For example, the volume of the mixed bacterial suspension and the additive can be preferably 1 mL.

[0080] In step S120 , the additives include a thickener, an emulsifier, and an antioxidant.

[0081] As a further preferred embodiment, the thickener is any one of gum arabic at a concentration of 0.3%, gum arabic at a concentration of 0.6%, sodium alginate at a concentration of 0.3%, sodium alginate at a concentration of 0.6%, agar at a concentration of 0.3%, and agar at a concentration of 0.6%. For example, the thickener may preferably be gum arabic at a concentration of 0.3%;

[0082] As a further preferred embodiment, the emulsifier is any one of 0.2% Tween 20, 0.4% Tween 20, 0.2% Tween 80, and 0.4% Tween 80. For example, the emulsifier may preferably be 0.4% Tween 80.

[0083] As a further preferred embodiment, the antioxidants include ascorbic acid at a concentration of 0.03%, ascorbic acid at a concentration of 0.06%, tea polyphenols at a concentration of 0.03%, tea polyphenols at a concentration of 0.06%, 2,6-di-tert-butyl-p-cresol at a concentration of 0.03%, and 2,6-di-tert-butyl-p-cresol at a concentration of 0.06%. For example, the antioxidant may preferably be ascorbic acid at a concentration of 0.06%.

[0084] The preparation method disclosed in the present invention is simple, and the obtained composite biocontrol agent is liquid, can be stored, is convenient to transport, is not likely to lose its biocontrol effect under adverse conditions, and has a long survival time.

[0085] Another aspect of the present disclosure provides an application of a composite biocontrol agent, wherein the composite biocontrol agent described above is used to control at least one of gray mold, penicillium, black spot, and aspergillus in fruits and vegetables.

[0086] The liquid composite biocontrol agent of this embodiment can prevent and control a variety of post-harvest diseases of fruits and vegetables, has a high inhibition rate, is easy to rehydrate, and is convenient to transport.

[0087] The preparation and application of the composite biocontrol agent will be described below with reference to specific examples:

[0088] It should be noted that the Meyerozyma aribbica, Metschnikowia zizyphicola Y-3, and Pichia pastoris used in the following examples are antagonistic bacteria screened by the applicant in the early stage. For specific deposit information, please refer to the above record.

[0089] Example 1

[0090] This example uses the effect of carbon source on mixed bacterial culture as an example to illustrate the results:

[0091] Meyerozyma caribbica, Metschnikowia zizyphicola Y-3, and Pichia rarassimilans were used as a mixed seed liquid in a mass ratio of 1:2:3. The purified mixed seed liquid was inoculated into a liquid culture medium at a 2% inoculum size, and cultured in a shaking incubator at 28°C and 180 rpm for 48 h. A bacterial suspension of 1×108 cfu / mL was prepared with sterile water using a hemocytometer method for standby use to form a mixed bacterial suspension.

[0092] Among them, 15g / L YNB was used as the basic nitrogen source, and 10g / L of different carbon sources were added as the basal culture medium. The different carbon sources were glucose, molasses, lactose, sucrose, dextrin, and soluble starch, and they were screened; after selecting the optimal carbon source, the carbon source concentration was screened, which were 5g / L, 10g / L, 15g / L, 20g / L, and 25g / L respectively.

[0093] Effects of different carbon sources and optimal carbon source concentrations on mixed bacterial culture Figure 2 When sucrose was used as the carbon source, the viable bacterial count in the fermentation broth was significantly higher than with other carbon sources, reaching 8.77 lg cfu / mL. Therefore, sucrose was selected as the carbon source for the mixed fermentation broth. The highest viable bacterial count, reaching 8.82 lg cfu / mL, was achieved at a sucrose concentration of 15 g / L.

[0094] Example 2

[0095] This example uses the effect of nitrogen source on mixed bacterial culture as an example to illustrate the results:

[0096] The method of this example is the same as that of Example 1, wherein the basal culture medium uses 15 g / L sucrose as the carbon source, and 15 g / L of different nitrogen sources are added for nitrogen source screening. The different nitrogen sources are yeast extract powder, yeast extract, beef extract, tryptone, ammonium sulfate, ammonium chloride, and urea. After selecting the optimal nitrogen source, the nitrogen source concentration is screened, and the concentrations are 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 25 g / L, respectively.

[0097] Effects of different types of nitrogen sources and optimal nitrogen source concentrations on mixed bacterial culture Figure 3 When yeast extract powder was used as the nitrogen source, the viable bacterial count in the fermentation broth was significantly higher than with other nitrogen sources, reaching 9.03 lg cfu / mL. Therefore, yeast extract powder was selected as the nitrogen source for the mixed fermentation broth. The highest viable bacterial count, reaching 9.33 lg cfu / mL, was achieved at a yeast extract powder concentration of 15 g / L.

[0098] Example 3

[0099] This example uses the effect of inorganic salts on mixed bacterial culture as an example to illustrate the results:

[0100] The method of this example is the same as that of Example 1, wherein the optimal concentration of carbon source and the optimal concentration of nitrogen source are used as the basal culture medium, and 5 g / L of 7 inorganic salts are added to screen the optimal inorganic salts. That is, the liquid culture medium includes the basal culture medium and inorganic salts, wherein the inorganic salts are potassium dihydrogen phosphate, dipotassium hydrogen phosphate, manganese sulfate, magnesium sulfate, zinc sulfate, sodium chloride, and potassium sulfate. Finally, the optimal inorganic salt is selected for screening the inorganic salt concentration, and the concentrations are 0.1 g / L, 0.5 g / L, 1 g / L, 3 g / L, 5 g / L, 7 g / L, and 9 g / L, respectively. Each treatment is repeated 3 times.

[0101] Effects of different types of inorganic salts and two optimal inorganic salt concentrations on mixed bacterial culture Figure 4 As shown, when dipotassium hydrogen phosphate and sodium chloride are used as inorganic salts, the viable bacterial count in the fermentation broth is significantly higher than that of other inorganic salts, reaching 9.17 lg cfu / mL and 9.14 lg cfu / mL, respectively. Therefore, dipotassium hydrogen phosphate and sodium chloride are selected as inorganic salts for mixed fermentation broth culture. The viable bacterial count is highest when the concentration of dipotassium hydrogen phosphate is 1 g / L, reaching 9.30 lg cfu / mL, while the viable bacterial count is highest when the concentration of sodium chloride is 1 g / L, reaching 9.20 lg cfu / mL.

[0102] Example 4

[0103] This example uses the optimization of the optimal culture medium composition for mixed bacterial culture as an example to illustrate the results:

[0104] Based on the above research results, we determined the optimal levels of the carbon source (A), nitrogen source (B), first inorganic salt (C), and second inorganic salt (D). A four-factor, three-level response surface optimization experiment was designed (see Table 1). Based on the analysis, the optimal medium composition for mixed culture was determined. The experimental design and results are shown in Table 2. In Table 2, A indicates sucrose as the carbon source, B indicates yeast extract powder as the nitrogen source, C indicates sodium chloride as the first inorganic salt, and D indicates dipotassium hydrogen phosphate as the second inorganic salt. Table 1 shows that the highest survival rate was observed in group 29, at 9.335 lg cfu / mL. The analysis of variance is shown in Table 3. When viable cell count was used as the response, the model P < 0.01, indicating that the quadratic model was extremely significant. The lack-of-fit term, P = 0.0569, was not significant, indicating that the regression equation had a high degree of fit and the mathematical model was stable. This model can be used to predict viable cell counts in mixed culture. From the P value, we can see that the linear terms A, B, C, D, the quadratic terms AB, AD, BC, CD, A 2 、B 2 、C 2 and D 2The effect on the viable bacterial survival rate of the inoculant was extremely significant (P<0.01), while AC and BD had no significant effect (P>0.05). According to the F value, the influence of each factor on the viable bacterial count was judged to be sucrose, yeast extract powder, sodium chloride, and potassium dihydrogen phosphate, indicating that sucrose had the most significant effect on the viable bacterial count.

[0105] Table 1L 12 (4 3 )Response surface experiment table

[0106]

[0107] Table 2 Response surface optimization design and experimental results

[0108]

[0109] Table 3 Variance analysis table of regression equation

[0110]

[0111]

[0112] It should be noted that in Table 3, SS represents the sum of squares, DF represents the degrees of freedom, MS represents the mean square, Pr>F represents the probability of no significant effect, ** represents very significant, and * represents significant.

[0113] The response surface experimental results were subjected to multiple linear regression and binomial fitting to obtain the regression equation for Y, as shown in the following relationship (1).

[0114] Y=9.32-0.13A-0.096B-0.025C-0.042D-0.042AB+0.012AC-0.047AD-0.035BC-0.016BD-0.04CD-0.12A 2 -0.1B 2 -0.057C 2 -0.061D 2 (1)

[0115] In formula (1), A represents the concentration of sucrose, B represents the concentration of yeast extract powder, C represents the concentration of dipotassium hydrogen phosphate, and D represents the concentration of sodium chloride.

[0116] Example 5

[0117] This example uses the effect of physical factors on mixed bacterial culture as an example to illustrate the results:

[0118] The method of this example is the same as that of Example 1, wherein sucrose is used as the carbon source at a concentration of 15 g / L, yeast extract powder is used as the nitrogen source at a concentration of 15 g / L, and dipotassium hydrogen phosphate and sodium chloride are used as the inorganic salts, wherein the concentrations of dipotassium hydrogen phosphate and sodium chloride are both 1 g / L. The difference is that the pH of the culture is set to 5, 6, 7, 8, and 9 respectively. The results of the mixed culture are shown in the figure below. Figure 5 As shown in (a), the inoculation amounts are 0.5%, 1%, 2%, 3%, and 4%, respectively. The results of mixed bacterial culture are shown in FIG. Figure 5 As shown in (b), the culture temperatures were set to 20°C, 24°C, 28°C, 32°C, and 36°C, respectively. The results of the mixed bacterial culture were as follows: Figure 5 As shown in (c), the culture time is set to 24h, 36h, 48h, 60h, and 72h respectively. The results of mixed bacterial culture are as follows Figure 5 As shown in (d) in .

[0119] It should be understood that the experiments under the above different conditions are all single-factor experiments. When the pH value is optimized, the temperature is 28°C, the inoculation size is 1%, and the incubation time is 48h. When the incubation temperature is optimized, the pH is 7, the inoculation size is 1%, and the incubation time is 48h. When the incubation time is optimized, the pH is 7, the inoculation size is 1%, and the temperature is 28°C. When the inoculation size is optimized, the temperature is 28°C, the pH is 7, and the incubation time is 48h.

[0120] Effects of different pH values, inoculation amounts, culture temperatures and culture times on mixed bacterial culture Figure 5 As shown in the results, it can be seen that the viable bacteria count is the highest when the pH value is 7, which can reach 9.38lg·cfu / mL, the viable bacteria count is the highest when the inoculation amount is 1%, which can reach 9.39lg·cfu / mL, the viable bacteria count is the highest when the culture temperature is 24℃, which can reach 9.41lg·cfu / mL, and the viable bacteria count is the highest when the culture time is 60h, which can reach 9.44lg·cfu / mL.

[0121] In summary, according to Examples 1-5, during the preparation of the composite bacterial agent, the optimal carbon source is preferably sucrose, with a preferred concentration of 15 g / L. The optimal nitrogen source is preferably yeast extract powder, with a preferred concentration of 15 g / L. Dipotassium hydrogen phosphate and sodium chloride are preferred inorganic salts, with both sodium chloride and dipotassium hydrogen phosphate concentrations preferably being 1 g / L. The pH during the culture process is preferably 7, the inoculum size is preferably 1%, the culture temperature is preferably 24°C, and the culture time is preferably 60 h. Based on the above preferred conditions, the preferred conditions for preparing the liquid bacterial agent are further described. For details, please refer to the following examples.

[0122] Example 6

[0123] This example uses the effects of different additives on composite biocontrol agents as an example to illustrate the results:

[0124] A single-factor optimization study was conducted to determine the optimal thickeners (0.3% gum arabic, 0.6% gum arabic, 0.3% sodium alginate, 0.6% sodium alginate, 0.3% agar, and 0.6% agar), the optimal emulsifiers (0.2% Tween 20, 0.4% Tween 20, 0.2% Tween 80, and 0.4% Tween 80), and the optimal antioxidants (0.03% ascorbic acid, 0.06% ascorbic acid, 0.03% tea polyphenols, 0.06% tea polyphenols, 0.03% 2,6-di-tert-butyl-p-cresol, and 0.06% 2,6-di-tert-butyl-p-cresol) by adding 1 mL of different additives to the mixed bacterial suspension. The viable bacterial count was measured on day 7, using the viable bacterial count as the evaluation indicator.

[0125] Effects of different thickeners, emulsifiers and antioxidants on liquid bacterial agents Figure 6 The viable bacterial count was highest when 0.3% gum arabic was used as the thickener, reaching 8.89 lg cfu / mL; the viable bacterial count was highest when 0.4% Tween-80 was used as the emulsifier, reaching 8.97 lg cfu / mL; and the viable bacterial count was highest when 0.06% ascorbic acid was used as the antioxidant, reaching 8.94 lg cfu / mL.

[0126] Furthermore, based on the above research results, the optimal levels of gum arabic (A), Tween-80 (B), and ascorbic acid (C) were obtained, and a three-factor, three-level response surface optimization experiment was designed, as shown in Table 4. Based on the analysis results, the optimal preparation conditions for the liquid composite microbial inoculant were finally obtained.

[0127] The experimental design and results are shown in Table 5, and the variance analysis is shown in Table 6. Table 5 shows that the highest survival rate is in Group 14, which is 9.144 lg cfu / mL. Table 6 shows that when the number of viable bacteria is the response value, the model P < 0.01, indicating that the quadratic equation model is extremely significant. The lack of fit term P = 0.1757 > 0.05 is not significant, indicating that the regression equation has a high degree of fit and the mathematical model is stable. This mathematical model can be used to predict the number of viable bacteria in liquid inoculants. As shown in the P value, the linear terms A, B, the quadratic terms AB, BC, and A 2 and B 2 The effect of AC and C on the survival rate of live bacteria in the inoculum was extremely significant (P<0.01). The primary term C had a significant effect on the survival rate of live bacteria in the inoculum (P<0.05). 2 The effect was not significant (P>0.05). According to the F value, the order of influence of each factor on the viable cell count was Tween-80, gum arabic and ascorbic acid, indicating that Tween-80 had the most significant effect on the viable cell count.

[0128] Table 4L9(33 )Response surface experiment table

[0129]

[0130] Table 5 Response surface optimization design and experimental results

[0131]

[0132]

[0133] Table 6 Variance analysis table of regression equation

[0134]

[0135] It should be noted that in Table 6, SS represents the sum of squares, DF represents the degrees of freedom, MS represents the mean square, Pr>F represents the probability of no significant effect, ** represents very significant, and * represents significant.

[0136] The response surface experimental results were subjected to multiple linear regression and binomial fitting to obtain the regression equation for Y, as shown in formula (2). In the formula, A represents the concentration of gum arabic, B represents the concentration of Tween-80, and C represents the concentration of ascorbic acid.

[0137] Y=9.14-8.75×10 -3 A+0.011B+7.5×10 -3 C+0.017AB-5.5×

[0138] 10 -3 AC+0.014BC-0.023A 2 -0.025B 2 -6.4×10 -3 C 2 (2)

[0139] In summary, the optimal preparation process for this inoculant, predicted by the response surface analysis, is to inoculate 1% mixed seed liquid of M. caribbica:M. zizyphicola:P. rarassimilans in a 1:2:3 ratio into a medium containing 12.49 g / L sucrose, 13.3 g / L yeast extract, 0.88 g / L dipotassium phosphate, and 0.95 g / L sodium chloride at a pH of 7. The mixture is then shaken and cultured at 24°C and 180 rpm for 60 hours. The fermentation broth is then mixed with a protective agent solution consisting of 0.27% gum arabic, 0.49% Tween-80, and 0.07% tea polyphenols in a 1:1 ratio to produce a liquid inoculant.

[0140] Furthermore, based on the optimal conditions determined in the above examples, the in vivo inhibitory effects of liquid microbial agents at different concentrations on different fruits and vegetables are described below:

[0141] Example 7

[0142] This example uses liquid microbial agents of different concentrations to illustrate the in vivo inhibitory effects on strawberry gray mold and blue mold:

[0143] Healthy strawberry fruits were taken and the surface of the fruit was disinfected with 2% NaClO. Then wounds with a diameter of 4 mm and a depth of 4 mm were made at the equator of the fruit. One wound was made per fruit. 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of liquid bacterial agent, and sterile water was added as a control. 2 hours later, 1×10 4 Incubate 20 μL of B. cinerea and P. expansum at 26°C at a concentration of 100 cfu / mL. After 7 days, measure lesion diameters and calculate inhibition rates. Twenty fruits were used for each treatment. Repeat the experiment three times.

[0144] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 7 As shown in Figure 2, with the increase of concentration, the inhibition rate of strawberry gray mold and blue mold increased. 5 cfu / mL, the inhibition rates were 35.23%, 31.36%, and 1×10 6 cfu / mL is 50%, 48.31%, 1×10 7 cfu / mL were 73.48%, 73.3%, and 1×10 9 cfu / mL were 83.33% and 84.75%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 85.61% and 86.44%, respectively, and the inhibition effect was the best at this time.

[0145] Example 8

[0146] This example uses liquid microbial agents of different concentrations to illustrate the in vivo inhibitory effects on pear gray mold and black spot:

[0147] Take pear healthy fruit and treat it as in Example 7, with 4 wounds per fruit. Add liquid bacterial agent 2 hours later and inoculate 1×10 4 cfu / mL of Botrytis cinerea and 1×10 5 Incubate 20 μL of A. tenuissima at 26°C for 7 days. Measure lesion diameter and calculate inhibition rate. Ten fruits were used for each treatment. Repeat the experiment three times.

[0148] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 8 As shown in Figure 2, with the increase of concentration, the inhibition rate of pear gray mold and black spot increased. 5 cfu / mL, the inhibition rates were 38.1%, 41.19%, and 1×10 6 cfu / mL is 50.14%, 53.1%, 1×10 7 cfu / mL were 68.91%, 69.05%, and 1×10 9 cfu / mL were 84.87% and 83.1%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 88.52% and 84.05%, respectively, and the inhibition effect was the best at this time.

[0149] Example 9

[0150] This example uses liquid microbial agents of different concentrations to illustrate the in vivo inhibitory effects on gray mold and black spot of winter jujube:

[0151] Take healthy winter jujube fruits and treat them as in Example 7. Each fruit has one wound. Add liquid bacterial agent for 2 hours and inoculate 1×10 4 cfu / mL of Botrytis cinerea and 1×10 5 Incubate 20 μL of A. tenuissima at 26°C for 7 days. Measure lesion diameter and calculate inhibition rate. Ten fruits were used for each treatment. Repeat the experiment three times.

[0152] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 9 As shown in Figure 2, with the increase of concentration, the inhibition rate of gray mold and black spot of winter jujube increased. 5 cfu / mL, the inhibition rates were 36.11%, 30.56%, and 1×10 6 cfu / mL is 58.33%, 47.22%, 1×10 7 cfu / mL were 72.22%, 63.89%, and 1×10 9 cfu / mL were 80.56% and 69.44%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 87.5% and 76.39%, respectively, and the inhibition effect was the best at this time.

[0153] Example 10

[0154] This example uses the in vivo inhibitory effects of liquid microbial agents of different concentrations on gray mold and penicillium mold in kiwifruit as an example to illustrate:

[0155] Take healthy kiwifruit, disinfect the surface of the fruit with 2% NaClO, and make wounds with a diameter of 4 mm and a depth of 4 mm at the equator of the fruit. Each fruit should have two wounds. Add 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of liquid bacterial agent, and sterile water was added as a control. 2 hours later, 1×10 4 Incubate 20 μL of B. cinerea and P. expansum at 26°C at a concentration of 100 cfu / mL. After 7 days, measure lesion diameters and calculate inhibition rates. Ten fruits were used for each treatment. The experiment was repeated three times.

[0156] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 10 As shown in Figure 2, with the increase of concentration, the inhibition rate of kiwifruit gray mold and blue mold increased. 5 cfu / mL, the inhibition rates were 44.17%, 39.8%, and 1×10 6 cfu / mL is 63.33%, 53.07%, 1×10 7 cfu / mL were 70%, 67.08%, and 1×10 9 cfu / mL were 83.75% and 84.28%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 86.88% and 87.22%, respectively, and the inhibition effect was the best at this time.

[0157] Example 11

[0158] This example uses liquid microbial agents of different concentrations to illustrate the in vivo inhibitory effects on blueberry aspergillus and gray mold:

[0159] Take healthy blueberry fruits, disinfect the fruit surface with 2% NaClO, and make a wound with a diameter of 4mm and a depth of 4mm at the equator of the fruit. Each fruit should have one wound. Add 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of liquid bacterial agent, and sterile water was added as a control. 2 hours later, 1×10 5 cfu / mL of Aspergillus niger and 1×10 4Incubate 20 μL of B. cinerea at 26°C with a concentration of 100 cfu / mL. After 7 days, measure the lesion diameter and calculate the inhibition rate. Ten fruits were used for each treatment. Repeat the experiment three times.

[0160] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 11 As shown in Figure 2, as the concentration increases, the inhibition rate of blueberry aspergillus and gray mold increases. 5 cfu / mL, the inhibition rates were 29.66%, 27.5%, and 1×10 6 cfu / mL is 51.69%, 47.5%, 1×10 7 cfu / mL were 70.34%, 69.17%, and 1×10 9 cfu / mL were 80.51% and 75%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 83.05% and 84.17%, respectively, and the inhibition effect was the best at this time.

[0161] Example 12

[0162] This example uses liquid microbial agents of different concentrations to illustrate the in vivo inhibitory effects on cucumber aspergillus and gray mold:

[0163] Take healthy cucumber fruits, disinfect the fruit surface with 2% NaClO, and make wounds with a diameter of 4 mm and a depth of 4 mm at the equator of the fruit. Each fruit should have two wounds. Add 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of liquid bacterial agent, and sterile water was added as a control. 2 hours later, 1×10 5 cfu / mL of Aspergillus niger (A. niger) and 1×10 4 Incubate 20 μL of B. cinerea at 26°C with a concentration of 100 cfu / mL. After 7 days, measure the lesion diameter and calculate the inhibition rate. Ten fruits were used for each treatment. Repeat the experiment three times.

[0164] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 12 As shown in Figure 2, with the increase of concentration, the inhibition rate of cucumber aspergillus and gray mold increased. 5 cfu / mL, the inhibition rates were 31.78%, 35.58%, and 1×10 6 cfu / mL is 43.93%, 49.04%, 1×10 7cfu / mL were 56.54%, 58.65%, and 1×10 9 cfu / mL were 59.35% and 67.79%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 69.16% and 77.88%, respectively, and the inhibition effect was the best at this time.

[0165] Example 13

[0166] This example uses different concentrations of liquid microbial agents to illustrate the inhibitory effect on apple blue mold:

[0167] Healthy apples were treated as in Example 7, with four wounds per fruit. Two hours after adding the liquid inoculum, 20 μL of 1×10⁴ cfu / mL of Penicillium expansum was inoculated into each wound. The wounds were incubated at 26°C. After 7 days, the diameter of the lesions was measured and the inhibition rate was calculated. Ten fruits were used for each treatment. The experiment was repeated three times.

[0168] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 13 As shown in Figure 2, the inhibition rate of apple blue mold increased with the increase of concentration. 5 cfu / mL, the inhibition rate was 31.29%, 1×10 6 cfu / mL is 36.5%, 1×10 7 cfu / mL was 48.77%, 1×10 9 cfu / mL was 60.74%, and at a concentration of 1×10 8 cfu / mL, the inhibition rate was 61.96%, and the inhibition effect was the best at this time.

[0169] Example 14

[0170] This example uses different concentrations of liquid microbial agents to illustrate the inhibitory effect on orange Penicillium mold:

[0171] Take healthy orange fruits and treat them as in Example 7, with one wound per fruit. Add liquid bacterial agent for 2 hours and inoculate 1×10 4 20 μL of Penicillium citrinum (CFU / mL) was incubated at 26°C. After 7 days, the lesion diameter was measured and the inhibition rate was calculated. Ten fruits were used for each treatment. The experiment was repeated three times.

[0172] Furthermore, after adding liquid bacterial agents of different concentrations, the statistical results of the inhibition rate are as follows: Figure 14 As shown in Figure 2, the inhibition rate of orange Penicillium mold disease increases with the increase of concentration. 5 cfu / mL, the inhibition rate was 31.82%, 1×10 6cfu / mL is 38.02%, 1×10 7 cfu / mL was 47.93%, 1×10 9 cfu / mL was 57.85%, and at a concentration of 1×10 8 cfu / mL, the inhibition rate was 63.22%, and the inhibition effect was the best at this time.

[0173] Example 15

[0174] This example uses liquid microbial agents of different concentrations to illustrate the in vivo inhibitory effects on tomato gray mold and blue mold:

[0175] Take healthy tomato fruits and treat them as in Example 7, with one wound per fruit. Add liquid bacterial agent 2 hours later and inoculate 1×10 4 Incubate 20 μL of B. cinerea and P. expansum at 26°C at a concentration of 100 cfu / mL. After 7 days, measure lesion diameters and calculate inhibition rates. Ten fruits were used for each treatment. The experiment was repeated three times.

[0176] Furthermore, after accessing different concentrations of M. caribbica suspension, the inhibition rate statistics are as follows Figure 15 As shown in Figure 2, with the increase of concentration, the inhibition rate of tomato gray mold and blue mold increased. 5 cfu / mL, the inhibition rates were 39.2%, 45.27%, and 1×10 6 cfu / mL is 47.2%, 54.05%, 1×10 7 cfu / mL were 72.8%, 71.62%, and 1×10 9 cfu / mL were 92% and 90.54%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 93.6% and 93.92%, respectively, and the inhibition effect was the best at this time.

[0177] In summary, when the concentration of liquid bacterial agent is 1×10 8 cfu / mL, the inhibition rate of pathogens in fruits and vegetables is the highest.

[0178] It is understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present disclosure, and the present disclosure is not limited thereto. Those skilled in the art may make various modifications and improvements without departing from the spirit and substance of the present disclosure, and such modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A composite biocontrol agent for preventing and controlling fruit and vegetable diseases, characterized in that: The composite biocontrol agent comprises: Meyerozyma caribbica, Metschnikowia zizyphicola Y-3, Pichia rarassimilans and additives; wherein, The Meyerozyma caribbica was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on June 12, 2023, with the deposit number being CGMCC No. 27604. The Metschnikowia zizyphicola Y-3 was deposited in the China Center for Type Culture Collection on October 17, 2016, with a deposit number of CCTCCNO: M 2016564. The Pichia rarassimilans was deposited in the General Microbiology Center of the China Culture Collection Administration on December 2, 2024, and its deposit number is CGMCC No.32875.

2. The composite biocontrol agent according to claim 1, characterized in that The mass ratio of the Meyerozyma caribbica, the Metschnikowia zizyphicola Y-3, and the Pichia rarassimilans is 1:2:

3.

3. The composite biocontrol agent according to claim 1, characterized in that The additives include thickeners, emulsifiers and antioxidants.

4. The composite biocontrol agent according to claim 3, characterized in that The thickener is any one of gum arabic with a concentration of 0.3%, gum arabic with a concentration of 0.6%, sodium alginate with a concentration of 0.3%, sodium alginate with a concentration of 0.6%, agar with a concentration of 0.3%, and agar with a concentration of 0.6%; and / or, The emulsifier is any one of Tween 20 with a concentration of 0.2%, Tween 20 with a concentration of 0.4%, Tween 80 with a concentration of 0.2%, and Tween 80 with a concentration of 0.4%; and / or The antioxidants include ascorbic acid with a concentration of 0.03%, ascorbic acid with a concentration of 0.06%, tea polyphenol with a concentration of 0.03%, tea polyphenol with a concentration of 0.06%, 2,6-di-tert-butyl-p-cresol with a concentration of 0.03%, and 2,6-di-tert-butyl-p-cresol with a concentration of 0.06%.

5. The composite biocontrol agent according to claim 4, characterized in that The thickener is 0.3% gum arabic; and / or The emulsifier is Tween 80 with a concentration of 0.4%; and / or, The antioxidant is ascorbic acid with a concentration of 0.06%.

6. A method for preparing the composite biocontrol agent according to any one of claims 1 to 5, characterized in that: The method comprises: Meyerozyma caribbica, Metschnikowiazizyphicola Y-3, and Pichia rarassimilans are used as a mixed seed liquid, the mixed seed liquid is purified and inoculated into a liquid culture medium, and a mixed bacterial suspension is formed through culturing and preparation; Additives are added to the mixed bacterial suspension to prepare a composite biocontrol agent.

7. The method according to claim 6, characterized in that The volume ratio of the mixed bacterial suspension to the additive is 1:

1.

8. The method according to claim 6, characterized in that The liquid culture medium comprises 10-20 g / L of a carbon source, 10-20 g / L of a nitrogen source, and 0.2-1.8 g / L of an inorganic salt.

9. The method according to claim 8, characterized in that The carbon source is sucrose, and the concentration of the sucrose is 15 g / L; and / or, The nitrogen source is yeast extract powder, and the concentration of the yeast extract powder is 15 g / L; and / or, The inorganic salts are dipotassium hydrogen phosphate and sodium chloride, the concentration of the dipotassium hydrogen phosphate is 1 g / L, and the concentration of the sodium chloride is 1 g / L; and / or, The inoculation amount of the mixed seed liquid is 0.5-4%, the pH value during the culture process is 5-9, the culture temperature is 20-36° C., and the culture time is 24-72 hours.

10. An application of a composite biocontrol agent, characterized in that: The composite biocontrol agent according to any one of claims 1 to 5 is used to prevent and control at least one of gray mold, penicillium, black spot and aspergillosis of fruits and vegetables.

Images