A method for cultivating plant endophytic fungus strain and its application

Abstract

The present invention relates to the technical field of strain cultivation, and specifically discloses a method for cultivating a plant endophytic fungal strain, wherein the method for cultivating the plant endophytic fungal strain is a method for cultivating Alternaria brassicae BB‑6, and the deposit number of Alternaria brassicae BB‑6 is: CCTCCNO: M20232214; the specific cultivation method comprises the following steps: Alternaria brassicae is first placed in a chitosan regulating liquid for uniform dispersion treatment according to a weight ratio of 2:7, and uniform dispersion is completed to obtain a chitosan-regulated Alternaria brassicae agent. In the cultivation of the plant endophytic fungal strain of the present invention, Alternaria brassicae BB‑6 is uniformly dispersed in a chitosan regulating liquid, then cultivated with a culture modifier, and then cultivated by a coordinated cultivation process of the two, and then an antibacterial agent is formed by an acetone solvent to inhibit Aspergillus flavus.

Description

Technical Field

[0001] The invention relates to the technical field of fungal strain cultivation, and in particular to a cultivation method and application of a plant endophytic fungal strain. Background Art

[0002] The endophytic fungi of Pseudococcus chinensis among the plant endophytic fungi are Alternaria alternata, Alternaria sp., Alternaria brassicae, Colletotrichum sublineola, Pichia kudriavzevii, Epicoccum sorghinum, and Phoma sp., among which Alternaria is the dominant genus of endophytic fungi of Pseudococcus chinensis.

[0003] The existing culture process of Alternaria brassicae strains is simple and the culture efficiency is low. The cultured antibacterial agent has poor antibacterial efficiency against Aspergillus flavus and the antibacterial effect is not lasting, which limits the culture efficiency. Summary of the invention

[0004] In view of the defects of the prior art, the object of the present invention is to provide a cultivation method and application of a plant endophytic fungus strain to solve the problems raised in the above background technology.

[0005] The present invention solves the technical problem by adopting the following technical solution:

[0006] The present invention provides a method for culturing a plant endophytic fungus strain, wherein the method for culturing the plant endophytic fungus strain is a method for culturing Alternaria brassicae BB-6, and the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214; the specific culturing method comprises the following steps:

[0007] Step 1: placing Alternaria brassicae in a chitosan regulating solution at a weight ratio of 2:7 and uniformly dispersing the mixture until the uniform dispersion is complete, thereby obtaining an Alternaria brassicae agent adjusted with chitosan;

[0008] Step 2: Cultivate the Alternaria brassicae agent adjusted with chitosan in a constant temperature incubator at 28°C for 5 days, then transfer to a culture modifier for cultivation for 6-10 days at a culture temperature of 30°C, and then terminate the cultivation.

[0009] Preferably, the rotation speed of the uniform dispersion treatment is 150-200 r / min, and the uniform dispersion time is 20-30 min.

[0010] Preferably, the preparation method of the chitosan regulating solution is:

[0011] S01: adding 4-7 parts of flaky talc and 1-3 parts of sodium lignin sulfonate solution to 10-15 parts of phosphate buffer solution, and then adding 1-2 parts of urea and 1-2 parts of nano-silica sol, stirring and reacting, stirring to obtain flaky talc;

[0012] S02: First, hydroxyapatite is mixed thoroughly with a 10% by mass potassium permanganate solution, then washed with water and dried;

[0013] S03: ball-milling the dried hydroxyapatite and flaky talc at a weight ratio of 3:1, washing with water, and drying to obtain a modified regulator;

[0014] S04: Add 3-5% sodium citrate solution to the total amount of chitosan solution into 5-7% chitosan solution by mass, stir and mix, then add 2-5% modification regulator to the total amount of chitosan solution, stir thoroughly, and obtain chitosan adjustment solution.

[0015] Preferably, the mass fraction of the sodium lignin sulfonate solution is 10-15%; the pH value of the phosphate buffer solution is 5.0; and the mass fraction of the sodium citrate solution is 10-15%.

[0016] Preferably, the stirring speed of the S01 stirring reaction treatment is 450-550 r / min, and the stirring time is 20-30 min.

[0017] Preferably, the rotation speed of the SO3 ball milling reaction is 1000-1500r / min, and the ball milling time is 1-2h.

[0018] Preferably, the preparation method of the culture modifier is:

[0019] S101: The montmorillonite is first mixed in a sufficient amount of a 5% by mass hydrochloric acid solution, and then washed with water, dried, and then reacted at a temperature of 210-230°C for 1-2h. After the reaction is completed, the temperature is then reduced to 55°C at a rate of 1-3°C / min for heat preservation;

[0020] S102: 4-7 parts of heat-insulated montmorillonite, 1-3 parts of sodium carboxymethyl cellulose, 1-2 parts of stearic acid, 2-5 parts of silane coupling agent and 6-10 parts of water are mixed and ball-milled at a ball-milling speed of 1000-1500 r / min for 1-2 hours. After the ball-milling is completed, the mixture is washed with water and dried to obtain a montmorillonite modifier;

[0021] S103: 3-5 parts of glucose, 3-7 parts of agar, 2-5 parts of sulfate, 1-3 parts of vitamins, 2-5 parts of montmorillonite modifier, and 6-10 parts of deionized water are stirred and mixed thoroughly to obtain a culture modifier.

[0022] Preferably, the silane coupling agent is silane coupling agent KH560.

[0023] Preferably, the sulfate is one or more combinations of magnesium sulfate, ferrous sulfate, and manganese sulfate, and the vitamin is one or more combinations of vitamin B, vitamin D, and riboflavin.

[0024] The invention also provides an application of a method for culturing a plant endophytic fungus strain in inhibiting aflatoxin.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] In the plant endophytic fungus strain culture of the present invention, Alternaria brassicae BB-6 is subjected to a chitosan regulating liquid uniform dispersion treatment, then subjected to a culture modifier culture treatment, subjected to a coordinated culture process of the two, and then subjected to an acetone solvent to form an antibacterial agent to inhibit Aspergillus flavus. The flaky talcum powder in the chitosan regulating liquid is combined with hydroxyapatite, and then the chitosan regulating liquid formed by the chitosan solution and the sodium citrate solution is prepared to moisten and disperse the strain, and the flaky structure of the flaky talcum powder carries the strain and cooperates with the hydroxyapatite for point dispersion, thereby facilitating the bacteria to be dispersed. The strain is better cultured and grown in the culture modifier, and the montmorillonite in the culture modifier is treated with hydrochloric acid solution and then reacted at a temperature of 210-230°C for 1-2h. After the reaction is completed, the temperature is reduced to 55°C at a rate of 1-3°C / min. The insulation treatment optimizes the activity efficiency of the montmorillonite, and then mixed with sodium carboxymethyl cellulose, stearic acid and silane coupling agent, and water for ball milling to improve the dispersion of the montmorillonite, thereby further enhancing the synergistic effect with the chitosan regulating solution, making the strain generation more perfect, and the obtained strain has more stable antibacterial performance. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The present embodiment provides a method for culturing an endophytic fungus strain of a plant. The method for culturing an endophytic fungus strain of the plant is a method for culturing Alternaria brassicae BB-6. The deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214. The depository is: China Center for Type Culture Collection. The recommended classification name is: Alternaria brassicae BB-6. The culture was received by China Center for Type Culture Collection on November 13, 2023 and registered. The viability of the culture was tested by this collection on November 20, 2023, and the result was viable. The specific culturing method comprises the following steps:

[0029] Step 1: placing Alternaria brassicae in a chitosan regulating solution at a weight ratio of 2:7 and uniformly dispersing the mixture until the uniform dispersion is completed, thereby obtaining an Alternaria brassicae agent adjusted with chitosan;

[0030] Step 2: Cultivate the Alternaria brassicae agent adjusted with chitosan in a constant temperature incubator at 28°C for 5 days, then transfer to a culture modifier for cultivation for 6-10 days at a culture temperature of 30°C, and then terminate the cultivation.

[0031] The rotation speed of the uniform dispersion treatment in this embodiment is 150-200 r / min, and the uniform dispersion time is 20-30 min.

[0032] The preparation method of the chitosan regulating solution of the present embodiment is:

[0033] S01: adding 4-7 parts of flaky talc and 1-3 parts of sodium lignin sulfonate solution to 10-15 parts of phosphate buffer solution, and then adding 1-2 parts of urea and 1-2 parts of nano-silica sol, stirring and reacting, stirring to obtain flaky talc;

[0034] S02: First, hydroxyapatite is mixed thoroughly with a 10% by mass potassium permanganate solution, then washed with water and dried;

[0035] S03: ball-milling the dried hydroxyapatite and flaky talc at a weight ratio of 3:1, washing with water, and drying to obtain a modified regulator;

[0036] S04: Add 3-5% sodium citrate solution to the total amount of chitosan solution into 5-7% chitosan solution by mass, stir and mix, then add 2-5% modification regulator to the total amount of chitosan solution, stir thoroughly, and obtain chitosan adjustment solution.

[0037] The mass fraction of the sodium lignin sulfonate solution in this embodiment is 10-15%; the pH value of the phosphate buffer solution is 5.0; and the mass fraction of the sodium citrate solution is 10-15%.

[0038] The stirring speed of the S01 stirring reaction treatment in this embodiment is 450-550 r / min, and the stirring time is 20-30 min.

[0039] The rotation speed of the SO3 ball milling reaction in this embodiment is 1000-1500r / min, and the ball milling is 1-2h.

[0040] The preparation method of the culture modifier of this embodiment is:

[0041] S101: The montmorillonite is first mixed in a sufficient amount of a 5% by mass hydrochloric acid solution, and then washed with water, dried, and then reacted at a temperature of 210-230°C for 1-2h. After the reaction is completed, the temperature is then reduced to 55°C at a rate of 1-3°C / min for heat preservation;

[0042] S102: 4-7 parts of heat-insulated montmorillonite, 1-3 parts of sodium carboxymethyl cellulose, 1-2 parts of stearic acid, 2-5 parts of silane coupling agent and 6-10 parts of water are mixed and ball-milled at a ball-milling speed of 1000-1500 r / min for 1-2 hours. After the ball-milling is completed, the mixture is washed with water and dried to obtain a montmorillonite modifier;

[0043] S103: 3-5 parts of glucose, 3-7 parts of agar, 2-5 parts of sulfate, 1-3 parts of vitamins, 2-5 parts of montmorillonite modifier, and 6-10 parts of deionized water are stirred and mixed thoroughly to obtain a culture modifier.

[0044] The silane coupling agent of this embodiment is silane coupling agent KH560.

[0045] The sulfate in this embodiment is one or more combinations of magnesium sulfate, ferrous sulfate, and manganese sulfate, and the vitamin is one or more combinations of vitamin B, vitamin D, and riboflavin.

[0046] The present embodiment provides an application of a method for culturing a plant endophytic fungus strain in the inhibition of Aspergillus flavus.

[0047] Example 1: A method for culturing a plant endophytic fungus strain in this example, wherein the method for culturing the plant endophytic fungus strain is a method for culturing Alternaria brassicae BB-6, the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214, the depository is: China Center for Type Culture Collection, the recommended classification name is: Alternaria brassicae BB-6, the culture was received by China Center for Type Culture Collection on November 13, 2023, and registered; the viability of the culture was tested by this collection on November 20, 2023, and the result was survival; the specific culturing method comprises the following steps:

[0048] Step 1: placing Alternaria brassicae in a chitosan regulating solution at a weight ratio of 2:7 and uniformly dispersing the mixture until the uniform dispersion is completed, thereby obtaining an Alternaria brassicae agent adjusted with chitosan;

[0049] Step 2: The chitosan-adjusted Alternaria brassicae agent was cultured in a constant temperature incubator at 28°C for 5 days, and then transferred to a culture modifier for culture for 6 days at a culture temperature of 30°C, and the culture was terminated.

[0050] The rotation speed of the uniform dispersion treatment in this embodiment is 150 r / min, and the uniform dispersion time is 20 min.

[0051] The preparation method of the chitosan regulating solution of the present embodiment is:

[0052] S01: adding 4 parts of flaky talc powder and 1 part of sodium lignin sulfonate solution to 10 parts of phosphate buffer solution, and then adding 1 part of urea and 1 part of nano-silica sol, stirring for reaction treatment, and stirring to obtain flaky talc powder;

[0053] S02: First, hydroxyapatite is mixed thoroughly with a 10% by mass potassium permanganate solution, then washed with water and dried;

[0054] S03: ball-milling the dried hydroxyapatite and flaky talc at a weight ratio of 3:1, washing with water, and drying to obtain a modified regulator;

[0055] S04: Add 3% sodium citrate solution to the total amount of chitosan solution into 5% chitosan solution by mass, stir and mix, then add 2% modification regulator to the total amount of chitosan solution, stir thoroughly, and obtain chitosan adjustment solution.

[0056] The mass fraction of the sodium lignin sulfonate solution in this embodiment is 10%; the pH value of the phosphate buffer solution is 5.0; and the mass fraction of the sodium citrate solution is 10%.

[0057] The stirring speed of the S01 stirring reaction treatment in this embodiment is 450 r / min, and the stirring time is 20 min.

[0058] The rotation speed of the SO3 ball milling reaction in this embodiment is 1000r / min, and the ball milling is 1h.

[0059] The preparation method of the culture modifier of this embodiment is:

[0060] S101: The montmorillonite is first mixed in a sufficient amount of a 5% by mass hydrochloric acid solution, and then washed with water, dried, and then reacted at a temperature of 210°C for 1 hour. After the reaction is completed, the temperature is then reduced to 55°C at a rate of 1°C / min for heat preservation;

[0061] S102: 4 parts of heat-insulated montmorillonite, 1 part of sodium carboxymethyl cellulose, 1 part of stearic acid, 2 parts of silane coupling agent and 6 parts of water are mixed and ball-milled at a ball-milling speed of 1000 r / min for 1 hour. After the ball-milling is completed, the mixture is washed with water and dried to obtain a montmorillonite modifier;

[0062] S103: 3 parts of glucose, 3 parts of agar, 2 parts of sulfate, 1 part of vitamin, 2 parts of montmorillonite modifier, and 6 parts of deionized water are stirred and mixed thoroughly to obtain a culture modifier.

[0063] The silane coupling agent of this embodiment is silane coupling agent KH560.

[0064] The sulfate in this embodiment is magnesium sulfate, and the vitamin is vitamin B.

[0065] The present embodiment provides an application of a method for culturing a plant endophytic fungus strain in the inhibition of Aspergillus flavus.

[0066] Example 2: A method for culturing a plant endophytic fungus strain in this example, wherein the method for culturing the plant endophytic fungus strain is a method for culturing Alternaria brassicae BB-6, the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214, the depository is: China Center for Type Culture Collection, the recommended classification name is: Alternaria brassicae BB-6, the culture was received by China Center for Type Culture Collection on November 13, 2023, and registered; the viability of the culture was tested by this collection on November 20, 2023, and the result was survival; the specific culturing method comprises the following steps:

[0067] Step 1: placing Alternaria brassicae in a chitosan regulating solution at a weight ratio of 2:7 and uniformly dispersing the mixture until the uniform dispersion is completed, thereby obtaining an Alternaria brassicae agent adjusted with chitosan;

[0068] Step 2: The chitosan-adjusted Alternaria brassicae agent was cultured in a constant temperature incubator at 28°C for 5 days, and then transferred to a culture modifier for culture for 6 days at a culture temperature of 30°C, and the culture was terminated.

[0069] The rotation speed of the uniform dispersion treatment in this embodiment is 150 r / min, and the uniform dispersion time is 20 min.

[0070] The preparation method of the chitosan regulating solution of the present embodiment is:

[0071] S01: adding 4 parts of flaky talc powder and 1 part of sodium lignin sulfonate solution to 10 parts of phosphate buffer solution, and then adding 1 part of urea and 1 part of nano-silica sol, stirring for reaction treatment, and stirring to obtain flaky talc powder;

[0072] S02: First, hydroxyapatite is mixed thoroughly with a 10% by mass potassium permanganate solution, then washed with water and dried;

[0073] S03: ball-milling the dried hydroxyapatite and flaky talc at a weight ratio of 3:1, washing with water, and drying to obtain a modified regulator;

[0074] S04: Add 3% sodium citrate solution to the total amount of chitosan solution into 5% chitosan solution by mass, stir and mix, then add 2% modification regulator to the total amount of chitosan solution, stir thoroughly, and obtain chitosan adjustment solution.

[0075] The mass fraction of the sodium lignin sulfonate solution in this embodiment is 10%; the pH value of the phosphate buffer solution is 5.0; and the mass fraction of the sodium citrate solution is 10%.

[0076] The stirring speed of the S01 stirring reaction treatment in this embodiment is 450 r / min, and the stirring time is 20 min.

[0077] The rotation speed of the SO3 ball milling reaction in this embodiment is 1000r / min, and the ball milling is 1h.

[0078] The preparation method of the culture modifier of this embodiment is:

[0079] S101: The montmorillonite is first mixed in a sufficient amount of a 5% by mass hydrochloric acid solution, and then washed with water, dried, and then reacted at a temperature of 210°C for 1 hour. After the reaction is completed, the temperature is then reduced to 55°C at a rate of 1°C / min for heat preservation;

[0080] S102: 4 parts of heat-insulated montmorillonite, 1 part of sodium carboxymethyl cellulose, 1 part of stearic acid, 2 parts of silane coupling agent and 6 parts of water are mixed and ball-milled at a ball-milling speed of 1000 r / min for 1 hour. After the ball-milling is completed, the mixture is washed with water and dried to obtain a montmorillonite modifier;

[0081] S103: 3 parts of glucose, 3 parts of agar, 2 parts of sulfate, 1 part of vitamin, 2 parts of montmorillonite modifier, and 6 parts of deionized water are stirred and mixed thoroughly to obtain a culture modifier.

[0082] The silane coupling agent of this embodiment is silane coupling agent KH560.

[0083] The sulfate in this embodiment is ferrous sulfate, and the vitamin is vitamin D.

[0084] The present embodiment provides an application of a method for culturing a plant endophytic fungus strain in the inhibition of Aspergillus flavus.

[0085] Example 3: A method for culturing a plant endophytic fungus strain in this example, wherein the method for culturing the plant endophytic fungus strain is a method for culturing Alternaria brassicae BB-6, the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214, the depository is: China Center for Type Culture Collection, the recommended classification name is: Alternaria brassicae BB-6, the culture was received by China Center for Type Culture Collection on November 13, 2023, and registered; the viability of the culture was tested by this collection on November 20, 2023, and the result was survival; the specific culturing method comprises the following steps:

[0086] Step 1: placing Alternaria brassicae in a chitosan regulating solution at a weight ratio of 2:7 and uniformly dispersing the mixture until the uniform dispersion is completed, thereby obtaining an Alternaria brassicae agent adjusted with chitosan;

[0087] Step 2: The chitosan-adjusted Alternaria brassicae agent was cultured in a constant temperature incubator at 28°C for 5 days, and then transferred to a culture modifier for culture for 8 days at a culture temperature of 30°C, and the culture was terminated.

[0088] The rotation speed of the uniform dispersion treatment in this embodiment is 170r / min, and the uniform dispersion time is 25min.

[0089] The preparation method of the chitosan regulating solution of the present embodiment is:

[0090] S01: adding 5.5 parts of flaky talc and 2 parts of sodium lignin sulfonate solution to 12.5 parts of phosphate buffer solution, and then adding 1.5 parts of urea and 1.5 parts of nano-silica sol, stirring for reaction, and stirring to obtain flaky talc;

[0091] S02: First, hydroxyapatite is mixed thoroughly with a 10% by mass potassium permanganate solution, then washed with water and dried;

[0092] S03: ball-milling the dried hydroxyapatite and flaky talc at a weight ratio of 3:1, washing with water, and drying to obtain a modified regulator;

[0093] S04: Add 4% sodium citrate solution to the chitosan solution with a mass fraction of 6%, stir and mix well, then add 3.5% of the total amount of the chitosan solution to the modified regulator, stir thoroughly, and obtain a chitosan adjusted solution.

[0094] The mass fraction of the sodium lignin sulfonate solution in this embodiment is 12.5%; the pH value of the phosphate buffer solution is 5.0; and the mass fraction of the sodium citrate solution is 12.5%.

[0095] The stirring speed of the S01 stirring reaction treatment in this embodiment is 500 r / min, and the stirring time is 25 min.

[0096] The rotation speed of the SO3 ball milling reaction in this embodiment is 1250 r / min, and the ball milling is 11.5 h.

[0097] The preparation method of the culture modifier of this embodiment is:

[0098] S101: The montmorillonite is first mixed in a sufficient amount of a 5% by mass hydrochloric acid solution, and then washed with water, dried, and then reacted at a temperature of 220°C for 1.5 hours. After the reaction is completed, the temperature is then reduced to 55°C at a rate of 2°C / min for heat preservation;

[0099] S102: 5.5 parts of heat-insulated montmorillonite, 2 parts of sodium carboxymethyl cellulose, 1.5 parts of stearic acid, 3.5 parts of silane coupling agent and 8 parts of water are mixed and ball-milled at a ball-milling speed of 1250 r / min for 1.5 h. After the ball-milling is completed, the mixture is washed with water and dried to obtain a montmorillonite modifier;

[0100] S103: 4 parts of glucose, 5 parts of agar, 3.5 parts of sulfate, 2 parts of vitamins, 3.5 parts of montmorillonite modifier, and 8 parts of deionized water are stirred and mixed thoroughly to obtain a culture modifier.

[0101] The silane coupling agent of this embodiment is silane coupling agent KH560.

[0102] The sulfate in this embodiment is manganese sulfate, and the vitamin is riboflavin.

[0103] The present embodiment provides an application of a method for culturing a plant endophytic fungus strain in the inhibition of Aspergillus flavus.

[0104] Comparative Example 1.

[0105] The difference from Example 3 is that no chitosan regulating solution treatment was used.

[0106] Comparative Example 2.

[0107] The difference from Example 3 is that no modifying regulator is added to the chitosan regulating solution.

[0108] Comparative Example 3.

[0109] The difference from Example 3 is that the preparation method of the modified regulator is different, and dry hydroxyapatite treatment is not used.

[0110] Comparative Example 4.

[0111] The difference from Example 3 is that the preparation method of the modified regulator is different: no flaky talc powder is used for treatment.

[0112] Comparative Example 5.

[0113] The difference from Example 3 is that urea and nano-silica sol are not added in the preparation of the flaky talc.

[0114] Comparative Example 6.

[0115] The difference from Example 3 is that no montmorillonite modifier is added to the culture modifier.

[0116] Comparative Example 7.

[0117] The difference from Example 3 is that the montmorillonite modifier is replaced by montmorillonite.

[0118] The bacterial agents obtained by culturing in Examples 1-3 and Comparative Examples 1-7 were mixed with acetone solvent at a weight ratio of 2:5 to prepare an antibacterial agent, and the antibacterial agent was dropped into Aspergillus flavus at a weight ratio of 1:6 for antibacterial treatment. The antibacterial effect of the cultured bacterial agent on mold was tested, and the cultured bacterial agent of Example 3 without solvent was tested as a control group. The performance test was as follows; It can be seen from Examples 1-3 and Comparative Examples 1-7 and the control group that the products of Examples 1-3 have a persistent antibacterial rate against Aspergillus flavus from the moment of measurement to 72 hours, and the antibacterial rate is as high as 99% or more. At the same time, if no acetone solvent is added, the antibacterial effect of the cultured bacterial agent will also deteriorate;

[0119] It was found from comparative examples 1-7 that the antibacterial rate of the product showed a significant deterioration trend when the chitosan regulating solution was not used and the montmorillonite modifier was not added to the culture modifier, and the antibacterial persistence was significantly deteriorated. The two were used in synergy to enhance the effect together.

[0120] In the preparation of chitosan regulating liquid, the preparation methods of the modified regulating agent are different, dry hydroxyapatite treatment is not used, flaky talc powder is not used, urea and nano-silica sol are not added in the preparation of flaky talc powder, and montmorillonite modifier is replaced by montmorillonite. The performance of the products tends to deteriorate. The modified regulating agent prepared by the method of the present invention is combined with the montmorillonite modifier of the present invention, and the performance effect of the product is the most significant.

[0121] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0122] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for cultivating a plant endophytic fungus strain, characterized in that: The culturing method of the plant endophytic fungus strain is a culturing method of Alternaria brassicae BB-6, and the preservation number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214; the specific culturing method comprises the following steps: Step 1: placing Alternaria brassicae in a chitosan regulating solution at a weight ratio of 2:7 and uniformly dispersing the mixture until the uniform dispersion is completed, thereby obtaining an Alternaria brassicae agent adjusted with chitosan; Step 2: Cultivate the brassica Alternaria strain regulated by chitosan in a constant temperature incubator at 28°C for 5 days, and then transfer to a culture modifier for cultivation for 6-10 days at a culture temperature of 30°C. When the cultivation is completed, The preparation method of the culture modifier is: S101: The montmorillonite is first mixed in a sufficient amount of a 5% by mass hydrochloric acid solution, and then washed with water, dried, and then reacted at a temperature of 210-230°C for 1-2h. After the reaction is completed, the temperature is then reduced to 55°C at a rate of 1-3°C / min for heat preservation; S102: 4-7 parts of heat-insulated montmorillonite, 1-3 parts of sodium carboxymethyl cellulose, 1-2 parts of stearic acid, 2-5 parts of silane coupling agent and 6-10 parts of water are mixed and ball-milled at a ball-milling speed of 1000-1500 r / min for 1-2 hours. After the ball-milling is completed, the mixture is washed with water and dried to obtain a montmorillonite modifier; S103: 3-5 parts of glucose, 3-7 parts of agar, 2-5 parts of sulfate, 1-3 parts of vitamins, 2-5 parts of montmorillonite modifier, and 6-10 parts of deionized water are stirred and mixed thoroughly to obtain a culture modifier; The preparation method of the chitosan regulating solution is: S01: adding 4-7 parts of flaky talc and 1-3 parts of sodium lignin sulfonate solution to 10-15 parts of phosphate buffer solution, and then adding 1-2 parts of urea and 1-2 parts of nano-silica sol, stirring and reacting, stirring to obtain flaky talc; S02: First, hydroxyapatite is mixed thoroughly with a 10% by mass potassium permanganate solution, then washed with water and dried; S03: ball-milling the dried hydroxyapatite and flaky talc at a weight ratio of 3:1, washing with water, and drying to obtain a modified regulator; S04: Add 3-5% sodium citrate solution to the total amount of chitosan solution into 5-7% chitosan solution by mass, stir and mix, then add 2-5% modification regulator to the total amount of chitosan solution, stir thoroughly, and obtain chitosan adjustment solution.

2. The method for culturing a plant endophytic fungus strain according to claim 1, characterized in that: The rotation speed of the uniform dispersion treatment is 150-200r / min, and the uniform dispersion time is 20-30min.

3. The method for culturing a plant endophytic fungus strain according to claim 1, characterized in that: The mass fraction of the sodium lignin sulfonate solution is 10-15%; the pH value of the phosphate buffer solution is 5.0; and the mass fraction of the sodium citrate solution is 10-15%.

4. The method for culturing a plant endophytic fungus strain according to claim 3, characterized in that: The stirring speed of the S01 stirring reaction treatment is 450-550r / min, and the stirring time is 20-30min.

5. The method for culturing a plant endophytic fungus strain according to claim 3, characterized in that: The rotation speed of the SO3 ball milling reaction is 1000-1500r / min, and the ball milling is 1-2h.

6. The method for culturing a plant endophytic fungus strain according to claim 1, characterized in that: The silane coupling agent is silane coupling agent KH560.

7. The method for culturing a plant endophytic fungus strain according to claim 1, characterized in that: The sulfate is one or more combinations of magnesium sulfate, ferrous sulfate, and manganese sulfate, and the vitamin is one or more combinations of vitamin B and vitamin D.

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