Bacillus amyloliquefaciens B2 and preparation method and application of suspending agent of bacillus amyloliquefaciens B2

Abstract

The invention discloses a bacillus amyloliquefaciens B2 suspending agent and a preparation method and application thereof, the bacillus amyloliquefaciens is named as B2, the preservation number is CGMCC No.30634, and the bacillus amyloliquefaciens B2 suspending agent prepared from the bacillus amyloliquefaciens B2 suspending agent can be used for preventing and treating potato scab. By optimizing the formula of the fermentation culture medium and various process parameter conditions, B2 fermentation liquor of which the concentration is greater than or equal to 8 billion / mL is obtained; the bacillus amyloliquefaciens B2 suspending agent with the concentration of 2 billion / mL is researched and developed through auxiliary agent screening and compatibility testing of auxiliary agents. Field experiments show that the 2 billion / mL bacillus amyloliquefaciens B2 suspending agent has a good biocontrol effect on potato scab, and the yield of potatoes and the income of farmers are obviously increased.

Description

Technical Field

[0001] The invention relates to the technical field of potato scab prevention and treatment, and in particular to a preparation method and application of Bacillus amyloliquefaciens B2 and a suspension concentrate thereof. Background Art

[0002] Potato scab (Streptomyces scabies) is a soil-borne and seed-borne disease caused by the pathogen Streptomyces scabies, which belongs to the actinomycete soil microorganism. The pathogen invades the potato lenticels, producing brown spots on the tuber epidermis in the early stage of the disease, which gradually expand and cause necrosis of the tissues around the infection point. The tuber surface becomes rough and the texture becomes corky. In the later stage, the center is sunken or raised to form a scab-like hard plaque. It is common in the world's main potato producing areas. There are reports of the disease in North America, Europe, Asia and other places. It was once regarded as the fourth largest disease in potato production. It is common in China's potato production bases. In recent years, the occurrence of the disease in my country has gradually increased. After the disease occurs, although the lesions are limited to the cortex, the diseased potatoes are not resistant to storage and have an ugly appearance. After the disease is infected, the quality of potatoes is greatly affected, resulting in a decrease in commodity value. Generally, the yield can be reduced by 10-30%, and some plots even have a yield reduction of more than 40%. At present, the prevention and control of potato scab mainly relies on chemical pesticides, but there are still problems in the prevention and control process, such as unstable effects, resistance of pathogens to multiple fungicides, impact on the ecological environment and safety of agricultural products. The development and use of biological pesticides to prevent and control potato scab is expected to solve the above-mentioned problems.

[0003] As an important biological control strain, Bacillus amyloliquefaciens inhibits or kills pathogens through interspecific competition, lysis, antagonism, etc., thereby achieving the effect of biological control, and also has the effect of promoting plant growth. Plant diseases that have been reported to be controlled by Bacillus amyloliquefaciens include: tobacco bacterial wilt, tomato gray mold, cotton wilt, watermelon wilt, anthrax, etc. Therefore, developing a Bacillus amyloliquefaciens biopesticide for controlling potato scab disease is a technical problem that those skilled in the art urgently need to solve. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of Bacillus amyloliquefaciens B2 and its suspension concentrate in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect provides a Bacillus amyloliquefaciens, wherein the Bacillus amyloliquefaciens is named B2 and has a deposit number of CGMCC No.30634.

[0007] The second aspect is to provide the use of the above-mentioned Bacillus amyloliquefaciens in the preparation of a preparation for preventing and treating potato scab.

[0008] Furthermore, the potato scab control preparation is a Bacillus amyloliquefaciens B2 suspension.

[0009] Furthermore, the suspension is 2-3 billion CFU / ml of Bacillus amyloliquefaciens B2 suspension.

[0010] Furthermore, the suspension is a 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension.

[0011] The third aspect is to provide a Bacillus amyloliquefaciens B2 suspension, comprising the following components in weight percentage: 30-50wt% of Bacillus amyloliquefaciens B2 liquid; 1-10wt% of ProDisCHK dispersant; 1-10wt% of ProDis2076 emulsifying dispersant; 0.5-5wt% of Langtai LT-980 suspension stabilizer; 1-5wt% of antifreeze agent; 0.1-0.5wt% of defoaming agent; and the balance of process water.

[0012] The fourth aspect is to provide a method for preparing the above-mentioned Bacillus amyloliquefaciens B2 suspension, comprising the following steps:

[0013] Step 1: Activation of bacteria;

[0014] Step 2: Inoculate the activated strain into the seed culture medium, and perform shaking culture at 30-32° C. and a shaking speed of 200-250 rpm for 18-24 hours to obtain a seed solution;

[0015] Step 3, inoculating the seed solution obtained in step 2 into the fermentation medium at a volume ratio of 1-3%; fermenting and culturing for 48-64 hours under shaking conditions at 30-32° C. and a rotation speed of 200-250 rpm, and stopping the fermentation and culturing when the spore rate reaches more than 90%, thereby obtaining a fermentation liquid of Bacillus amyloliquefaciens B2;

[0016] Step 4, removing the culture medium residue in the fermentation liquid obtained in step 3;

[0017] Step 5, mixing the weighed auxiliary agent and process water, stirring evenly with a shear emulsifier at high speed, and then sand milling with a sand mill for 2-3 hours, and then adding the Bacillus amyloliquefaciens B2 fermentation liquid obtained by the step 4, shearing and stirring evenly, and filtering to obtain the Bacillus amyloliquefaciens B2 suspension.

[0018] Furthermore, the specific steps of strain activation are: streak Bacillus amyloliquefaciens B2 on a PDA plate culture medium, culture at 36°C for 24 hours, pick out a single colony on a PDA plate culture medium, and culture at 36°C for 24 hours to obtain an activated strain.

[0019] Furthermore, the preparation method of the seed culture medium is specifically as follows: prepare a PDB liquid culture medium according to potato: glucose: water = 10g: 1g: 50mL, sterilize by high pressure wet heat, cool down, and set aside.

[0020] Furthermore, the preparation method of the fermentation medium is specifically as follows: 1.0% cottonseed meal, 1.5% peanut meal, 0.35% NaCl, and MnSO 4 ·H 2 Add 0.8% O into water, stir to mix evenly, sterilize, cool down and set aside.

[0021] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0022] The present invention finds that Bacillus amyloliquefaciens B2 has an obvious ability to antagonize potato scab (Streptomyces scabies) on a plate, kills pathogenic bacteria through the antibacterial substances produced by itself, and has a significant prevention and control effect on pathogens at a low concentration.

[0023] The present invention obtains a B2 fermentation broth with a concentration of ≥ 8 billion / mL by optimizing the fermentation medium formula and various process parameter conditions; and develops a 2 billion / mL Bacillus amyloliquefaciens B2 suspension concentrate through auxiliary agent screening and auxiliary agent compatibility testing.

[0024] The 2 billion / mL Bacillus amyloliquefaciens B2 suspension of the present invention is a live microbial pesticide, which has the characteristics of strong selectivity, safety to the human and animal environment, wide source of raw materials, not easy to produce drug resistance, no killing of natural enemy insects, and little impact on the ecology, and meets the requirements of modern society for agricultural production and pesticides. Field tests show that the 2 billion / mL Bacillus amyloliquefaciens B2 suspension of the present invention has a good biocontrol effect on potato scab and significantly improves potato yield and farmers' income. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention shows the antagonistic effect of Bacillus amyloliquefaciens B2 on potato scab.

[0026] Figure 2 This is the control effect of the 2 billion CFU / mL Bacillus amyloliquefaciens B2 suspension of the present invention in a plot test. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0028] Example 1

[0029] 1. Isolation and screening of Bacillus amyloliquefaciens B2

[0030] 1. Preparation of experimental materials

[0031] 1.1 Soil sample collection: Soil samples were collected from the rhizosphere of eggplants in a vegetable greenhouse in Pudong New District, Shanghai.

[0032] 1.2 Culture medium

[0033] NA medium: peptone 1%, beef powder 0.3%, NaCl 0.5%, agar 2%, pH 7.0-7.2

[0034] NB medium: peptone 1%, beef powder 0.3%, NaCl 0.5%, pH 7.0-7.2;

[0035] Fermentation medium: 2% cottonseed meal, 2% corn starch, 0.25% yeast powder, MgSO 4 0.05%、K 2 HPO 4 0.2%.

[0036] 1.3 Experimental equipment

[0037] Mortar, oven, vortex oscillator, coating rod, constant temperature incubator, shaker.

[0038] 2. Strain Screening

[0039] 2.1 Strain Isolation

[0040] Place the soil sample in a sterilized mortar and grind it with a grinding rod until the soil particles are uniform. Place the soil in an oven at 80°C for 30 minutes, weigh 1g of the soil sample and add it to 9ml of sterile water. Vortex it thoroughly. Take 1ml of the above solution and add it to 9ml of sterile water. Mix it on a vortex to form a 10 -2 of soil dilution, and so on up to 10 -3 , 10 -4 , 10 -5 . Take 10 -2 , 10 -3 , 10 -4 , 10 -5 Add 100ul of the soil dilution to the NA culture medium separation plate and spread the dilution evenly with a spreading rod.

[0041] 2.2 Cultivation and purification

[0042] The above separation plate was placed in a constant temperature incubator at 37°C for 24 hours until a single colony grew out, the colonies with morphological characteristics of Bacillus colonies were marked and single colonies were picked out in time to NA culture medium plates, purified by streak separation method and transferred to a constant temperature incubator at 37°C, the single colonies after purification and culture were transferred to the test tube slant of NA culture medium and cultured at 37°C for 24 hours.

[0043] 2.3 Fermentation culture

[0044] Add 100ml NB medium to each 500ml flask, and use an inoculation loop to pick up a small amount of Bacillus amyloliquefaciens B2 cells and inoculate them into the flask. Cultivate the flask at 37°C and 200r / min shaking for 24h as a seed bottle, inoculate the seed liquid into the fermentation medium (100ml / 500ml) at a ratio of 5%, and culture the flask at 37°C and 200r / min shaking for 60h.

[0045] 2. Identification of Bacillus amyloliquefaciens B2

[0046] The Bacillus amyloliquefaciens strain B2 in the present invention is detected and identified by the Institute of Microbiology, Chinese Academy of Sciences. According to comprehensive analysis of experimental data such as bacterial morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, gyrB gene sequence, etc., and with reference to Bergey's Handbook of Systematic Bacteria and relevant research papers in International Journal of Systematic and Evolutionary Microbiology, the identification result is confirmed to be: Bacillus amyloliquefaciens.

[0047] 2.1 Bacterial morphology, physiological and biochemical characteristics

[0048] According to Bergey's Manual of Systematic Bacteriology, the bacterial morphology, physiological and biochemical characteristics of the strains were determined, as shown in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] 2.216S rRNA gene identification

[0053] The gyrB gene sequence of Bacillus amyloliquefaciens B2 is shown in SEQ ID NO.1, and the 16SrRNA gene sequence of Bacillus amyloliquefaciens B2 is shown in SEQ ID NO.2.

[0054] Example 2 Test on the antagonistic effect of strain B2 on potato scab

[0055] Source of potato scab for testing: The pathogenic strain was collected from diseased plants with a scab incidence rate of more than 50% in the main potato producing area of ​​Ordos, Inner Mongolia. It was separated and purified by Shanghai Wanlihua Biotechnology Co., Ltd. and identified as Streptomyces through morphology, physiological and biochemical characteristics and 16S rDNA sequence.

[0056] Activation of strains: streak Bacillus amyloliquefaciens B2 (CGMCC No. 30634) stored at -50°C on a PDA plate medium (its components and weight ratios are: 200 g potato, 20 g glucose, 20 g agar powder, 1000 mL water), culture at 36°C for 24 h, pick out single colonies and place them on a PDA plate medium, culture at 36°C for 24 h to obtain an activated strain.

[0057] Test method: Mix the bacterial liquid of potato scab pathogen with NA medium cooled to 45℃ and put it on the plate. After the plate solidifies, pick a single colony of Bacillus amyloliquefaciens B2 and inoculate it on both sides of the plate containing the scab pathogen. Set up a blank control (no B2 strain inoculation). Place the inoculated plate at a constant temperature of 30℃ for culture. When the blank control is about to grow all over the culture dish, measure the diameter of the inhibition zone. The antagonistic effect is expressed by the diameter of the inhibition zone. The test results are shown in Table 2.

[0058] Results (Table 2, Figure 1 ) The inhibition bandwidth of Bacillus amyloliquefaciens B2 strain against potato scab pathogen is 11-15 mm, indicating that the Bacillus amyloliquefaciens B2 of the present invention has a significant inhibitory effect on potato scab pathogens and has the potential for preventing and controlling potato scab biocontrol.

[0059] Table 2

[0060] deal with Diameter of inhibition zone (mm) Antibacterial index Pathogen of scab in Hailaer 13.1 +++ Blank control — —

[0061] Note: The antibacterial index is "+" when the antibacterial diameter is 1-5mm, the antibacterial index is "++" when the antibacterial diameter is 6-10cm, and the antibacterial index is "+++" when the antibacterial diameter is 11-15mm.

[0062] Example 3 Preparation of Bacillus amyloliquefaciens B2 fermentation broth

[0063] (1) Activation of bacteria: The same as the method of activating bacteria in Example 1.

[0064] (2) Preparation of seed culture medium: PDB liquid culture medium (its components and their weight ratios are: 200 g potato, 20 g glucose, 1000 mL water) was prepared according to conventional methods, 100 mL of PDB culture medium was placed in a 500 mL Erlenmeyer flask, and sterilized by high pressure wet heat. After the temperature dropped to room temperature, an inoculation loop of the strain activated in step (1) was inoculated into each flask, and shaking culture was carried out at 30° C. and a shaking speed of 200 rpm for 24 hours to obtain seed liquid.

[0065] (3) Preparation of fermentation medium: 1.0% cottonseed meal, 1.5% peanut meal, 0.35% NaCl, and 0.5% MnSO 4 ·H 2 Add 0.8% O into water and stir to mix well to obtain the fermentation medium; divide into 1000mL conical flasks, 200mL per bottle; sterilize at 121℃ for 30 minutes, then cool to below 40℃ for use.

[0066] Fermentation culture conditions: 2 mL of the seed solution obtained in step (2) is inoculated into the fermentation medium obtained in step (3) at a volume ratio of 1%; the fermentation culture is carried out under shaking conditions at 30° C. and 200 rpm for 48 h, and then samples are taken from the conical flask every 30 minutes for microscopic examination, the spores and the total number of bacteria in the field of view are counted, and the spore rate is calculated (spore rate (%) = number of mature spores / (number of mature spores+number of bacteria)×100); the fermentation culture is stopped when the spore rate reaches more than 90%; the fermentation culture is carried out for about 60 h to obtain the fermentation liquid of Bacillus amyloliquefaciens B2.

[0067] Example 4 Preparation of Bacillus amyloliquefaciens B2 suspension

[0068] (1) The fermentation broth of Bacillus amyloliquefaciens B2 obtained in Example 2 was centrifuged using a WZ30 horizontal screw centrifuge with the parameters set at a main shaft speed of 3000 and a secondary shaft speed of 1000 to remove the culture medium residue of the fermentation broth of Bacillus amyloliquefaciens B2. The spore quantity was 7 to 8 billion CFU / ml.

[0069] (2) The formula of Bacillus amyloliquefaciens B2 suspension concentrate includes the following components in weight percentage: 30-50wt% of Bacillus amyloliquefaciens B2 hair liquid; 1-10wt% of ProDisCHK dispersant; 1-10wt% of ProDis2076 emulsifying dispersant; 0.5-5wt% of Langtai LT-980 suspension stabilizer; 1-5wt% of antifreeze agent; 0.1-0.5wt% of defoaming agent; and the balance of process water.

[0070] (3) The process flow is as follows: the weighed auxiliary agent and process water are mixed, the mixture is stirred evenly at high speed by a shear emulsifier, and then sand-milled for 2 to 3 hours by a sand mill. Then, the fermentation liquid of Bacillus amyloliquefaciens B2 is added, sheared and stirred evenly, and then filtered (100 mesh) to obtain a 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension.

[0071] Table 3 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension concentrate test items

[0072]

[0073] Example 5: Plot test of the efficacy of 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension on potato scab

[0074] Experimental location: The community experiment in Shanghai was located in the Sunqiao greenhouse, in accordance with local agricultural operation standards;

[0075] Test target: Potato scab;

[0076] Crop variety: Zhongshu No. 1;

[0077] Test agents: 200 million CFU / g Trichoderma wettable powder, 6 billion CFU / ml Bs-3 suspension, 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension, 40% carbendazim suspension;

[0078] Table 4 Experimental design of test drugs

[0079]

[0080]

[0081] Plot arrangement: experimental drugs, control drugs and blank controls are arranged in random blocks;

[0082] Plot size and replication: 6 potatoes per plot, 3 replications;

[0083] Application method: root irrigation;

[0084] Time and number of application: first time: March 14; second time: March 24; third time: April 3; fourth time: April 13.

[0085] Pathogen inoculation: 24 hours after the first application, the solid pathogen of potato scab was diluted with water to prepare 1×10 7 cfu / mL of spore suspension, 20mL per injection.

[0086] Survey method, time and frequency:

[0087] When potatoes are harvested, the incidence of scab disease on each potato is investigated and the grading standards are:

[0088] Level 0, potato skin is healthy;

[0089] Level 1: The potato skin is basically healthy, with 1 to 2 sporadic spots, the area of ​​which does not exceed 1 / 4 of the potato skin surface area;

[0090] Level 2: There are 3 to 5 lesions on the potato skin, covering an area of ​​1 / 4 to 1 / 3 of the potato skin surface;

[0091] Level 3: There are 5 to 10 lesions on the potato skin, covering an area of ​​1 / 3 to 1 / 2 of the potato skin surface area;

[0092] Level 4: Severe disease, with more than 10 lesions or the area of ​​lesions exceeding 1 / 2 of the potato peel surface area.

[0093] Pharmacodynamics calculation method:

[0094] The control effect is calculated according to the following formula:

[0095]

[0096] The results of the plot test showed that the use of Ba-2, Bs-3 and Trichoderma could alleviate the condition of potato scab to a certain extent. At the same multiple of use, the effect of Ba-2 was better than that of Bs-3 and Trichoderma. The control effect of Ba-2 100 times treatment on scab was as high as 65.54%, which was significantly better than other treatments. The control effect decreased with the decrease of the dosage. The control effects of Trichoderma and Bs-3 100 times on scab were 43.19% and 45.69% respectively, which were higher than chemical drugs (40.35%), but the difference in the control effect between the two was not significant (Table 5, Figure 2 ).

[0097] Applying Ba-2 (100 times application) one week before the early stage of potato stem formation and applying it 4 times every 7-10 days can greatly reduce the disease of potato scab.

[0098] Table 5

[0099] deal with disease finger Prevention effect / % Trichoderma 100 times 26.19 43.19bc Trichoderma 200 times 28.30 38.17abc Trichoderma 400 times 32.70 28.75a Ba-2100 times 15.82 65.54d Ba-2200 times 26.64 42.12bc Ba-2400 times 30.89 32.36ab Bs-3100 times 25.03 45.69c Bs-3200 times 30.71 32.93ab Chemical drug control 27.42 40.35bc Pathogen Control 46.06 /

[0100] Example 6 Large-area efficacy test of 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension on potato scab

[0101] Experimental location: Hailar Farm, Inner Mongolia;

[0102] Trial time: October 2022;

[0103] Test area: 150 mu was treated with Bacillus amyloliquefaciens preparation, and the rest was treated with chemical drugs;

[0104] Variety: McCann No. 7;

[0105] Usage: (1) Furrow application, diluted 100 times; (2) Leaf spraying before flowering, diluted 100 times;

[0106] Table 6. Demonstration effect of Bacillus amyloliquefaciens B2 preparation and conventional chemical treatment on potato scab in Hailar Farm

[0107] deal with Average incidence Average disease index Relative efficacy (compared with chemical drugs) Conventional control (copper preparations) 91.19％ 42.05 - Bacillus amyloliquefaciens preparation 21.59％ - 75.22％

[0108] Note: Each treatment is carried out in a Z-shaped pattern with 12 points, and each point is 1m 2 Potato plot measurement.

[0109] The incidence of potato scab disease was higher when chemical drugs were used than when Bacillus amyloliquefaciens was used, and the disease index was more serious. During the observation process, potatoes treated with Bacillus amyloliquefaciens were better than those treated with chemical drugs in terms of severity and quantity of disease.

[0110] Example 7 Demonstration test of improving potato quality and yield with 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension

[0111] Experimental location: Daheihe Township, Siziwang Banner;

[0112] Trial time: May 2022;

[0113] Test area: 20 mu;

[0114] Potato seed information: The potato variety used in the demonstration plot is Xisen No. 6 (smooth skin).

[0115] Table 7 Demonstration of the use of Bacillus amyloliquefaciens B2 suspension on potatoes

[0116]

[0117] The demonstration results are shown in Table 8-9 below:

[0118] Table 8 Potato (Xisen No. 6) yield statistics (survey area: 3m per treatment) 2 )

[0119]

[0120] Table 9 Potato (Xisen No. 6) Benefit Statistics

[0121]

[0122] From the potato promotion demonstration in 2022, it can be seen that the application of 2 billion CFU / ml Bacillus amyloliquefaciens B2 suspension has a significant effect on increasing potato production. Compared with the control, the rate of large potatoes increased by 17.3%, the yield increased by 3.5%, and the actual income per mu of farmers increased by about 550 yuan.

[0123] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens, characterized in that The Bacillus amyloliquefaciens is named as B2 and has a preservation number of CGMCC No.30634.

2. Use of Bacillus amyloliquefaciens as claimed in claim 1 in preparing a preparation for preventing and treating potato scab.

3. The use according to claim 2, characterized in that: The potato scab prevention and treatment preparation is a Bacillus amyloliquefaciens B2 suspension.

4. The use according to claim 3, characterized in that: The suspension is 2-3 billion CFU / ml of Bacillus amyloliquefaciens B2 suspension.

5. The use according to claim 4, characterized in that: The suspension is 2 billion CFU / ml of Bacillus amyloliquefaciens B2 suspension.

6. A Bacillus amyloliquefaciens B2 suspension concentrate, characterized in that The invention comprises the following components in weight percentage: 30-50wt% of Bacillus amyloliquefaciens B2 hair liquid; 1-10wt% of ProDisCHK dispersant; 1-10wt% of ProDis2076 emulsifying dispersant; 0.5-5wt% of Langtai LT-980 suspension stabilizer; 1-5wt% of antifreeze agent; 0.1-0.5wt% of defoaming agent; and the balance of process water.

7. The preparation method of the Bacillus amyloliquefaciens B2 suspension concentrate as claimed in claim 6, characterized in that, The steps include: Step 1: Activation of bacteria; Step 2: Inoculate the activated strain into the seed culture medium, and perform shaking culture at 30-32° C. and a shaking speed of 200-250 rpm for 18-24 hours to obtain a seed solution; Step 3, inoculating the seed solution obtained in step 2 into the fermentation medium at a volume ratio of 1-3%; fermenting and culturing for 48-64 hours under shaking conditions at 30-32° C. and a rotation speed of 200-250 rpm, and stopping the fermentation and culturing when the spore rate reaches more than 90%, thereby obtaining a fermentation liquid of Bacillus amyloliquefaciens B2; Step 4, removing the culture medium residue in the fermentation liquid obtained in step 3; Step 5, mixing the weighed auxiliary agent and process water, stirring evenly with a shear emulsifier at high speed, and then sand milling with a sand mill for 2-3 hours, and then adding the Bacillus amyloliquefaciens B2 fermentation liquid obtained by the step 4, shearing and stirring evenly, and filtering to obtain the Bacillus amyloliquefaciens B2 suspension.

8. The preparation method according to claim 7, characterized in that: The specific steps of strain activation are: streak Bacillus amyloliquefaciens B2 on a PDA plate culture medium, culture at 36°C for 24 hours, pick out a single colony and culture it on a PDA plate culture medium at 36°C for 24 hours to obtain an activated strain.

9. The preparation method according to claim 7, characterized in that: The preparation method of the seed culture medium is specifically as follows: preparing a PDB liquid culture medium according to potato: glucose: water = 10 g: 1 g: 50 mL, sterilizing by high pressure wet heat, cooling, and setting aside.

10. The preparation method according to claim 7, characterized in that: The preparation method of the fermentation medium is specifically as follows: adding 1.0% cottonseed cake powder, 1.5% peanut cake powder, 0.35% NaCl and 0.8% MnSO4·H2O into water according to weight percentage, stirring and mixing evenly, sterilizing, cooling and setting aside.

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