A compound microbial agent, a method for its on-site preparation and application, and use thereof

Abstract

The application discloses a kind of compound microbial inoculant and its method and application of present use.The application discloses a bacillus proteolytic XZT198 with accession number CCTCC M 20232321.The compound microbial inoculant disclosed by the application comprises the bacillus proteolytic XZT198 and bacillus velezensis XY40-1 with accession number CCTCC NO:M 2022342.The method disclosed by the application is to use microbial inoculant water and fertilizer integrated drip irrigation system, which can be used in field fermentation, maximize the biological activity of compound microbial inoculant, and avoid the loss of microbial inoculant activity in traditional microbial inoculant preparation process and transportation process.The compound microbial inoculant provided by the application uses crop straw, inferior fruits and vegetables and brown sugar as fermentation substrate, which is simple to operate, low in raw material cost, can greatly reduce the cost per mu in agricultural production, green and environmentally friendly, practical, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of green and efficient agricultural production, and specifically relates to a composite bacterial agent and a method and application of preparing and using the same. Background Art

[0002] In agricultural production, chemical pesticides can easily cause soil compaction, leading to the death of soil microorganisms, causing environmental pollution, a series of food safety issues, and serious harm to human health. With increasing demands for crop yield and quality, many technologies and processes related to agricultural production are undergoing continuous innovation, improvement, and development, and fertilizer is a key component of these technological improvements. Currently, agricultural fertilizers have evolved into the third generation, with microbial fertilizers being the main representative.

[0003] The primary functional component of microbial fertilizers is biologically active bacteria. Leveraging their degradation activity, they promote the efficient circulation of various nutrients, reduce the use of chemical fertilizers, and improve crop production efficiency. Microbial fertilizers also utilize the metabolic activity of various active bacteria and their metabolites to achieve benefits such as stress resistance, disease prevention, and growth promotion. Microbial fertilizers can reduce the use of chemical fertilizers and pesticides, contributing to increased crop yields, improved agricultural product quality, and ecological conservation. They are economical, environmentally friendly, and safe, and have broad application prospects in modern agricultural production.

[0004] However, the current market for microbial inoculants is diverse and of varying quality. The primary functional component of microbial inoculants is the active bacteria within them, so the activity of the strain significantly impacts their effectiveness. After fermentation, the activity gradually decreases. Microbial inoculants are categorized into three dosage forms: powders, granules, and liquids. Both powders and granules require drying the fermentation broth into a powder, a process that reduces bacterial activity and results in suboptimal efficacy. Therefore, enhancing and maintaining the biological activity of microbial inoculants to ensure their effectiveness is a pressing issue. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a Bacillus proteolyticus XZT198, which has an antagonistic effect on plant Ralstonia solanacearum.

[0006] The second object of the present invention is to provide a composite bacterial agent, which aims to reduce the use of chemical pesticides, promote crop growth, improve soil conditions, reduce the impact of continuous cropping obstacles and enhance crop resistance.

[0007] The third object of the present invention is to provide a method and application for preparing and using the above-mentioned composite bacterial agent immediately, so as to retain the activity of the composite bacterial agent to the greatest extent and improve the effect of the composite bacterial agent.

[0008] To achieve the above objectives, the present invention provides the following technical solutions:

[0009] The present invention first provides a Bacillus proteolyticus XZT198, which was deposited in the China Center for Type Culture Collection in Wuhan, Hubei on November 24, 2023, with a deposit number of CCTCC M 20232321. The strain has a good antagonistic effect on plant Ralstonia solanacearum.

[0010] The present invention also provides a composite bacterial agent, comprising Bacillus proteolyticus XZT198 and Bacillus velezensis XY40-1 with a preservation number of CCTCC NO: M2022342.

[0011] The Bacillus Velezii XY40-1 was deposited in the China Center for Type Culture Collection on March 29, 2022, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M 2022342.

[0012] Furthermore, in the composite bacterial agent, the total effective viable count of Bacillus proteolyticus XZT198 and Bacillus velezensis XY40-1 is 5.5 to 6.5 billion CFU / ml.

[0013] The composite bacterial agent is an original bacterial liquid that has not been concentrated.

[0014] The present invention also provides a method for preparing and using the composite bacterial agent, comprising the following steps:

[0015] 1) Strain activation: Bacillus proteolyticus XZT198 and Bacillus velez XY40-1 were inoculated onto LB solid medium, respectively, and cultured at a constant temperature of 28-30° C. for 22-24 hours to obtain activated Bacillus proteolyticus XZT198 and Bacillus velez XY40-1;

[0016] 2) Fermentation seed culture: Activated Bacillus proteolyticus XZT198 and Bacillus velezensis XY40-1 were inoculated into seed culture medium, respectively, and cultured at 35-37° C. and 170-190 rpm on a shaker for 22-24 hours, and then mixed according to a preset volume ratio to obtain a mixed seed solution;

[0017] 3) On-site fermentation: Add the mixed seed liquid to a simple fermentation tank in the integrated water and fertilizer drip irrigation system of the microbial agent at a preset inoculum volume, add radish dregs, brown sugar, PQQ, and lactopeptide, and ferment under film at room temperature for 7-10 days, mixing every two days to obtain a composite microbial agent;

[0018] 4) Transfer and storage: After fermentation is completed, the composite bacterial agent in the simple fermentation tank is transferred through a pipeline to the fertilizer and water bacterial agent storage tank of the bacterial agent, water and fertilizer integrated irrigation system;

[0019] 5) Drip irrigation application: The composite bacterial agent in the fertilizer and water agent storage tank is drained into the fertilizer and water compound dilution equipment of the bacterial agent water and fertilizer integrated irrigation system, diluted with water, and introduced into the drip irrigation equipment at a set rate to complete drip irrigation.

[0020] Furthermore, in step 1), the formula of the LB solid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, and a pH of 7.2-7.3.

[0021] Furthermore, in step 2), the preset volume ratio is 2:1; the formula of the seed culture medium is: 20 g / L peptone, 10 g / L sodium chloride, 10 g / L yeast extract, 50 mmol / L zinc sulfate, 1000 nmol / LPQQ (pyrroloquinoline quinone), and the pH is 7.2-7.3.

[0022] Furthermore, in step 3), the preset inoculation amount is 0.15% to 0.25%; the mixed seed liquid is a late logarithmic fermentation liquid; the concentration of the PQQ after addition is 800 to 1000 nmol / L, the concentration of the lactose peptide after addition is 0.2 to 0.4%, the amount of vegetable waste and brown sugar used is 8 to 12% of the volume of the simple fermentation tank, the ratio of brown sugar to vegetable waste used is 11:10 to 13:10, and river water or tap water is used to make up the difference.

[0023] Furthermore, in step 4), the integrated microbial agent, water and fertilizer drip irrigation system includes a simple fermentation tank, a fertilizer and water microbial agent storage tank, a fertilizer and water compound dilution device, and a drip irrigation system; the simple fermentation tank is connected to the fertilizer and water microbial agent storage tank through a pipeline, the fertilizer and water microbial agent storage tank is connected to the fertilizer and water compound dilution device through a pipeline, and the fertilizer and water compound dilution device is connected to the drip irrigation system.

[0024] Furthermore, in step 5), the dilution ratio of water is 1:(50-100), and the rate of adding fertilizer water to the compound dilution equipment is 1.8-2.1 L / h.

[0025] Specifically, in actual use, as needed, in the six rounds of drip irrigation in one growing season of the crop, feeding and re-fermentation are carried out in the simple fermentation tank before the third and fifth rounds of drip irrigation.

[0026] Furthermore, the feed needs to be supplemented with brown sugar, mixed seed liquid and water. The feeding amount of brown sugar is 2% to 3% of the volume of the simple fermentation tank. The inoculation amount of the mixed seed liquid is 0.075 to 0.125% when supplementing. River water or tap water is used to make up the difference.

[0027] The application of the complex microbial agent in the field of microbial agents or microbial fertilizers.

[0028] Advantages of the present application:

[0029] (1) The present application first discovers the antagonistic effect of Bacillus proteolyticus on Ralstonia solanacearum, and the complex microbial agent prepared by using Bacillus proteolyticus and Bacillus velezensis has the effects of improving soil conditions, promoting crop growth, and preventing disease occurrence, especially the high-efficiency prevention effect of Bacillus proteolyticus on Ralstonia solanacearum of peppers, tomatoes, and tobacco.

[0030] (2) The present application realizes on-site fermentation and compounding of the microbial agent by constructing a microbial agent water and fertilizer integrated drip irrigation system, and the obtained microbial liquid has high activity concentration and strong timeliness, and can be directly used after fermentation, thereby reducing the loss and function reduction in the shelf period.

[0031] (3) The microbial agent water and fertilizer integrated drip irrigation system provided by the present application can reduce fertilizer and water consumption, save labor and effort, and by applying the technology of the present application, the labor cost per mu can be reduced by 40% to 60%, and the production quality can be improved by more than 30%. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a plate front colony morphology of Bacillus proteolyticus XZT198;

[0033] Figure 2 It is an evolutionary tree analysis of Bacillus proteolyticus XZT198;

[0034] Figure 3 It is an antagonistic effect diagram of Bacillus proteolyticus XZT198 on Ralstonia solanacearum, wherein A is Ralstonia solanacearum, and B is the confrontation culture effect diagram of Bacillus proteolyticus XZT198 and Ralstonia solanacearum;

[0035] Figure 4 It is a front view of a microbial agent water and fertilizer integrated drip irrigation system except for a drip irrigation system;

[0036] Figure 5 It is a plane schematic diagram of a microbial agent water and fertilizer integrated drip irrigation system;

[0037] Figure 6 It is a field effect diagram after drip irrigation of a present-compound present-use complex microbial agent. DETAILED DESCRIPTION

[0038] Example 1 Isolation and screening of bacteria

[0039] The isolation and screening method of Bacillus proteolyticus XZT198 of the present invention comprises the following steps: collecting soil from the Tibetan Plateau, isolating and purifying bacteria in the soil by using a dilution spread plate method, using Ralstonia solanacearum in pepper as a target bacterium, and screening a strain capable of inhibiting the growth of Ralstonia solanacearum in pepper through a plate confrontation experiment. Figure 2 This is the colony morphology of Bacillus proteolyticus XZT198 on the front of the plate.

[0040] Example 2 Identification of bacteria

[0041] (1) The morphological, cultural, physiological, and biochemical characteristics of this strain are as follows:

[0042] Strain XZT198 is a Gram-positive bacterium. After three days of culture on LB medium, the strain is off-white, opaque, moist, with radially shaped edges and a shiny, wrinkle-free surface, making it easily picked up with an inoculating loop. Strain XZT198 can liquefy gelatin, reduce nitrates, and hydrolyze glucose, maltose, and starch. However, the indole test is negative.

[0043] The results of strain 16S rRNA gene sequence determination are shown in SEQ ID: 1.

[0044] The strain was streaked and purified on an LB plate, and the 16S rRNA gene was amplified using bacterial universal primers (sequences shown in SEQ ID: 2 and SEQ ID: 3). The PCR product was sent for sequencing and the obtained gene sequence was compared and analyzed in NCBI. The results showed that the similarity between strain XZT198 and Bacillus proteolyticus reached 99.93%, and its morphological characteristics and physiological and biochemical properties were most similar to those of Bacillus proteolyticus. Therefore, based on the 16S rRNA gene sequence determination and morphological characteristics, physiological and biochemical characteristics, this strain can be classified as Bacillus proteolyticus.

[0045] The strain was named Bacillus proteolyticus XZT198, with the preservation number CCTCC M 20232321, and was deposited in the China Center for Type Culture Collection in Wuhan, Hubei on November 24, 2023.

[0046] Example 3 Antagonistic Experiment of Bacillus proteolyticus XZT198 against Ralstonia solanacearum

[0047] The plate confrontation method was used: Ralstonia solanacearum was inoculated in the center of the PDA plate, and then the strain Bacillus proteolyticus XZT198 was inoculated 2 cm away from the center of the culture medium using the cross method; the control group was inoculated with pathogens but not Bacillus proteolyticus XZT198, and cultured at a constant temperature of 28°C. After the pathogens in the control group covered the plate, the width of the inhibition zone was measured, and each group was treated 6 times. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter * 100%. One of the treatment groups was compared with the control group as follows. Figure 3 shown.

[0048] Table 1 Inhibitory effect of Bacillus proteolyticus XZT198 on Ralstonia solanacearum

[0049]

[0050] The experimental results in Table 1 show that Bacillus proteolyticus XZT198 can effectively inhibit the growth of Ralstonia solanacearum, with an inhibition efficiency of 82.3%.

[0051] Example 4: Application of Composite Bacterial Agent Prepared Immediately

[0052] This example will apply the compound microbial agent to the drip irrigation of pepper crops in 50 mu of greenhouse vegetable fields from planting to harvesting. The specific process is as follows:

[0053] 1) Strain activation: Bacillus proteolyticus XZT198 and Bacillus velez XY40-1 with the accession number of CCTCC M 20232321 were inoculated into LB medium, respectively, and cultured at 28° C. for 24 h to obtain activated Bacillus proteolyticus and Bacillus velez.

[0054] 2) Fermentation seed culture: The activated Bacillus proteolyticus and Bacillus velezensis were inoculated into LB liquid medium, shaken at 180 rpm and 37°C for 24 h, and then mixed in a 2:1 volume ratio to obtain a mixed seed solution;

[0055] 3) On-site fermentation: add 10ml of mixed seed liquid at an inoculum volume of 18L 3 In a simple fermentation tank, add radish, brown sugar, PQQ, and lactose peptide, ferment under film covering at room temperature for 8 days, and stir and mix once every two days to obtain a composite bacterial agent;

[0056] The concentration of lactose peptide was 0.3%, and the amount of vegetable waste was 0.45m 3 , the input amount of brown sugar is 0.55m 3 , the PQQ input amount is 10mmol.

[0057] 4) Transfer the composite bacterial agent in the simple fermentation tank to the fertilizer bacterial agent storage tank through a pipeline;

[0058] 5) The compound bacterial agent is then drained into the fertilizer-water compound dilution equipment, diluted 50 to 100 times with water, and then introduced into the drip irrigation equipment to complete drip irrigation of 50 mu of facility vegetable fields, with a drip irrigation volume of 1 to 2 tons per mu.

[0059] The microbial agent prepared in this example was tested, and it was found that the effective viable bacteria count in the original bacterial solution of the undiluted microbial agent was 6 billion CFU / ml.

[0060] Drip irrigation was carried out for 6 rounds according to actual conditions during the planting period, flowering period, early fruiting period and mid-fruiting period.

[0061] After planting, soil samples were taken from the fields to measure basic physical and chemical properties, microbial composition, and the incidence of common soil-borne diseases of peppers. The results are shown in Tables 2 and 3. Yield statistics and quality analysis were then conducted on the harvested peppers, with the results shown in Table 4.

[0062] The schematic diagram of the microbial agent water and fertilizer integrated drip irrigation system is as follows: Figure 6 As shown, the front view of the part other than the drip irrigation system is as follows Figure 5 shown.

[0063] Comparative Example 1

[0064] Peppers were planted on a facility vegetable plot of the same area as in Example 4, and drip irrigation was performed using a 5000-fold dilution of 25% chloramphenicol wettable powder at a rate of 1-2 tons per mu, with the same number of drip irrigations as in Example 4.

[0065] After planting, soil samples were taken from the fields to measure basic physical and chemical properties, microbial composition, and the incidence of common soil-borne diseases of peppers. The results are shown in Tables 2 and 3. Yield statistics and quality analysis were then conducted on the harvested peppers, with the results shown in Table 4.

[0066] Comparative Example 2

[0067] Peppers were planted on a field of the same area as in Example 4, and drip irrigation was performed using 100 million CFU / mL Bacillus subtilis microbial agent fertilizer. The drip irrigation amount was 1-2 tons / mu, and the number of drip irrigations was the same as in Example 4.

[0068] After planting, soil samples were taken from the fields to measure basic physical and chemical properties, microbial composition, and the incidence of common soil-borne diseases of peppers. The results are shown in Tables 2 and 3. Yield statistics and quality analysis were then conducted on the harvested peppers, with the results shown in Table 4.

[0069] Comparative Example 3

[0070] Peppers were planted on a field of the same area as in Example 4, and drip irrigation was performed using clean water at a rate of 1-2 tons per mu and the same number of drip irrigations as in Example 4.

[0071] After planting, soil samples were taken from the fields to measure basic physical and chemical properties, microbial composition, and the incidence of common soil-borne diseases of peppers. The results are shown in Tables 2 and 3. Yield statistics and quality analysis were then conducted on the harvested peppers, with the results shown in Table 4.

[0072] Table 2 Changes in soil physical and chemical properties under different treatments

[0073]

[0074] The results of soil chemical properties and nutrient content measurements showed that the soil hydrolyzable nitrogen, available phosphorus, and available potassium contents of Example 4 were 103.8 mg / kg, 221.8 mg / kg, and 565.3 mg / kg, respectively, all significantly higher than those of the three comparative examples, and significantly higher than those of Comparative Example 3 by 17.2%, 41.4%, and 75.1%, respectively. The soil exchangeable calcium and exchangeable magnesium contents of Comparative Example 2 were the highest among the four groups, followed by Example 4, which had an exchangeable calcium content of 18.1 cmol / kg and an exchangeable magnesium content of 5.73 cmol / kg. There was no significant difference in soil pH between Example 4 and Comparative Examples 1, 2, and 3. Overall, Example 4, i.e., the composite bacterial agent treatment, significantly promoted the improvement of soil nitrogen, phosphorus, and potassium mineral nutrients, and significantly improved soil physical and chemical properties.

[0075] Table 3 Soil microbial composition and incidence of common soil-borne diseases under different treatments

[0076]

[0077] The results of the study on soil microbial composition and the incidence of common soil-borne diseases under different treatments showed that the number of soil bacteria in the composite microbial agent treatment group in Example 4 was 21.6×10 6 CFU / g is significantly higher than that of the other three comparative examples. This may be because the functional bacteria in the composite microbial agent accelerate the transformation of soil mineral nutrients and thus promote the growth and reproduction of bacteria; the number of soil fungi in Example 4 and Comparative Example 2 (Bacillus subtilis microbial agent fertilizer) is significantly reduced compared with Comparative Example 3 (clear water control), with a reduction of more than 42.9%, which may be related to the functional bacteria in the microbial agent. The Bacillus Velez in Example 4 and the Bacillus subtilis in Comparative Example 2 have antagonistic effects on some fungi, thereby inhibiting the reproduction of fungi; the content of soil actinomycetes is the highest in Comparative Example 2, followed by Example 4. Both groups are treated with the addition of functional bacteria, which shows that the functional bacteria also have a promoting effect on the reproduction of soil actinomycetes.

[0078] In terms of pepper disease prevention and control, Example 4 (composite bacterial agent treatment) can effectively prevent and control pepper blight, root rot and bacterial wilt. Among them, the incidence of pepper blight in the composite bacterial agent treatment group of Example 4 is reduced by 93.4%, 94.3% and 95.5% respectively compared with Comparative Example 1 (wettable powder of chloramphenicol), Comparative Example 2 (Bacillus subtilis agent) and Comparative Example 3 (clear water). The incidence of pepper root rot in Example 4 is reduced by 80.8%, 81.9% and 90.4% respectively compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3; Example 4, i.e., the composite bacterial agent treatment has a very obvious effect on the prevention and control of pepper bacterial wilt, and the incidence of pepper bacterial wilt is reduced by 88.1%, 87.1% and 89.9% respectively compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3.

[0079] In conclusion, the composite microbial agent can significantly affect the structure of soil microbial populations, and its control effect on pepper blight, bacterial wilt and root rot is significantly better than other treatments.

[0080] Table 4 Changes in pepper yield and quality under different treatments

[0081]

[0082] The results of pepper yield and quality changes under different treatments showed that the pepper yield of Example 4, i.e., the composite agent treatment, reached 2585.0 kg / mu, which was significantly increased by 26.2%, 17.5%, and 40.6% compared with Comparative Examples 1, 2, and 3, respectively. The vitamin C, soluble protein, and soluble sugar content of the pepper fruit of Example 4 were 1.49 mg / g, 16.83 mg / g, and 40.7 mg / g, respectively, which were significantly higher than those of the three groups of comparative examples. The vitamin C content was significantly increased by 10.4%, 8.8%, and 13.7% compared with Comparative Examples 1, 2, and 3, respectively. The soluble protein content was significantly increased by 17.7%, 8.6%, and 38.3% compared with Comparative Examples 1, 2, and 3, respectively. The soluble sugar content was significantly increased by 17.3%, 7.1%, and 27.2% compared with Comparative Examples 1, 2, and 3, respectively. This shows that composite agent treatment can significantly increase pepper yield and improve the nutritional and flavor quality of pepper fruit.

[0083] As can be seen, the ready-to-use microbial inoculant provided by the present invention not only significantly outperformed the pesticide and water control groups, but also significantly outperformed the control group treated with the Bacillus subtilis inoculant. Freshly prepared and used microbial inoculants can significantly maintain their biological activity, significantly impacting their effectiveness in practical applications.

[0084] Example 5

[0085] The costs of Example 4 and Comparative Examples 1 to 3 were statistically analyzed, and the results are shown in the following table:

[0086] Table 5 Costs required under different treatments (yuan / mu)

[0087]

[0088]

[0089] Comparative Example 1:

[0090] Apply 6 times, labor cost 15 yuan / mu*6=90 yuan, water cost 20 yuan*6=120 yuan, water and electricity cost 10*6=60 yuan,

[0091] The price of CP wettable powder is 110 yuan / mu*6=660 yuan;

[0092] Comparative Example 2:

[0093] Apply 6 times, labor cost 15 yuan / mu*6=90 yuan, water cost 20 yuan*6=120 yuan, water and electricity cost 10*6=60 yuan,

[0094] Bacillus subtilis microbial agent: 140 yuan / mu*6=840 yuan;

[0095] Comparative Example 3:

[0096] Apply 6 times, labor cost 15 yuan / mu*6=90 yuan, water cost 20 yuan*6=120 yuan, water and electricity cost 10*6=60 yuan;

[0097] Example 4:

[0098] Apply 6 times, labor cost 15 yuan / mu*6=90 yuan, water cost 20 yuan*6=120 yuan, water and electricity cost 10*6=60 yuan,

[0099] Fermentation culture medium base material 24+5+5=34 yuan / mu, microbial agent 30*3=90 yuan / mu.

[0100] Cost analysis data shows that the cost per mu of application using the composite microbial agent (Example 4) is only 394 yuan, which is a 60.3% reduction compared to the application of the chemical pesticide solanacyl wettable powder (Comparative Example 1) and a 64.5% reduction compared to the application of the microbial fertilizer Bacillus subtilis agent (Comparative Example 2). This shows that the ready-to-use composite microbial agent of the present invention can significantly reduce the use of chemical pesticides and fertilizers, lower agricultural production costs, and thus improve agricultural economic benefits.

Claims

1. A strain of Bacillus proteolyticus ( Bacillus proteolyticus ) XZT198, characterized in that The Bacillus proteolyticus XZT198 was deposited in the China Center for Type Culture Collection on November 24, 2023, with the deposit number being CCTCCNO: M 20232321.

2. A method for preparing and using the composite bacterial agent containing Bacillus proteolyticus XZT198 according to claim 1, comprising the following steps: 1) Strain activation: Bacillus proteolyticus XZT198 and Bacillus velezinoffii ( Bacillus velezensis ) XY40-1 were inoculated on LB solid medium and cultured at 28-30°C for 22-24 hours to obtain activated Bacillus proteolyticus XZT198 and Bacillus velezensis XY40-1; 2) Fermentation seed culture: Activated Bacillus proteolyticus XZT198 and Bacillus velezensis XY40-1 were inoculated into seed culture medium, incubated at 35-37°C, 170-190 rpm on a shaker for 22-24 hours, and then mixed according to a predetermined volume ratio to obtain a mixed seed solution. 3) On-site fermentation: Add the mixed seed liquid at a preset inoculum volume to a simple fermentation tank in the integrated water and fertilizer drip irrigation system. Add radish dregs, brown sugar, pyrroloquinoline quinone (PQQ), and lactopeptide. Ferment under film at room temperature for 7-10 days, stirring every two days to obtain a composite inoculum. 4) Transfer and storage: After fermentation is completed, the composite bacterial agent in the simple fermentation tank is transferred through a pipeline to the fertilizer and water bacterial agent storage tank in the bacterial agent, water and fertilizer integrated drip irrigation system; 5) Drip irrigation: Drain the compound bacterial agent in the fertilizer and water agent storage tank into the fertilizer and water compound dilution device in the bacterial agent, water and fertilizer integrated drip irrigation system, add water to dilute it, and then pass it into the drip irrigation equipment at a set rate to complete drip irrigation; In the composite bacterial agent, the total effective viable count of Bacillus proteolyticus XZT198 and Bacillus velez XY40-1 is 5.5 to 6.5 billion CFU / ml; Bacillus velez XY40-1 was deposited in the China Center for Type Culture Collection on March 29, 2022, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2022342.

3. The method according to claim 2, characterized in that In step 1), the formula of the LB solid medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15 g / L agar, with a pH of 7.2-7.

3.

4. The method according to claim 2, characterized in that In step 2), the preset volume ratio is 2:1; the formula of the seed culture medium is: 20 g / L peptone, 10 g / L sodium chloride, 10 g / L yeast extract, 50 mmol / L zinc sulfate and 1000 nmol / L pyrroloquinoline quinone (PQQ), with a pH of 7.2-7.

3.

5. The method according to claim 2, characterized in that In step 3), the preset inoculum size is 0.15% to 0.25%; the mixed seed solution is a late logarithmic fermentation solution; the concentration of the pyrroloquinoline quinone (PQQ) after addition is 800 to 1000 nmol / L; the concentration of the lactose peptide after addition is 0.2 to 0.4%, the amount of vegetable waste and brown sugar used is 8 to 12% of the volume of the simple fermentation tank, the ratio of brown sugar to vegetable waste used is (11 to 13):10, and river water or tap water is used to make up the difference.

6. The method according to claim 2, characterized in that The microbial agent, water and fertilizer integrated drip irrigation system includes a simple fermentation tank, a fertilizer and microbial agent storage tank, a fertilizer and water compound dilution device and a drip irrigation device; the simple fermentation tank is connected to the fertilizer and microbial agent storage tank through a pipeline, the fertilizer and microbial agent storage tank is connected to the fertilizer and water compound dilution device through a pipeline, and the fertilizer and water compound dilution device is connected to the drip irrigation system; in step 5), the water dilution ratio is 1:(50~100), and the rate of addition to the drip irrigation device is 1.8~2.1L / h.

7. The method according to claim 2, characterized in that In actual use, as needed, in the six rounds of drip irrigation in a crop growing season, feeding and re-fermentation are carried out in the simple fermentation tank before the third and fifth rounds of drip irrigation; the feeding needs to be supplemented with brown sugar, mixed seed liquid and water, the feeding amount of brown sugar is 2-3% of the volume of the simple fermentation tank, and the inoculation amount of mixed seed liquid when feeding is 0.075%-0.125%, and river water or tap water is used to make up the difference.

8. Use of the composite bacterial agent obtained by the method according to any one of claims 2 to 7 in the preparation of microbial fertilizer.

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