Bacillus type compound microbial agent as well as preparation method and application thereof

Abstract

The invention discloses a bacillus type compound microbial agent as well as a preparation method and application thereof, and relates to the technical field of agricultural biology. Comprising the following steps: S1, strain selection and culture; s11, strain screening: screening bacillus subtilis Z-14 and paenibacillus mucilaginosus HB-02 which are vigorous in growth vigor and resistant to stress and heat, respectively carrying out streak inoculation and constant-temperature culture overnight on the screened strains on an NA culture medium, selecting activated single colonies, transferring the activated single colonies into an NB culture medium, and carrying out shake culture overnight to prepare a bacterial suspension; and S12, activating bacteria, namely activating the bacteria by using an NA culture medium, and culturing at constant temperature overnight. The compound microbial agent disclosed by the invention can be used for effectively preventing and treating the occurrence of wheat root rot, and the effect of the compound microbial agent is better than that of single use of Z-14 microbial agent and HB-02 microbial agent; field experiments show that the control effect of a compound fungicide treatment group on the wheat root rot reaches 81.8%, and the control effect is obviously better than that of a single fungicide.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to a Bacillus type composite microbial agent and a preparation method thereof. Background Art

[0002] A composite microbial agent refers to one or more beneficial microorganisms and microbial carriers, and contains a certain number of live bacteria. It has the functions of quickly replenishing beneficial bacteria in the soil, inhibiting harmful bacteria, regulating soil pH, improving soil fertility, enhancing plant resistance to diseases and pests, and promoting plant growth. It can well overcome the shortcomings of the application of a single agent.

[0003] For example, Li Xueping et al. reported a composite bacterial agent BP5 composed of Brevibacterium frigoritolerans, Bacillus cereus, and Bacillus subtilis. After field application, it can achieve a 68% prevention effect on lily wilt disease and can effectively improve the quality of lilies and soil quality.

[0004] However, these composite microbial agents are all made up of equal proportions, and their optimal ratio is still unclear, which limits the strains from exerting their synergistic effects to the maximum extent. At the same time, the field application effect and optimal dosage are not clear, which hinders their large-scale demonstration and promotion and application. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a Bacillus type composite microbial agent and a preparation method thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A bacillus is a composite bacillus, and the composite bacillus comprises:

[0008] Bacillus subtilis, the deposit number is CGMCC No.20821; the deposit center is: General Microbiology Center of China Microorganism Culture Collection Administration; the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is September 25, 2020;

[0009] Paenibacillus mucilaginosus, the deposit number is CGMCC No.24636; the deposit center is: General Microbiology Center of China Culture Collection Administration; the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is April 6, 2022.

[0010] A method for preparing a Bacillus type composite microbial agent comprises the following steps:

[0011] S1: strain selection and cultivation;

[0012] S11: Screen out Bacillus subtilis Z-14 and Paenibacillus colloids HB-02 with vigorous growth, stress resistance and heat resistance, and streak the screened strains on NA medium, culture them at a constant temperature overnight, pick out activated single colonies and transfer them to NB medium for shaking culture overnight to prepare bacterial suspension;

[0013] S12: Bacterial activation, using NA medium for bacterial activation and culturing at a constant temperature overnight;

[0014] S13: Collection and preparation of bacterial cells: inoculate the activated single colony into NB medium, shake culture overnight, inoculate the culture into fresh NB medium at 10% inoculum, shake culture for 48 hours, centrifuge the fermentation broth, recover the bacterial cells, and dilute with sterile water;

[0015] S2: Preparation of composite bacterial agent;

[0016] S21: The mixing ratio is determined, and a test is conducted. Based on the test results, the optimal mixing ratio of the Z-14 bacterial agent and the HB-02 bacterial agent is determined;

[0017] S22: Prepare bacterial suspension, adjust the live bacterial content of Z-14 and HB-02 mixed in proportion to 1.0×10 10 cfu / mL and prepared composite bacterial suspension.

[0018] Preferably: in S11, the live bacteria content of the bacterial suspension is controlled to be 1×10 8 cfu / mL or 1×10 10 cfu / mL.

[0019] Preferably, the specific ingredients of the NA medium are: 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, and 1000mL water;

[0020] The pH of NA medium was controlled at 7.0-7.2.

[0021] Preferably: the specific ingredients of the NB culture medium are: 5g beef extract, 10g peptone, 5g NaCl, and 1000mL water;

[0022] The pH of NB medium was controlled at 7.0-7.2.

[0023] Preferably, in S13, the centrifugal speed is controlled at 8000 rpm and the centrifugal time is controlled at 10 minutes.

[0024] Preferably: in S13, the mixture is diluted with sterile water to a viable bacterial content of 1×10 8 cfu / mL or 1×10 10 cfu / mL.

[0025] Preferably: in S21, the specific method of conducting the test includes:

[0026] Pot test: A pot test was conducted by setting up a blank control group, a pathogen group and different treatment groups to test the control effect and growth-promoting effect of each treatment on wheat root rot;

[0027] Field test: Field tests were conducted under actual farmland conditions to determine the effects of the composite microbial agent on wheat yield, plant height, ear number, ear number and soil enzyme activity;

[0028] Effect analysis and ratio optimization: Combined with the results of potted and field tests, the effects of different proportions of composite microbial agents were analyzed to ultimately determine the optimal mixing ratio.

[0029] Preferably, in S21, the optimal mixing ratio is 2:1 of Z-14 and HB-02.

[0030] Preferably, in S1, the temperature of the constant temperature overnight culture and the shaking culture are both controlled at 30°C.

[0031] Preferably: in the pathogen group, the pathogen culture medium is PDA culture medium, and the PDA culture medium is specifically: 200g potato, 20g glucose, 15-20g agar, 1000mL water. Peel the potato, cut into small pieces, add water and boil for 0.5h, filter with double gauze, take the filtrate, add sugar, and add water to make up to 1000mL;

[0032] After the sterilized PDA medium is melted, pour it onto a plate, and streptomycin sulfate is added to make the final concentration 40 μg / mL; the pathogenic bacteria piece is inoculated in the center of the PDA plate, and cultured at 26°C until the hyphae fill the plate for use.

[0033] The beneficial effects of the present invention are:

[0034] 1. The composite microbial agent of the present invention can effectively prevent and control the occurrence of wheat root rot, and the effect is better than using Z-14 agent and HB-02 agent alone; field experiments show that the control effect of the composite agent treatment group on wheat root rot reached 81.8%, which is significantly better than the effect of a single agent.

[0035] 2. The inoculation of Z-14 and HB-02 bacterial agents of the present invention can significantly increase wheat yield, and the growth-promoting effect is significant; the composite bacterial agent treatment group is higher than the control group in all test indicators, and has the best yield-increasing effect, with an increase of 18.19% over the control group.

[0036] 3. The composite microbial agent of the present invention has the functions of improving soil fertility, regulating soil physical and chemical properties, increasing soil enzyme activity, and improving the cultivation effect of wheat; it improves soil quality by supplementing beneficial bacteria in the soil, inhibiting harmful bacteria, and improving soil microenvironment.

[0037] 4. The biological control of the present invention is green and environmentally friendly, and pathogens are not easy to develop drug resistance; compared with chemical fungicides, the use of composite microbial agents will not cause environmental pollution and agricultural product pesticide residue problems, and meets the requirements of green agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention provides a flow chart of a method for preparing a Bacillus-type composite microbial agent. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0040] Embodiment 1:

[0041] A Bacillus type composite microbial agent, and a method for preparing, testing and applying the agent, specifically comprising:

[0042] 1 Materials and methods

[0043] 1.1 Preparation of test materials

[0044] The wheat planting soil samples were taken from the wheat planting field of Hebei Agricultural University Farm. The wheat variety used in the experiment was Jimai 418, produced in Baoding, and was uncoated.

[0045] 1.2 Culture medium

[0046] PDA medium: 200g potato, 20g glucose, 15-20g agar, 1000mL water. Peel the potato, cut into small pieces, add water and boil for 0.5h, filter with double-layer gauze, take the filtrate, add sugar, and add water to make up to 1000mL.

[0047] NA medium: 5 g beef extract, 10 g peptone, 5 g NaCl, 15-20 g agar, 1000 mL water, pH 7.0-7.2.

[0048] NB medium: 5 g beef extract, 10 g peptone, 5 g NaCl, 1000 mL water, pH 7.0-7.2.

[0049] 1.3 Pathogen culture

[0050] The wheat root rot pathogen Cochliobolus sativus was stored by the Agricultural Microbiology Laboratory of the College of Life Sciences of Hebei Agricultural University. Sterile PDA medium was melted and poured onto a plate, and streptomycin sulfate was added to make the final concentration 40 μg / mL. The pathogenic bacteria sheet was inoculated in the center of the PDA plate and cultured at 26°C until the mycelium filled the plate for later use.

[0051] 1.4 Bacterial culture

[0052] A small amount of bacterial moss was picked from the NA slant with a sterilized bamboo stick and inoculated onto a fresh NA medium plate by streaking. The plate was cultured at 30°C overnight. An activated single colony was picked and inoculated into NB medium. The plate was cultured at 30°C overnight with shaking. The culture was inoculated into fresh NB medium at 10% inoculum and cultured at 30°C with shaking for 48 h. The fermentation broth was centrifuged at 8000 rpm for 10 min. The recovered bacteria were diluted with sterile water to prepare a plate with a viable bacterial content of about 1×10 8 cfu / mL or 1×10 10 cfu / mL of bacterial suspension, and detect the viable bacteria content using a bacterial counting plate.

[0053] 1.5 Effect of microbial agents on the prevention and control of wheat root rot

[0054] The pot experiment was set up with 5 treatments, namely blank control group CK0, pathogen group CK1, Z-14 treatment group Z14, HB-02 treatment group HB02, Z-14 and HB-02 composite treatment group ZHB (Z-14 and HB-02 mixed 1:1), and each treatment was set up with 5 replicates. A flower pot with a diameter of 12.9 cm and a height of 12.4 cm was taken, and 12 wheat seeds were evenly sown in each pot. CK0 was not inoculated with pathogens and antagonistic bacteria. CK1 was inoculated with wheat root rot bacteria, and wheat seeds were placed on pathogen bacterial discs (diameter 7mm), with 1 wheat seed placed on each bacterial disc. The way of inoculating pathogens in Z14, HB02 and ZHB treatment groups was the same as CK1, and 20 mL of Z-14, HB-02 and mixed bacterial suspension were irrigated respectively, cultured at room temperature, and the control effect of antagonistic bacteria was investigated 20 days after planting. The plant growth status test was carried out on a plant-by-plant basis, with a sampling volume of 20 plants, and then the average value was calculated. Grading standards for the severity of wheat root rot: Level 0: No disease; Level 1: The outer leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is less than 1 / 2 of the leaf sheath; Level 2: The outer leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is greater than 1 / 2 of the leaf sheath; Level 3: The inner leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is less than 1 / 2 of the leaf sheath; Level 4: The inner leaf sheath at the base of the wheat plant stem turns dark brown, and the length of the lesion is greater than 1 / 2 of the leaf sheath; Level 5: Root rot and plant death. Disease index = [∑ (number of diseased plants at each level × representative value) / total number of plants surveyed × representative value of the most severe disease level] × 100. Control effect = [(disease index of the control group - disease index of the treatment group) / disease index of the control group] × 100.

[0055] 1.6 Field test on the growth-promoting effect of microbial agents

[0056] A wheat planting plot was selected at the Experimental Farm of Hebei Agricultural University, with an area of ​​about 800m 2 , divided into 4 small pieces, each about 200m 2 , respectively blank control group CK0, Z-14 bacterial agent treatment group Z14, HB-02 bacterial agent treatment group HB02, Z-14 and HB-02 composite bacterial agent treatment group ZHB (Z-14 bacterial agent and HB-02 bacterial agent 1:1 mixture). The control group was not inoculated with bacterial agents; the other three groups were all treated with a live bacterial content of about 1.0×10 10 cfu / mL of the inoculant, the inoculation amount was 20mL / kg of seeds; in the green period, the inoculant was inoculated by irrigation, and the concentration of the inoculant was 1.0×10 8 cfu / mL, the inoculation dose is 1000mL / m 2No fertilizer was applied to the control group and the treatment group, mechanical sowing was used, and the management method was the same as that of general fields. In the milky stage of wheat, the 5-point sampling method was adopted for sampling, and 3 consecutive rows were sampled at each point, and 0.4m was sampled from each row. The plant height and average number of grains per ear were investigated and counted (30 plants at each random sampling point, and 150 plants were sampled in each group). After the seeds were dried, the thousand-grain weight was weighed, and the yield per square meter was determined by single threshing and harvesting.

[0057] 1.7 Optimization of compound microbial agent ratio

[0058] Z-14 and HB-02 were mixed in different proportions, and the treatment group with a live bacteria content ratio of 3:1 between Z-14 and HB-02 was set as TR1, 2:1 was set as TR2, 1:1 was set as TR3, 1:2 was set as TR4, and 1:3 was set as TR5. The blank control group without added bacteria was set as CK0, and the pathogen group with added pathogens was set as CK1. The disease prevention and growth promotion effects of different proportions of composite bacteria were tested. The disease prevention effect detection method was the same as 1.5, and the growth promotion effect detection method was the same as 1.6.

[0059] 1.8 Soil enzyme activity determination

[0060] During the field test of the effect of composite microbial agent on promoting wheat growth, the rhizosphere soil of wheat in different treatment groups was selected to detect the biological enzyme activity at the jointing stage of wheat. The sucrase activity was determined by colorimetry, and the mass (mg) of glucose produced in 1.0g of soil after 1 day was used to express the sucrase activity, and mg·g -1 ·d -1 Urease activity was determined by colorimetry, and the mass (mg) of NH3-N generated by 1.0g of soil after 1 day was used to express urease activity, expressed in mg·g -1 ·d -1 The alkaline phosphatase activity was determined by disodium phenyl phosphate colorimetry, and the phosphatase activity was expressed as mg·g P2O5 in 1.0 g of soil after 1 day. -1 ·d -1 The activity of catalase was determined by potassium permanganate titration, and the activity of catalase was expressed as mg·g H2O2 consumed per minute by 1.0 g soil. -1 ·min -1 express.

[0061] 1.9 Data Processing

[0062] Microsoft Excel 2010 was used to draw pictures and make tables, SPSS statistics 21 statistical software was used for data analysis, ANOVA was used for one-way analysis of variance, and Duncan's method was used for multiple comparisons.

[0063] 2 Results and analysis

[0064] 2.1 Potted plant experiment to test the effect of bacterial agent on wheat root rot

[0065] This study detected the control effect of Z-14, HB-02 and their composite agents on wheat root rot. The results of the pot test are shown in the following table. The root length, root weight and aboveground weight of the composite agent treatment group ZHB were higher than those of the blank control group CK0, but the aboveground height was slightly lower than CK0. There was no significant difference between all indicators and CK0, but they were significantly higher than the pathogen control group CK1. The root length, root weight and aboveground height of the Z14 treatment group were lower than those of the CK0 treatment group, but the difference was not significant, and the aboveground weight was significantly lower than that of the CK0 treatment group. The root length, aboveground height and aboveground weight of the HB02 treatment group were significantly lower than those of the CK0 treatment group, and the root weight was also lower than that of the CK0 treatment group, but the difference was not significant. The control effect of the ZHB treatment group on wheat root rot reached 81.8%, which was significantly different from the 50.1% of the HB02 treatment group, and not significantly different from the 63.0% of the Z14 treatment group. In summary, the application of compound bacterial agents can effectively prevent and control the occurrence of wheat root rot, and the effect is better than the application of Z-14 and HB-02 agents alone.

[0066] Effect of Bacillus preparation on wheat root rot

[0067]

[0068] 2.2 Growth-promoting effect of Bacillus preparations on wheat

[0069] During the milky stage of wheat, the growth-promoting effect of Bacillus preparations on wheat was analyzed by investigating indicators such as plant height, number of ears, number of grains per ear, thousand-grain weight and yield. Compared with the blank control group CK0, the above indicators of wheat in the Z14, HB02 and ZHB treatment groups increased. There were no significant differences in each indicator between the Z14 treatment group and CK0. There was a significant difference in yield between the HB02 treatment group and CK0, but the differences in other indicators were not significant. Compared with CK0, there were significant differences in plant height and yield between ZHB and CK0, but the differences in other indicators were not significant. The ZHB treatment group was higher than the Z14 and HB02 treatment groups in all the detected indicators, but the differences in each indicator were not significant. Compared with CK0, the Z14, HB02 and ZHB treatment groups increased yield by 7.51%, 14.69% and 18.19%, respectively (as shown in the following table). Inoculation with Z-14 and HB-02 bacterial agents can significantly increase wheat yield, and the growth-promoting effect is significant, but the effect of inoculation with composite bacterial agents is the best.

[0070] Detection of the growth-promoting effect of Bacillus preparations at the wheat milk stage

[0071]

[0072]

[0073] 2.3 Control effect of compound Bacillus preparation on wheat root rot

[0074] This study tested the control effect of different proportions of Z-14 and HB-02 composite bacterial agents on wheat root rot. The results of the pot test are shown in the following table. The root length, root weight, aboveground height, and aboveground weight of the TR2 and TR3 groups were higher than those of the CK0 group, but the difference was not significant. The TR2 group was higher than the TR3 group, but the difference was not significant. The root weight of the TR1 group was significantly lower than that of the CK0 group, and the other indicators were not significantly different. The TR4 group was significantly lower than the CK0 group in all indicators, and the TR5 group was lower than the CK0 group in all indicators, but the difference was not significant. In the detected root length, root weight, aboveground height, aboveground weight and other indicators, the treatment groups with the addition of composite bacterial agents were significantly higher than the CK1 group, indicating that the field Bacillus preparation can significantly inhibit the occurrence of wheat root rot. TR2 had the best effect on the prevention and control of wheat root rot, with a control effect of 82.7%, followed by the TR3 group, with a control effect of 79.1%, which was consistent with the test results of the above four test indicators. TR1 had the lowest control effect, which was 45.9%, followed by TR4, which was 53.6%. In summary, among the applications of composite microbial agents with different addition ratios, TR2 had the best control effect on wheat root rot and could effectively control the occurrence of wheat root rot.

[0075] Effect of compound Bacillus preparation on wheat root rot

[0076]

[0077]

[0078] 2.4 Effect of compound Bacillus preparation on wheat growth promotion

[0079] During the milky stage of wheat, the growth-promoting effect of the composite Bacillus preparation on wheat was analyzed by investigating indicators such as plant height, number of spikes, number of grains per spike, thousand-grain weight and yield (as shown in the table below). Compared with the blank control group CK0, the plant height of the TR2, TR3 and TR5 treatment groups increased significantly, and the plant height of the TR1 and TR4 treatment groups also increased, but the difference was not significant. The number of spikes in the TR2 and TR4 treatment groups was significantly higher than that in the control group, and the number of spikes in the other treatment groups was also higher than that in the control group, but the difference was not significant. The number of grains per spike and thousand-grain weight of each treatment group with added bacterial agents were higher than those of the CK0 group, but the difference was not significant. The yield of each treatment group with added bacterial agents was significantly higher than that of the CK0 group. The yield of the TR2 treatment group was the highest, with an increase of 25.99% over the control group, followed by TR3, with an increase of 22.43% over the control group, and the lowest yield was the TR4 group, with an increase of 13.77% over the control group. In summary, the TR2 treatment group had the best yield-increasing effect and a significant growth-promoting effect.

[0080] Detection of the growth-promoting effect of compound Bacillus preparation at the wheat milk stage

[0081]

[0082]

[0083] 2.5 Effects of composite Bacillus preparation on biological enzyme activities in wheat rhizosphere soil

[0084] The growth-promoting effect of the composite Bacillus preparation on wheat was detected through field trials. At the jointing stage of wheat, the rhizosphere soil of wheat in different treatment groups was selected to detect the activities of sucrase, urease, alkaline phosphatase and catalase. The results are shown in the following table. Compared with the blank control group CK0, the sucrase activity of each treatment group added with the composite bacterial agent increased, among which the increase in the TR5 treatment group was not significant, and the increase in the other groups was significant. The sucrase activity of the TR2 group increased the most, reaching 36.64%, followed by the TR4 group, which was 32.24%. The urease activity of each treatment group added with the composite bacterial agent increased to varying degrees, among which the increase in the TR3 group was not significant, and the increase in the other groups was significant. The urease activity of the TR2 group increased the most, reaching 34.69%. The alkaline phosphatase activity of each treatment group added with the composite bacterial agent increased, the increase in the TR1 group and the TR5 group was not significant, and the increase in the other groups was significant. The enzyme activity of the TR2 group was the highest, increasing by 36.51%. The catalase activity of each treatment group with the addition of composite bacteria increased, with significant increases in the TR2 and TR4 groups, and insignificant increases in the other groups. The TR4 group increased the most, reaching 54.14%, and the TR2 group increased by 43.95%. In summary, the TR2 group had the most significant increases in sucrase, urease, and alkaline phosphatase activities, and the catalase activity was second only to the TR4 group, indicating that the TR2 group had the most significant effect on the enzyme activity of the wheat rhizosphere soil.

[0085] Effects of compound Bacillus preparation on biological enzyme activities in wheat rhizosphere soil

[0086]

[0087] 3 Conclusion

[0088] The application of composite microbial agents can effectively prevent and control the occurrence of wheat root rot, and the effect is better than the application of Z-14 and HB-02 alone. Inoculation of Z-14 and HB-02 can significantly increase wheat yield, and the growth-promoting effect is significant, but the effect of inoculation of composite microbial agents is the best. In the application of composite microbial agents with different addition ratios, the TR2 group has the best effect on the prevention and control of wheat root rot, and the control effect of wheat root rot reaches 82.7%, which can effectively prevent and control the occurrence of wheat root rot. The TR2 treatment group has the best yield-increasing effect, which is 25.99% higher than that of the control group. The activities of sucrase, urease and alkaline phosphatase in the TR2 group increased most significantly, and the catalase activity was second only to the TR4 group, indicating that the TR2 group has the most significant effect on the enzyme activity of wheat rhizosphere soil.

[0089] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A Bacillus, characterized in that It is a composite Bacillus, which includes: Bacillus subtilis, the deposit number is CGMCC No.20821; the deposit center is: General Microbiology Center of China Microorganism Culture Collection Administration; the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is September 25, 2020; Paenibacillus mucilaginosus, the deposit number is CGMCC No.24636; the deposit center is: General Microbiology Center of China Culture Collection Administration; the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is April 6, 2022.

2. A method for preparing a Bacillus type composite microbial agent, characterized in that: The steps include: S1: strain selection and cultivation; S11: Screen out Bacillus subtilis Z-14 and Paenibacillus colloids HB-02 with vigorous growth, stress resistance and heat resistance, and streak the screened strains on NA medium, culture them at a constant temperature overnight, pick out activated single colonies and transfer them to NB medium for shaking culture overnight to prepare bacterial suspension; S12: Bacterial activation, using NA medium for bacterial activation and culturing at a constant temperature overnight; S13: Collection and preparation of bacterial cells: inoculate the activated single colony into NB medium, shake culture overnight, inoculate the culture into fresh NB medium at 10% inoculum, shake culture for 48 hours, centrifuge the fermentation broth, recover the bacterial cells, and dilute with sterile water; S2: Preparation of composite bacterial agent; S21: The mixing ratio is determined, and a test is conducted. Based on the test results, the optimal mixing ratio of the Z-14 bacterial agent and the HB-02 bacterial agent is determined; S22: Prepare bacterial suspension, adjust the live bacterial content of Z-14 and HB-02 mixed in proportion to 1.0×10 10 cfu / mL and prepared composite bacterial suspension.

3. The method for preparing a Bacillus type composite microbial agent according to claim 2, characterized in that: In S11, the live bacteria content of the bacterial suspension is controlled to be 1×10 8 cfu / mL or 1×10 10 cfu / mL.

4. The method for preparing a Bacillus type composite microbial agent according to claim 2, characterized in that: The specific components of the NA medium are: 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, and 1000mL water; The pH of NA medium was controlled at 7.0-7.2; The specific components of the NB culture medium are: 5 g beef extract, 10 g peptone, 5 g NaCl, and 1000 mL water; The pH of NB medium was controlled at 7.0-7.

2.

5. The method for preparing a Bacillus type composite microbial agent according to claim 2, characterized in that: In S13, the centrifugal speed is controlled at 8000 rpm and the centrifugal time is controlled at 10 minutes.

6. The method for preparing a Bacillus type composite microbial agent according to claim 2, characterized in that: In S13, the mixture was diluted with sterile water to a viable bacterial content of 1×10 8 cfu / mL or 1×10 10 cfu / mL.

7. The method for preparing a Bacillus type composite microbial agent according to claim 6, characterized in that: In S21, the specific method of conducting the test includes: Pot test: A pot test was conducted by setting up a blank control group, a pathogen group and different treatment groups to test the control effect and growth-promoting effect of each treatment on wheat root rot; Field test: Field tests were conducted under actual farmland conditions to determine the effects of the composite microbial agent on wheat yield, plant height, ear number, ear number and soil enzyme activity; Effect analysis and ratio optimization: Combined with the results of potted and field tests, the effects of different proportions of composite microbial agents were analyzed to ultimately determine the optimal mixing ratio.

8. The method for preparing a Bacillus type composite microbial agent according to claim 2, characterized in that: In S21, the optimal mixing ratio is 2:1 for Z-14 and HB-02.

9. The method for preparing a Bacillus type composite microbial agent according to claim 2, characterized in that: In the pathogen group, the pathogen culture medium is PDA culture medium, which is specifically: 200g potato, 20g glucose, 15-20g agar, 1000mL water. Peel the potato, cut into small pieces, add water and boil for 0.5h, filter with double-layer gauze, take the filtrate, add sugar, and add water to make up to 1000mL; After the sterilized PDA medium is melted, pour it onto a plate, and streptomycin sulfate is added to make the final concentration 40 μg / mL; the pathogenic bacteria piece is inoculated in the center of the PDA plate, and cultured at 26°C until the hyphae fill the plate for use.

10. A Bacillus type composite microbial agent, characterized in that: Prepared according to the preparation method according to any one of claims 2 to 9.

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