Compound microbial growth-promoting and anti-disease agent, preparation method and application thereof

Abstract

The application provides a composite microbial growth-promoting and disease-resistant bactericide, a preparation method and application thereof. The composite microbial growth-promoting and disease-resistant bactericide is prepared by mixing B24 bactericide of Pseudomonas putida and 79-1 bactericide of Bacillus subtilis. Since there is no obvious antagonism between the two, the growth-promoting effect of the B24 bactericide of Pseudomonas putida can be utilized, and the antagonism of the 79-1 bactericide of Bacillus subtilis to J-9, B152 and 617-3 of Fusarium during the growth of crops can be utilized, so that the rapid growth of crops can be effectively promoted.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology application and biofertilizer, and in particular relates to a compound microbial growth-promoting and disease-resistant agent, its preparation method and application. Background Technology

[0002] The comprehensive development and utilization of growth-promoting bacteria has been a research focus in recent years. The development of growth-promoting bacteria resources can not only have an immediate effect, but also reduce the use of pesticides and improve soil ecological conditions. It is an important way to promote the development of ecological agriculture and the restoration of the ecological environment. Summary of the Invention

[0003] The purpose of this invention is to provide a multifunctional, synergistic compound microbial growth promoter and disease-resistant agent. This compound microbial growth promoter and disease-resistant agent can simultaneously and efficiently degrade cellulose, degrade the main components of straw, inhibit soil-borne pathogenic fungi, and promote plant growth. Consequently, it can promote the growth of crops such as cabbage, potato, wheat, cucumber, broccoli, and tomato. At the same time, it has a significant antagonistic effect on pathogenic fungi such as Fusarium J-9, B152, and 617-3 that crops face during growth, thus helping to prevent and control crop diseases.

[0004] To achieve the above-mentioned objectives, this invention provides a compound microbial growth-promoting and disease-resistant agent, which is obtained by mixing and formulating Priestella megaterium B24 inoculum and / or Bacillus subtilis 79-1 inoculum and additives (yeast powder, peptone, NaCl).

[0005] As a further improvement of the present invention, the Priestella megaterium B24 inoculum includes Priestella megaterium B24 and the fermentation product of Priestella megaterium B24. The *Priestia megaterium* B24 is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named as *Priestia megaterium*, with accession number CGMCC No. 33700, deposit date March 3, 2025, and address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] As a further improvement of the present invention, the Bacillus subtilis 79-1 inoculum includes Bacillus subtilis 79-1 and the fermentation product of Bacillus subtilis 79-1; The Bacillus subtilis 79-1 inoculum is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named as Bacillus subtilis, with accession number CGMCC No. 35474, deposit date July 31, 2025, and address of the depository institution: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] As a further improvement of the present invention, the pathogenic fungi of Bacillus subtilis 79-1 include Fusarium strains J-9, B152, and 617-3 that infect crops such as wheat and vegetables.

[0008] Among them, after 3 days of in-situ treatment of corn straw with Bacillus subtilis 79-1 inoculum agent, the degradation rate of the main components of the straw, hemicellulose, can reach 12.65%, cellulose, and lignin can reach 13.14%.

[0009] As a further improvement of the present invention, the mixing ratio of the Priestella megaterium B24 inoculant and / or Bacillus subtilis 79-1 inoculant and yeast powder is: 40-60 parts of the yeast powder, 80-120 parts of the Priestella megaterium B24 and 80-120 parts of the Bacillus subtilis 79-1.

[0010] As a further improvement of the present invention, the compound microbial growth-promoting and disease-resistant agent is a solid microbial agent or a liquid microbial agent.

[0011] To achieve the above-mentioned objective, the present invention also provides a method for preparing a compound microbial growth-promoting and disease-resistant agent, comprising the following steps: Preparation of bacterial expansion culture medium; Single strains of Priestella megaterium B24 and Bacillus subtilis 79-1 were inoculated into the bacterial expansion medium and fermented at 37°C for 24-48 h. The fermentation products of a single strain of *Priestella megaterium* B24 and the fermentation products of a single strain of *Bacillus subtilis* 79-1 are mixed and formulated to prepare the composite microbial growth promoter and antibacterial agent.

[0012] As a further improvement of the present invention, the preparation of the bacterial expansion culture medium includes: mixing 100 g of peptone, 50 g of yeast powder and 100 g of sodium chloride, adjusting the pH to 7.0, sterilizing at 121°C for 20 min, and preparing 10 L of the bacterial expansion culture medium.

[0013] To achieve the above-mentioned objectives, the present invention also provides an application of compound microbial growth-promoting and disease-resistant agents, which is the application of the aforementioned compound microbial growth-promoting and disease-resistant agents in the crop growth process.

[0014] As a further improvement of the present invention, the application of the compound microbial growth-promoting and disease-resistant agent in the crop growth process includes: diluting the compound microbial growth-promoting and disease-resistant agent with water 10 to 50 times and then irrigating the roots of the crops.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects: The compound microbial growth-promoting and disease-resistant agent of the present invention is made by mixing Priestella megaterium B24 inoculant and Bacillus subtilis 79-1 inoculant. Since there is no obvious antagonistic effect between the two, the growth-promoting effect of Priestella megaterium B24 inoculant can be applied at the same time as the antagonistic effect of Bacillus subtilis 79-1 inoculant on Fusarium J-9, B152 and 617-3 in the crop growth process, thereby effectively promoting the rapid growth of crops. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 This is a phylogenetic tree diagram of the 16S rRNA gene sequence of Priestella giantiflora B24 according to the present invention; Figure 2 This is a phylogenetic tree construction diagram of the 16S rRNA gene sequence of Bacillus subtilis 79-1 of this invention; Figure 3 This is a colony morphology diagram of Priestella giantiflora B24, which is the subject of this invention. Figure 4 This is a colony morphology diagram of Bacillus subtilis 79-1 of the present invention; Figure 5 This is a diagram of the non-antagonistic colony morphology of Priestella megaterium B24 and Bacillus subtilis 79-1 of the present invention. Figure 6 This is a growth curve of Priestella giantiflora B24 at different pH values. Figure 7 This is a growth curve of Bacillus subtilis 79-1 under different pH values. Figure 8 This invention presents the IAA yield curves of *Priestella megaterium* B24 within different pH ranges. Figure 9 This is a diagram showing the antagonism between *Priestella megaterium* B24 and / or *Bacillus subtilis* 79-1 and *Fusarium*. Figure 10 This is a curve of Bacillus subtilis 79-1 degrading cellulose and producing reducing sugars according to the present invention; Figure 11 This invention relates to a diagram showing how Priestella megaterium B24 and / or Bacillus subtilis 79-1 promote crop growth. Figure 12 This is a degradation diagram of the lignocellulose component of corn straw by Bacillus subtilis 79-1 of this invention. Detailed Implementation

[0018] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] It should also be noted that the specific embodiments or implementation methods described below are a series of optimized settings listed by the present invention to further explain the specific content of the invention, and these settings can be combined or used in conjunction with each other.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] It should be noted that: unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and culture media involved are all commercially available; and unless otherwise specified, the experimental methods involved are all conventional methods.

[0022] In this application, the compound microbial growth-promoting and disease-resistant agent is obtained by mixing and formulating *Priscilla megaterium* B24 inoculum and / or *Bacillus subtilis* 79-1 inoculum and yeast powder. Specifically, the compound microbial growth-promoting and disease-resistant agent provided by this invention, based on colony forming units (CFU / mL), is mainly prepared from the following raw materials (by weight): 1.8%-2.2% of the weight of microbial cells in the compound microbial growth-promoting and disease-resistant agent; 2.5% of the additives; and the fermentation product of the compound microbial growth-promoting and disease-resistant agent in the nutrient solution; wherein, the compound microbial growth-promoting and disease-resistant agent is prepared by mixing *Priscilla megaterium* B24 inoculum and *Bacillus subtilis* 79-1 inoculum.

[0023] The Priestella megaterium B24 inoculum includes Priestella megaterium B24 and the metabolites of Priestella megaterium B24. like Figure 8 As shown, *Priestella megaterium* B24 is a rhizosphere growth-promoting bacterium with a stable and efficient ability to secrete indole-3-acetic acid (IAA), producing 30-101 mg / L of IAA after 12 hours.

[0024] Furthermore, such as Figure 6As shown, Priestella megaterium B24 is a strain that can tolerate a wide range of pH levels and adapts to both acidic and alkaline environments.

[0025] The *Priestella megaterium* B24 is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 33700, deposit date: March 3, 2025, address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0026] The 16S rRNA gene sequence of *Priscilla megaterium* B24 was sequenced, and the sequence is shown in SEQ ID NO. 1; the phylogenetic tree diagram of the 16S rRNA gene sequence of *Priscilla megaterium* B24 is shown below. Figure 1 .

[0027]

[0028] The Bacillus subtilis 79-1 inoculant includes Bacillus subtilis 79-1 and its metabolites; furthermore, in this application, the pathogenic fungi inhibited by Bacillus subtilis 79-1 include Fusarium strains J-9, B152, and 617-3 that infect crops such as wheat and vegetables.

[0029] Bacillus subtilis 79-1 is a strain that can tolerate a wide range of pH levels, adapting to both acidic and alkaline environments; for example... Figure 7 As shown.

[0030] The Bacillus subtilis 79-1 contained in this invention is a strain with both highly efficient cellulose degradation and broad-spectrum antifungal functions. It can degrade cellulose into reducing sugars within 12-60 hours, with a reducing sugar content of 37-100 mg / L. Figure 10 After 3 days of in-situ treatment of corn stalks, the degradation rates of the main components of the stalks—hemicellulose, cellulose, and lignin—reached 12.65%, 13.14%, and 20.06%, respectively. Figure 12 It has an inhibition rate of 49%-77% against a variety of pathogenic fungi, as shown in Table 1.

[0031] Table 1. Inhibition rates of *Priscilla megaterium* B24 inoculant, *Bacillus subtilis* 79-1, and compound microbial growth-promoting and disease-resistant agents against *Fusarium*.

[0032] The Bacillus subtilis 79-1 inoculum is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 35474, deposit date: July 31, 2025, address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0033] The 16S rRNA gene sequence of Bacillus subtilis 79-1 was sequenced, and the sequence is shown in SEQ ID NO. 2; the phylogenetic tree diagram of the 16S rRNA gene sequence of Bacillus subtilis 79-1 is shown below. Figure 2 .

[0034]

[0035] The mixing ratio of the *Priscilla megaterium* B24 inoculant and / or *Bacillus subtilis* 79-1 inoculant and yeast powder is: 40-60 parts of the yeast powder, 80-120 parts of the *Priscilla megaterium* B24, and 80-120 parts of the *Bacillus subtilis* 79-1.

[0036] In this application, the compound microbial growth-promoting and disease-resistant agent is a solid or liquid microbial agent.

[0037] This invention also provides a method for preparing a compound microbial growth-promoting and disease-resistant agent, used to prepare the aforementioned compound microbial growth-promoting and disease-resistant agent. The method for preparing the compound microbial growth-promoting and disease-resistant agent includes the following steps: Preparation of bacterial expansion culture medium; Single strains of Priestella megaterium B24 and Bacillus subtilis 79-1 were inoculated into the bacterial expansion medium and fermented at 37°C for 24-48 h. The fermentation products of a single strain of *Priestella megaterium* B24 and the fermentation products of a single strain of *Bacillus subtilis* 79-1 are mixed and formulated to prepare the composite microbial growth promoter and antibacterial agent.

[0038] Furthermore, the preparation of the bacterial expansion culture medium includes: mixing 100 g of peptone, 50 g of yeast powder, and 100 g of sodium chloride, adjusting the pH to 7.0, sterilizing at 121°C for 20 min, and preparing 10 L of the bacterial expansion culture medium.

[0039] Specifically, a single strain of *Priscilla megaterium* B24 was inoculated onto a bacterial expansion medium and fermented at 37°C for 48 h. The colony morphology of *Priscilla megaterium* B24 was then observed. Figure 3 .

[0040] A single strain of Bacillus subtilis 79-1 was inoculated onto a bacterial expansion medium and fermented at 37°C for 48 h. The colony morphology of Bacillus subtilis 79-1 was observed. Figure 4 .

[0041] Furthermore, pure cultures of *Priscilla megaterium* B24 and *Bacillus subtilis* 79-1 were cross-inoculated onto bacterial solid culture medium and incubated at 37°C for 24–48 h. The colony morphology of both cultures was then observed. Figure 5 .

[0042] The following description will provide a detailed explanation of the compound microbial growth-promoting and disease-resistant agent provided in this application through specific embodiments.

[0043] Preparation and verification of Priestella megaterium B24 Biological identification of *Priscilla megaterium* strain B24 was performed. Specifically, the biological identification process was as follows: pure *Priscilla megaterium* B24 was inoculated into LB liquid medium (Luria-Bertani medium) and cultured at 37°C with shaking until the logarithmic growth phase. 1 ml of bacterial culture was collected. The collected culture was sent to Suzhou Genewiz Biotechnology Co., Ltd., where it was amplified and sequenced using universal bacterial 16S rRNA primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The obtained sequencing results were compared with sequences in databases, demonstrating high homology with *Priscilla megaterium*. Its phylogenetic tree is shown below. Figure 1 As shown; sequencing results are shown in SEQ ID NO:1.

[0044] like Figure 3 As shown, the colonies of Priestella giantiflora B24 are yellowish-white, opaque, slightly wrinkled, and have slightly smooth edges.

[0045] The effect of pH on the growth characteristics of Priestella megaterium B24 was verified by adjusting the pH of the culture medium.

[0046] Specifically, *Priscilla megaterium* strain B24 was inoculated at a 1% inoculation ratio into LB liquid medium at pH 4.0, 4.5, 5.5, 6.5, 7.0, 7.5, 8.5, 9.5, and 10.0, respectively, and cultured at 37°C for 24 h. Furthermore, the absorbance (OD) of the culture medium was measured during the culture period. 600 To determine the growth status of *Priscilla megaterium* B24 based on absorbance values, refer to [reference needed]. Figure 6 It can be seen that *Priestella megaterium* strain B24 grows well at pH values ​​of 4.5–10.0, but hardly grows at pH 4.0; thus, *Priestella megaterium* strain B24 has a broad spectrum of acid and alkali adaptability.

[0047] Furthermore, the IAA-producing characteristics of *Priscilla megaterium* strain B24 were further verified. The specific verification process included: 1. Activation of Priestella megaterium strain B24 Take out pure culture of Priestella giantiflora B24 from -80℃, use a 10 μL sterile inoculation loop to take one loopful of bacterial solution and streak it on an LB agar plate, and place it in a constant temperature and humidity incubator at 37℃ for 12-24h.

[0048] Once *Priestella megaterium* B24 has grown, pick a single colony and transfer it to LB liquid medium. Incubate at 37°C and 180 rpm / min in a shaker until the bacterial concentration reaches OD24.600 =0.6-1.0 for later use.

[0049] 2. Prepare the colorimetric solution Preparation of PC colorimetric solution: Weigh 12g of FeCl3 and dissolve it in 300mL of distilled water. Slowly add 429.7mL of 98% H2SO4. After cooling, make up to 1L. The determination range is 0.3-20mg / L. S 2 Preparation of colorimetric solution: Dissolve 4.5g of FeCl3 in 300mL of distilled water, slowly add 587.4mL of 98% H2SO4, and after cooling, make up to 1L. The determination range is 5-200 mg / L.

[0050] 3. Configure standard curves Prepare two sets of IAA series concentration standard solutions.

[0051] The concentrations of IAA solution group 1 were: 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, and 17.5 mg / L; The concentrations of IAA solution in group 2 were: 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, and 175.0 mg / L.

[0052] Take 4 mL of the above IAA solution, add 4 mL of PC colorimetric solution to group 1, and add S to group 2. 2 4 mL of colorimetric solution was allowed to stand in the dark for 30-60 minutes; the OD was then immediately measured using a spectrophotometer. 530 The values ​​were zeroed with distilled water and repeated three times to obtain data for creating standard curves, namely the standard curves for low-concentration IAA and high-concentration IAA.

[0053] 4. IAA content detection 100 µL of activated *Priscilla megaterium* B24 bacterial culture was transferred to 20 mL of LB liquid medium and cultured for 12 h to obtain the stock solution. The stock solution was then subjected to OD analysis. 600 For testing, sterile LB medium was used to adjust the concentration of each test strain to be equal. The OD of the diluted bacterial solution was then measured. 600 The value remains consistent between 0.5 and 1.0.

[0054] ① Take 100µL of the bacterial culture to be tested and inoculate it into 50ml of LB liquid medium without tryptophan and 50ml of LB liquid medium containing 100 mg / L tryptophan, and repeat 3 times. This is the experimental group; the control group is an equal volume of LB liquid medium and cultured for 6 days. ② Take 2 ml of bacterial culture / liquid from the experimental group and the control group into a 5 ml centrifuge tube, and add an equal volume of colorimetric solution according to the measurement range (if the IAA concentration is low, add PC colorimetric solution; if the IAA concentration is high, add S). 2 (Use colorimetric solution) and react in the dark for 30-60 minutes. Measure the absorbance at 530 nm using a UV spectrophotometer. A reddish color indicates the production of IAA; the deeper the color, the higher the concentration of IAA produced through metabolism.

[0055] 5. The effect of pH value on IAA yield Pure culture of *Priscilla megaterium* B24 was taken from -80℃, and 10-20 µL of the bacterial culture was transferred to 20 mL of LB medium and cultured for 8-12 h to prepare a seed culture. The OD value of the seed culture was measured using a UV spectrophotometer. 600 Detection of bacterial concentration.

[0056] A certain amount of seed culture was placed in LB medium containing different pH values, with and without tryptophan, and the initial bacterial concentration in the medium was adjusted to OD. 600 =0.1, fermented at 37℃ for 6 days, with samples taken every 12 hours to determine the amount of IAA produced. Figure 8 As shown.

[0057] Preparation and validation of Bacillus subtilis 79-1 Molecular biological identification of Bacillus subtilis strain 79-1.

[0058] Bacillus subtilis 79-1 pure culture was inoculated into LB liquid medium (Luria-Bertani medium) and cultured at 37°C with shaking until the logarithmic growth phase. One ml of bacterial culture was collected and sent to Suzhou Genewiz Biotechnology Co., Ltd. Amplification and sequencing were performed using universal bacterial 16S rRNA primers 27F: 5'-AGAGTTTGACCTGGCTAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. The obtained sequencing results were compared with sequences in databases, demonstrating high homology with Bacillus subtilis. The phylogenetic tree is shown below. Figure 2 As shown; sequencing results are shown in SEQ ID NO:2.

[0059] like Figure 4 As shown, Bacillus subtilis 79-1 colonies are yellowish-white, opaque, with raised wrinkles on the surface and slightly wrinkled edges.

[0060] The effect of pH on the growth characteristics of Bacillus subtilis 79-1 was verified by adjusting the pH of the culture medium.

[0061] Bacillus subtilis strain 79-1 was inoculated at a 1% inoculation ratio into LB liquid medium with pH values ​​of 4.0, 4.5, 5.5, 6.5, 7.0, 7.5, 8.5, 9.5, and 10.0, respectively, and incubated at 37°C for 24 h. The absorbance (OD) of the culture medium was measured during the incubation period. 600 The growth status of bacteria can be determined based on absorbance values, such as... Figure 7 As shown, Bacillus subtilis strain 79-1 grows well at pH 4.0-9.5, but grows weakly at pH 10.0; this indicates that Bacillus subtilis strain 79-1 has a broad spectrum of acid and alkali adaptability.

[0062] Furthermore, the cellulose degradation characteristics of Bacillus subtilis strain 79-1 were verified. Culture medium preparation: CMC liquid medium (1L) contains 2.5 g dipotassium hydrogen phosphate, 2.5 g disodium hydrogen phosphate, 20 g sodium carboxymethyl cellulose, and 2.0 g peptone; CMC solid medium (1L) contains 2.5 g dipotassium hydrogen phosphate, 2.5 g disodium hydrogen phosphate, 20 g sodium carboxymethyl cellulose, 2.0 g peptone, and 15 g agar.

[0063] Pure Bacillus subtilis 79-1 culture was taken from -80℃, and 10 μL was added to LB medium using a pipette. The culture was allowed to grow to OD0.05. 600 =0.6-1.0, dilute the bacterial suspension to a dilution factor of 10. -5 10 -6 10 -7 10 -8 For each of the four gradients, take 100µL and spread it onto a CMC solid plate. Incubate at 37℃ for 24-48 hours, then stain with 1% Congo red for 15 minutes. After staining, wash 1-2 times with 1M sodium chloride for 15 minutes each time, and observe whether a clear zone forms around the colonies on the CMC solid plate. Figure 10 It can be seen that a transparent ring is generated on the CMC solid plate, which proves that Bacillus subtilis 79-1 has the ability to degrade cellulose.

[0064] Take 200 μL of the activated Bacillus subtilis 79-1 strain and place it in CMC liquid medium. Incubate at 37℃ and 180 rpm / min for 3 days. Take samples every 12 hours. Add 2 mL of 3,5-dinitrosalicylic acid solution (DNS chromogenic solution) to 1 mL of the sample, mix well, and place in boiling water at 99℃ for 2 min. Remove and cool in running cold water, then add distilled water to a final volume of 15 mL. Measure the absorbance at 540 nm using a UV spectrophotometer. Calculate the reducing sugar content based on the standard curve.

[0065] The verification of the antibacterial properties of Bacillus subtilis 79-1 specifically includes the following steps: 1. Preparation of inoculum Fusarium activation: Take out pure Fusarium J-9, B152, and 617-3 strains from -80℃, take one loopful with a 10 μL sterile disposable inoculation loop and place it in the center of a PDA plate (potato dextrose agar), and incubate in a constant temperature and humidity mold incubator at 30-37℃ for 3-5 days.

[0066] Activation of Bacillus subtilis 79-1 and Priscilla megaterium B24: Pure cultures of Bacillus subtilis 79-1 and Priscilla megaterium B24 were taken from -80℃ and a loopful was placed into LB broth using a 10 μL sterile disposable inoculation loop. The cultures were incubated at 37℃ in a shaker at 180 rpm / min until OD (open-circuit retrieval). 600 =0.6-1.0.

[0067] Preparation of compound bacterial culture: Activated Bacillus subtilis 79-1 and Priscilla megaterium B24 pure cultures were inoculated separately into 20 mL LB liquid medium and cultured at 37°C and 180 rpm / min until OD reached. 600 =1.0, mix evenly at a 1:1 ratio and set aside.

[0068] Select Fusarium J-9, B152, and 617-3 and inoculate them into the center of LB solid medium. Using a disposable sterile inoculation loop, take 1-2 loops of activated Bacillus subtilis 79-1, Priscilla megaterium B24, and a compound bacterial suspension of Bacillus subtilis 79-1 and Priscilla megaterium B24, and draw a square around the Fusarium J-9, B152, and 617-3 cultures 1-2 cm away. Place the culture in a 30-37℃ constant temperature and humidity mold incubator and incubate for 3-7 days, observing the results. Figure 9 As shown, *Priestella megaterium* strain B24 had no inhibitory effect on *Fusarium*; *Bacillus subtilis* strain 79-1 had an inhibitory effect on *Fusarium*; and the combined strain had an inhibitory effect on *Fusarium*.

[0069] Preparation and validation of compound microbial growth-promoting and disease-resistant agents 1. Experimental verification of the compound microbial growth-promoting and disease-resistant agent prepared by mixing Priestella megaterium B24 and Bacillus subtilis 79-1 in the prevention and control of crop contact infectious diseases.

[0070] This experiment was conducted in June 2025 at the Tianjin Jinghai Ziya Industrial Park Laboratory, with a blank control containing no added microbial agents.

[0071] The specific steps are as follows: Cucumbers were selected and divided into 5 groups, with 10 plants in each group. The groups were: a control group without any inoculant, a Fusarium J-9 inoculant experimental group, a Fusarium B152 inoculant experimental group, a Fusarium 617-3 inoculant experimental group, and a Priestella megaterium + Bacillus subtilis inoculant experimental group; and were incubated for 15 days.

[0072] Fusarium bacteria stored at -80℃ were picked and inoculated onto potato dextrose agar medium, and then 1×10⁻⁶ agar was prepared. 7~8 Spore quantity, used to infect crops.

[0073] After inoculation, the disease incidence of the crops was observed daily. Once the crops began to rot or their growth was significantly weaker than the control group, biological control was carried out using a compound microbial growth-promoting and disease-resistant agent prepared by mixing *Priscilla megaterium* B24 and / or *Bacillus subtilis* 79-1. The treatment effect was observed and statistically analyzed. Experimental results verified... Figure 3 As shown: Table 2. Effects of compound microbial growth-promoting and disease-resistant agents on crop growth.

[0074] As can be seen from Table 2, crops treated with pathogenic bacteria have shorter root lengths, shorter plant heights, lower fresh weights, and slower growth; while crops treated with a compound microbial growth-promoting and disease-resistant agent prepared by mixing Bacillus subtilis 79-1 and Priestella megaterium B24 showed improved growth and significantly optimized growth after biological control.

[0075] 2. Experimental verification of the compound microbial growth-promoting and disease-resistant agent prepared by mixing Priestella megaterium B24 and Bacillus subtilis 79-1 in the direction of crop growth.

[0076] This experiment was conducted in November 2024 at an experimental site in Dasi Town, Xiqing District, Tianjin, with tap water as a blank control.

[0077] The specific operating procedure is as follows: The experiment was conducted at the Qingning Village experimental base in Dasi Town, with four treatment groups: *Priscilla megaterium* B24, *Bacillus subtilis* 79-1, a control (tap water), and a compound microbial growth-promoting and disease-resistant agent prepared by mixing *Priscilla megaterium* B24 and *Bacillus subtilis* 79-1. These were applied to cash crops such as cabbage and broccoli. Each treatment was 4m long and 1m wide, with a 0.3m isolation strip between different treatments. The planting area also had 0.1m wide strips at the front and back to provide space for conventional agricultural operations.

[0078] 10 μL each of *Priscilla gigantea* B24 and *Bacillus subtilis* 79-1, stored at -80℃, were inoculated into 20 ml of bacterial LB medium and cultured overnight for 12 h to obtain seed culture. These were then transferred to propagation liquid medium at a 1% ratio and cultured on a shaker until the bacterial concentration reached OD0.05. 600 When the concentration is 1.0~1.5, a single inoculum of *Priscilla megaterium* B24 and *Bacillus subtilis* 79-1 is obtained. After mixing them in a 1:1 volume ratio, a compound microbial inoculum of *Priscilla megaterium* B24 and *Bacillus subtilis* 79-1 for promoting crop growth and disease resistance is obtained.

[0079] Dilute the compound microbial growth-promoting and disease-resistant agent with water 10-50 times and apply it to the crop roots. Repeat the application every 1-2 weeks, for a total of 2-3 applications, and observe the crop growth. Refer to Table 3 for crop growth data. Figure 11 As shown, the compound microbial growth-promoting and disease-resistant agent of *Priestella megaterium* B24 and *Bacillus subtilis* 79-1 has the effect of promoting crop growth and increasing yield.

[0080] Table 3. Effects of compound microbial growth-promoting and disease-resistant agents on crop growth.

[0081] In summary, the *Priestella giantiflora* B24 included in this invention is a rhizosphere growth-promoting bacterium with a stable and efficient ability to secrete indole-3-acetic acid (IAA), producing 30-101 mg / L of IAA after 12 hours. *Bacillus subtilis* 79-1 is a strain with both efficient cellulose degradation and broad-spectrum antifungal functions, capable of degrading cellulose into reducing sugars within 12-60 hours, with reducing sugar levels reaching 37-100 mg / L. After 3 days of in-situ treatment of corn straw, the degradation rates of the main components of the straw—hemicellulose, cellulose, and lignin—reached 12.65%, 13.14%, and 20.06%, respectively, with an inhibition rate of 49%-77% against various pathogenic fungi. Furthermore, the viable cell ratio of *Priestella giantiflora* B24 and strain 79-1 was 1:1; both strains can tolerate temperatures ≤60℃ and pH ranges of 4.5-10, exhibiting strong resistance.

[0082] The compound microbial growth-promoting and disease-resistant agent prepared by this invention, when applied to the root drench of crops at a mass ratio of 0.5-5‰, has a good growth-promoting effect on crops. The two strains of bacteria do not antagonize each other under symbiotic conditions and can complement each other's functions. Compared with single bacterial agents, the compound bacterial agent of this invention has more stable functional expression and better effect than single bacterial agents. The compound microbial growth-promoting and disease-resistant agent prepared by the method of this invention has the advantages of low production cost, convenient use, and good growth-promoting effect, and has broad application prospects and great economic value in the fields of biofertilizers.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound microbial growth-promoting and disease-resistant agent, characterized in that, It is obtained by mixing Priestella megaterium B24 inoculum, Bacillus subtilis 79-1 inoculum, yeast powder, peptone, NaCl, and the fermentation products of the inoculum in nutrient solution.

2. The compound microbial growth-promoting and antibacterial agent according to claim 1, characterized in that: The *Priestella megaterium* B24 inoculum includes *Priestella megaterium* B24 and the metabolites of *Priestella megaterium* B24. The *Priestella megaterium* B24 is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 33700, deposit date: March 3, 2025, address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

3. The compound microbial growth-promoting and disease-resistant agent according to claim 1, characterized in that: The Bacillus subtilis 79-1 inoculum includes Bacillus subtilis 79-1 and the metabolites of Bacillus subtilis 79-1; The Bacillus subtilis 79-1 inoculum is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 35474, deposit date: July 31, 2025, address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

4. The compound microbial growth-promoting and disease-resistant agent according to claim 3, characterized in that, The Bacillus subtilis 79-1 exhibits significant antagonistic / inhibitory effects against Fusarium J-9, B152, and 617-3, which infect crops such as wheat and vegetables.

5. The compound microbial growth-promoting and disease-resistant agent according to claim 1, characterized in that, The mixing ratio of the *Priscilla megaterium* B24 inoculant and / or *Bacillus subtilis* 79-1 inoculant and yeast powder is: 40-60 parts of the yeast powder, 80-120 parts of the *Priscilla megaterium* B24, and 80-120 parts of the *Bacillus subtilis* 79-1.

6. The compound microbial growth-promoting and disease-resistant agent according to claim 1, characterized in that, The compound microbial growth-promoting and disease-resistant agent is a solid or liquid microbial agent.

7. A method for preparing a compound microbial growth-promoting and disease-resistant agent, characterized in that, The preparation of the compound microbial growth-promoting and disease-resistant agent according to claim 1 includes the following steps: Preparation of bacterial expansion culture medium; Single strains of Priestella megaterium B24 and Bacillus subtilis 79-1 were inoculated into the bacterial expansion medium and fermented at 37°C for 24-48 h. The fermentation products of a single strain of *Priestella megaterium* B24 and the fermentation products of a single strain of *Bacillus subtilis* 79-1 are mixed and formulated to prepare the composite microbial growth promoter and antibacterial agent.

8. The preparation method of the compound microbial growth-promoting and disease-resistant agent according to claim 7, characterized in that: The preparation of the bacterial expansion culture medium includes: mixing 100 g of peptone, 50 g of yeast powder and 100 g of sodium chloride, adjusting the pH to 7.0, sterilizing at 121°C for 20 min, and preparing 10 L of the bacterial expansion culture medium.

9. The application of a compound microbial growth-promoting and disease-resistant agent, characterized in that, The application of the compound microbial growth-promoting and disease-resistant agent as described in claim 1 in the crop growth process.

10. The application of the compound microbial growth-promoting and antibacterial agent according to claim 9, characterized in that: This includes diluting the compound microbial growth-promoting and disease-resistant agent with water 10 to 50 times and then applying it to the roots of crops.

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