Composite microbial bacteria suspension, bacteria agent, tobacco seed coating agent, and preparation method and application thereof

Abstract

This invention relates to the field of microbial technology, and provides a composite microbial suspension comprising a *Streptococcus pyogenes* R3 suspension and a *Cyclocarya pallida* N4 suspension in a volume ratio of 1-2:1-2; the *Streptococcus pyogenes* R3 classification name is... Herbaspirillum sp. R3 was deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231757; the taxonomic name of Chlorella N4 is... Chryseobacterium endophyticum N4 was deposited on March 31, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025633. The aforementioned composite microbial suspension promotes nitrogen fixation and nitrogen conversion.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a composite microbial suspension, microbial agent, tobacco seed coating agent, its preparation method and application. Background Technology

[0002] In modern agricultural production, soil nutrient supply capacity is a crucial factor determining crop growth and yield. However, due to the physicochemical properties of the soil itself and the low utilization rate of nutrients, resources are wasted and the environment is polluted. These problems are particularly prominent in the utilization of key nutrients such as nitrogen and phosphorus.

[0003] Phosphorus is one of the three major nutrients required for crop growth, playing a crucial role in plant energy metabolism and root development. However, most phosphorus is adsorbed and fixed by the soil, accumulating in slow-release and highly stable forms. Only a small portion of phosphorus can be absorbed and utilized by plants and microorganisms, resulting in a relatively low content of available phosphorus in the soil that can be absorbed and utilized by crops. Nitrogen is also a key nutrient for crop growth and the nutrient with the largest input in fertilization. However, the utilization rate of nitrogen fertilizer has long been low, with more than 60% of nitrogen lost in various reactive nitrogen forms. This loss not only wastes resources but also increases the generation of greenhouse gases.

[0004] The low nitrogen and phosphorus use efficiency in soil not only restricts normal crop growth but also increases the negative impact of fertilizer use on the ecological environment. Improving the supply of available phosphorus in the soil, reducing nitrogen fertilizer loss, and supporting healthy crop growth by optimizing soil nutrient cycling efficiency have become core technical problems that modern agriculture urgently needs to solve. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial suspension, inoculant, tobacco seed coating agent, and their preparation method and application that can greatly improve nitrogen and phosphorus utilization efficiency, so as to solve the problem of the limited protective effect of endophytic bacteria in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a composite microbial suspension, comprising a suspension of *Streptococcus pyogenes* R3 and a suspension of *Chlorella vulgaris* N4;

[0008] The volume ratio of the *Streptococcus pyogenes* R3 suspension to the *Aureobacillus chrysogenum* N4 suspension is 1~2:1~2;

[0009] The *Streptococcus filiis* R3 suspension was prepared from *Streptococcus filiis* R3.

[0010] The R3 classification name of the *Streptococcus* is... Herbaspirillumsp. R3 was deposited on September 20, 2023 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231757;

[0011] The Chlorella N4 bacterial suspension was prepared from Chlorella N4.

[0012] The taxonomic name of the *Cyperus yunnanensis* N4 is: Chryseobacterium endophyticum N4 was deposited on March 31, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 2025633.

[0013] In one embodiment, the concentrations of both the *Streptococcus solani* R3 suspension and the *Chlorella vulgaris* N4 suspension are (1~9)×10⁻⁶. 8 cfu / mL.

[0014] Based on the same inventive concept, a composite microbial agent is also provided, which is made from the composite microbial suspension, composite carrier and protectant as described above.

[0015] Based on the same inventive concept, a method for preparing the composite microbial agent as described above is also provided, comprising the following steps:

[0016] Step (1): Mix the composite microbial suspension, carrier material and protectant to obtain a mixture;

[0017] Step (2): Mix the mixture with glutaraldehyde solution and react for 10-20 minutes to obtain a mixed solution;

[0018] Step (3): Spray dry the mixed solution to obtain the composite microbial agent;

[0019] The mass ratio of the composite microbial suspension, carrier material, and protectant is 1~2:1~2:0.1~0.2.

[0020] In one embodiment, the carrier material in step (1) includes a polyvinyl alcohol solution and a chitosan-montmorillonite nanocomposite; the mass concentration of the polyvinyl alcohol solution is 4~6wt%; the mass ratio of polyvinyl alcohol to chitosan-montmorillonite nanocomposite in the polyvinyl alcohol solution is 4~6:2~4;

[0021] The protective agent in step (1) is glycerol, and the mass concentration of the glycerol is 8~12wt%.

[0022] In one embodiment, the volume ratio of the mixture to the glutaraldehyde solution in step (2) is 15-25:1; the mass concentration of the glutaraldehyde solution is 0.5-1.5 wt%.

[0023] The reaction temperature in step (2) is 20~30℃.

[0024] In one embodiment, during the spray drying process in step (3), the temperature of the air inlet is 70~80℃, the temperature of the air outlet is 60~70℃, and the feed rate is 15~18mL / min.

[0025] Based on the same inventive concept, a tobacco seed coating agent is also provided, comprising the composite microbial agent and binder as described above, wherein the binder is gum arabic liquid, and the concentration of the gum arabic liquid is 5~15wt%.

[0026] In one embodiment, the coating layer of the tobacco seed coating agent has a thickness of 60~100μm.

[0027] Based on the same inventive concept, the application of the compound microbial agent or tobacco seed coating agent as described above in increasing the nitrogen and phosphorus content of tobacco planting soil and improving the growth traits of tobacco plants is also provided.

[0028] The present invention has the following technical effects and advantages:

[0029] This invention uses a combination of *Streptococcus pyogenes* R3 and *Aureobacillus yunnanensis* N4, which have phosphorus-solubilizing and nitrogen-fixing functions, to prepare a compound microbial suspension. This allows the bacteria to continuously release beneficial substances during seed germination, helping tobacco dissolve insoluble phosphorus sources in the soil, promoting nitrogen fixation and nitrogen conversion, thereby improving agronomic traits such as tobacco plant height and maximum leaf area. Based on this, a compound microbial agent and a tobacco seed coating agent were prepared.

[0030] This invention utilizes polyvinyl alcohol, chitosan, and montmorillonite nanocomposites to create a carrier material, and finally employs gum arabic, a natural binder, to enhance the stability of the coating layer. Polyvinyl alcohol possesses excellent film-forming properties and water solubility, forming a uniform, sustained-release system as the carrier matrix, and is non-toxic to the microorganisms. The amino groups of chitosan can form an interpenetrating network structure with the hydroxyl groups of polyvinyl alcohol through hydrogen bonds, extending the release cycle of the microbial agent. The montmorillonite nanocomposites can adsorb microbial metabolites (such as acid phosphatase) through ion exchange, achieving a dual sustained release of nutrients and enzymes. Gum arabic, as a natural binder, has moderate viscosity, allowing the composite microbial agent to adhere uniformly to the seed surface, forming a coating layer of controllable thickness. Furthermore, it exhibits good biodegradability and is environmentally friendly. This coating process significantly improves the stability and activity of the microorganisms in different environments.

[0031] Preservation Instructions

[0032] The classification name of *Streptococcus solani* R3 is: Herbaspirillumsp. R3 was deposited on September 20, 2023 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231757.

[0033] The classification name of Chlorella N4 is Chryseobacterium endophyticum N4 was deposited on March 31, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO:M 2025633. Attached Figure Description

[0034] Figure 1 The effects of different compound microbial agents on tobacco plant height;

[0035] Figure 2 The effects of different compound microbial inoculants on the maximum leaf area of ​​tobacco;

[0036] Figure 3 The effects of different compound microbial inoculants on the total dry weight of tobacco plant roots, stems and leaves.

[0037] Among them, different lowercase letters indicate significant differences between different treatments in the same period. P <0.05); where the bar chart for the same trait is marked as a, b, c or d respectively, it indicates that the difference in the same trait is significant; if the bar chart for the same trait is all a, it indicates that the difference between the two is not significant. Detailed Implementation

[0038] This invention provides a composite microbial suspension, comprising a suspension of *Streptococcus pyogenes* R3 and a suspension of *Chlorella vulgaris* N4;

[0039] The volume ratio of the *Streptococcus pyogenes* R3 suspension to the *Aureobacillus chrysogenum* N4 suspension is 1~2:1~2, preferably 1:1;

[0040] The *Streptococcus filiis* R3 suspension was prepared from *Streptococcus filiis* R3.

[0041] The taxonomic name of the *Streptococcus pyogenes* R3 is: Herbaspirillum sp. R3 was deposited on September 20, 2023 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231757;

[0042] The preparation method of the *Streptococcus pyogenes* R3 bacterial suspension is as follows: *Streptococcus pyogenes* R3 is inoculated into LB liquid medium and cultured for 24 h at a temperature of 28℃ and a rotation speed of 160 rpm to obtain fermentation broth. The fermentation broth is centrifuged at a rotation speed of 6000 rpm for 10 min, the supernatant is removed and the bacterial cells are collected. The bacterial cells are washed twice with sterile PBS buffer and then resuspended in sterile deionized water to obtain *Streptococcus pyogenes* R3 bacterial suspension.

[0043] The Chlorella N4 bacterial suspension was prepared from Chlorella N4.

[0044] The taxonomic name of the *Cyperus yunnanensis* N4 is: Chryseobacterium endophyticum N4 was deposited at the China Center for Type Culture Collection on March 31, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 2025633.

[0045] The method for preparing the Chlorella N4 bacterial suspension is as follows: Chlorella N4 is inoculated into LB liquid medium and cultured for 36 hours at 32°C and 180 rpm to obtain fermentation broth. The fermentation broth is centrifuged at 6000 rpm for 10 minutes, the supernatant is removed, and the bacterial cells are collected. The bacterial cells are washed twice with sterile PBS buffer and then resuspended in sterile deionized water to obtain the Chlorella N4 bacterial suspension.

[0046] In this invention, the concentrations of the *Streptococcus pyogenes* R3 suspension and the *Chlorella vulgaris* N4 suspension are independently (1~9)×10⁻⁶. 8 cfu / mL, preferably 5 × 10⁻⁶ 8 cfu / mL.

[0047] This invention provides a composite microbial agent, which is made using the composite microbial suspension, composite carrier and protectant as described above.

[0048] This invention provides a method for preparing a compound microbial agent, comprising the following steps:

[0049] Step (1): Mix the composite microbial suspension, carrier material and protectant to obtain a mixture;

[0050] Step (2): Mix the mixture with glutaraldehyde solution and react for 10-20 minutes to obtain a mixed solution;

[0051] The preferred reaction time is 15 min;

[0052] Step (3): Spray dry the mixed solution to obtain the composite microbial agent;

[0053] The composite microbial suspension mentioned in step (1) is the composite microbial suspension described above;

[0054] The mass ratio of the composite microbial suspension, carrier material, and protectant is 1~2:1~2:0.1~0.2, preferably 1:1:0.1.

[0055] In this invention, the carrier material in step (1) includes a polyvinyl alcohol solution and a chitosan-montmorillonite nanocomposite; the mass concentration of the polyvinyl alcohol solution is 4~6wt%, preferably 5wt%; the mass ratio of polyvinyl alcohol to chitosan-montmorillonite nanocomposite in the polyvinyl alcohol solution is 4~6:2~4, preferably 5:3;

[0056] The preparation method of the chitosan-montmorillonite nanocomposite is as follows: chitosan (CS) powder is added to 1% (v / v) glacial acetic acid solution at a mass fraction of 2% (w / v), and the mixture is magnetically stirred at room temperature for 2.5 h until it is completely dissolved to obtain a transparent and homogeneous chitosan solution; montmorillonite (MMT) powder is weighed at a mass ratio of CS:MMT = 2:1 (w / w), and the montmorillonite is added to a small amount of deionized water and ultrasonically dispersed for 25 min to allow it to be fully exfoliated. Montmorillonite dispersion was added dropwise to chitosan solution while stirring. Stirring continued at 35℃ for 3 hours to uniformly disperse the montmorillonite sheets and form a nanocomposite system with chitosan molecules, resulting in a homogeneous chitosan-montmorillonite nanocomposite sol. Subsequently, the pH of the CS-MMT nanocomposite sol was adjusted to 6.3 with 1 mol / L NaOH solution, allowed to stand for 30 minutes, and centrifuged to remove undispersed large particles, obtaining a supernatant. The supernatant was placed in a freeze-drying bottle and pre-frozen at -40℃ for 7 hours to completely freeze the system. Then, it was placed in a freeze dryer and sublimation dried under a vacuum of 20 Pa, with the shelf temperature slowly increased from -40℃ to 5℃ until the moisture was basically removed, resulting in a loose and porous chitosan-montmorillonite nanocomposite.

[0057] The protective agent in step (1) is glycerol, and the mass concentration of the glycerol is 8~12wt%, preferably 10wt%.

[0058] In this invention, the volume ratio of the mixture to the glutaraldehyde solution in step (2) is 15-25:1, preferably 20:1; the mass concentration of the glutaraldehyde solution is 0.5-1.5 wt%, preferably 1 wt%.

[0059] The reaction temperature in step (2) is 20~30℃, preferably 25℃.

[0060] In this invention, the temperature of the spray drying air inlet in step (3) is 70~80℃, the temperature of the air outlet is 60~70℃, and the feeding rate is 15~18mL / min;

[0061] During the spray drying process, the air inlet temperature is preferably 75°C, the air outlet temperature is preferably 65°C, and the feed rate is preferably 16.5 mL / min.

[0062] Based on the same inventive concept, a tobacco seed coating agent is also provided, comprising the composite microbial agent and binder as described above, wherein the binder is gum arabic liquid, and the concentration of the gum arabic liquid is 5-15 wt%. The coating layer of the tobacco seed coating agent has a thickness of 60-100 μm.

[0063] Based on the same inventive concept, the application of the compound microbial agent or tobacco seed coating agent as described above in increasing the nitrogen and phosphorus content of tobacco planting soil and improving the growth traits of tobacco plants is also provided.

[0064] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0065] In the examples, both *Streptococcus pyogenes* R3 and *Aureobacillus yunnanensis* N4 were endophytic bacteria screened from Huanghuazhan rice seeds in Zhouxi Town, Kaili City, Guizhou Province by Professor Li Juan's research group at the College of Agriculture, Hunan Agricultural University, and have been deposited at the China Center for Type Culture Collection.

[0066] Example 1: Isolation, screening, identification, and preservation of *Streptococcus pyogenes* R3 and *Chlorella vulgaris* N4

[0067] I. Preparation of Culture Medium

[0068] 1 / 2LB liquid culture medium: Weigh 5.0 g sodium chloride, 2.5 g yeast extract and 5.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, autoclave at 121°C for 30 minutes, and use after cooling.

[0069] NB liquid culture medium: Weigh 10.0 g of glucose, 3.0 g of beef extract and 5.0 g of peptone, and make up to 1000 mL with ultrapure water. Adjust the pH to 7.0, dispense into Erlenmeyer flasks, seal and sterilize by autoclaving at 121°C for 30 minutes. Use after cooling.

[0070] Phosphate-solubilizing bacteria screening medium: Weigh 5 g of tricalcium phosphate [Ca3(PO4)2], 10 g of glucose, 0.25 g of MgSO4·7H2O, 5 g of MgCl2·6H2O, 0.1 g of (NH4)2SO4, and 0.2 g of KCl. Make up to 1000 mL with ultrapure water, adjust the pH to 7.5, add 15 g of agar, and autoclave at 121℃ for 30 minutes. Cool to 50℃ and pour into plates.

[0071] Ashby nitrogen-fixing bacteria selection medium: Weigh 0.2 g KH2PO4, 10 g mannitol, 0.2 mL NaCl, 0.2 g MgSO4·7H2O, 5 g CaCO3, and 0.1 g CaSO4·2H2O, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0072] 1 / 2LB solid culture medium: Weigh 5.0 g of sodium chloride, 2.5 g of yeast extract and 5.0 g of tryptone, and make up to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g of agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0073] NB solid medium: Weigh 10.0 g glucose, 3.0 g beef extract and 5.0 g peptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0074] II. Bacterial Screening

[0075] Plump and healthy Huanghuazhan rice seeds from Zhouxi Town, Kaili City, Guizhou Province were selected. First, the seeds were soaked in 75% anhydrous ethanol for 10 minutes, then the ethanol was poured off, and the seeds were rinsed five times with sterile water. Next, they were soaked in 5% NaClO for 10 minutes, and then rinsed five times with sterile water. 100 μL of the sterile water from the final rinse was spread onto 1 / 2 LB solid culture medium and incubated in a constant temperature incubator for 5 days. The presence of bacterial growth on the medium was observed to ensure thorough sterilization of the rice seed surface.

[0076] Endophytic bacteria were isolated from rice seeds using 1 / 2 LB and NB liquid media. Surface-sterilized rice seeds were ground into powder in a sterile mortar with a small amount of liquid nitrogen. The rice seed powder was then inoculated into 250 mL of 1 / 2 LB and NB liquid media using a sterile weighing spoon. After 36 hours of incubation in a shaker (28 ℃, 180 rpm / min), 1 mL of the bacterial suspension was diluted to 1×10⁻⁶. 4 1×10 5 and 1×10 6After dilution, 100 μL of the bacterial suspension was spread onto the corresponding 1 / 2 LB and NB solid media, and incubated upside down in a constant temperature incubator (28 ℃). The growth of the strains on the plates was observed periodically. After 5 days of growth, strains with different morphological characteristics were selected from the plates using an inoculation loop and streaked onto the corresponding solid media to obtain purified single bacteria. After activation, the single bacteria were mixed with sterile glycerol at a ratio of 1:1, and 1.5 mL was aliquoted into 2 mL centrifuge tubes and stored at -80 ℃ for later use.

[0077] III. Analysis of the growth-promoting effects of endophytic bacteria

[0078] 3.1 Qualitative Analysis of IAA Functions

[0079] Two isolated endophytic bacteria strains were inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured in a shaker (28 ℃, 180 rpm / min) for 36 hours. 50 μL of the cultured bacterial suspension, plus 50 μL of Salkowski's colorimetric solution, was placed in the wells of a white ceramic plate. The positive control consisted of 50 μL of IAA (50 mg / L) plus 50 μL of Salkowski's colorimetric solution. After standing in the dark at a constant temperature for 30 minutes, the color changes were observed.

[0080] 3.2 Qualitative Analysis of Phosphorus Solubility

[0081] The two isolated endophytic bacteria were inoculated onto solid culture media used to test the phosphorus-solubilizing function of the strains. The presence of phosphorus-solubilizing zones on the plates was observed over 7 days. The presence of such zones indicated that the bacteria had phosphorus-solubilizing function.

[0082] 3.3 Qualitative Analysis of Nitrogen Fixation Function

[0083] The isolated endophytic bacteria were inoculated onto Ashby nitrogen-fixing bacteria selection medium, which is used to test the nitrogen-fixing function of the strains. Their growth was observed over 7 days, and growth indicated that the bacteria had nitrogen-fixing function.

[0084] The study found that two strains of bacteria simultaneously possessed phosphorus-solubilizing and nitrogen-fixing functions. The species and physiological functions of the isolated and screened bacteria were identified, and the results are shown in Table 1.

[0085] Table 1. Physiological functions and species identification of the strains

[0086]

[0087] Note: "+" indicates that this function is available; "-" indicates that this function is not available.

[0088] IV. Preservation of bacterial strains

[0089] Two bacterial strains were inoculated into 50 mL of LB liquid medium (pH 7.0) and activated for 24 h in a shaker (28℃, 180 rpm). The LB slant medium, which had been purchased in advance and stored under refrigeration, was taken out of the refrigerator and placed in a clean bench for UV sterilization for 1 h (room temperature).

[0090] Establish a flame-sterile zone by lighting the alcohol lamp inside the laminar flow hood. Holding the slant culture medium in your left hand and the inoculation loop in your right, first heat the inoculation loop over the flame until it is red-hot for about 30 seconds, then repeatedly heat the metal parts three times. After slightly cooling, dip the loop into an appropriate amount of LB bacterial suspension. When opening the tube, hold the stopper with your ring and little fingers, bring the tube opening close to the flame, and rotate the heated end to open. Insert the inoculation loop into the tube to the bottom of the LB slant culture medium, draw a straight line from bottom to top, then draw a continuous curved line from bottom to top to the top of the slant, ensuring the bacteria are evenly distributed on the slant surface. After removing the inoculation loop, quickly seal the tube near the flame and immediately sterilize the inoculation loop again by flame.

[0091] The inoculated bacterial tubes were placed in a 28°C incubator for 24 hours. After bacterial growth, they were sent to the China Center for Type Culture Collection in Wuhan, China. The taxonomic name of *Streptococcus solani* R3 is... Herbaspirillum sp. R3 was deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20231757. The taxonomic name of *Citrus japonicus* N4 is... Chryseobacterium endophyticum N4 was deposited on March 31, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 2025633.

[0092] Example 2

[0093] 1. Preparation method of compound microbial inoculant

[0094] 1.1 Material Preparation

[0095] (1) Preparation of *Streptococcus pyogenes* R3 suspension: *Streptococcus pyogenes* R3 was inoculated into LB liquid medium and cultured for 24 h at 28℃ and 160 rpm to obtain fermentation broth. The fermentation broth was centrifuged at 6000 rpm for 10 min, the supernatant was removed, and the bacterial cells were collected. The bacterial cells were washed twice with sterile PBS buffer and then resuspended in sterile deionized water to obtain a suspension with a concentration of 5 × 10⁻⁶. 8 A suspension of *Streptococcus pyogenes* R3 bacteria at cfu / mL is prepared for later use.

[0096] (2) Preparation of Chlorella N4 bacterial suspension: Chlorella N4 was inoculated into LB liquid medium and cultured at 32℃ and 180 rpm for 36 h to obtain fermentation broth. The fermentation broth was centrifuged at 6000 rpm for 10 min, the supernatant was removed, and the bacterial cells were collected. The bacterial cells were washed twice with sterile PBS buffer and then resuspended in sterile deionized water to obtain a concentration of 5×10⁻⁶. 8 A suspension of *C. chrysogenum* N4 bacteria at cfu / mL is prepared for later use.

[0097] (3) Preparation of 5wt% polyvinyl alcohol solution: Soak 50g of polyvinyl alcohol (PVA) powder in 950mL of deionized water, heat and stir in a water bath at 65℃ until completely dissolved, cool to room temperature and filter the membrane on the top of the solution through a 20-mesh sieve to obtain a 5wt% polyvinyl alcohol solution, which is stored in a plastic container for later use.

[0098] Preparation of 1wt% glutaraldehyde solution: Take 10mL of 25wt% glutaraldehyde stock solution and add it to 240mL of water. Mix well to obtain a 1wt% glutaraldehyde solution.

[0099] (4) Preparation of chitosan-montmorillonite nanocomposite: Take chitosan (CS) powder and add it to 1% (v / v) glacial acetic acid solution at a mass fraction of 2% (w / v). Stir magnetically for 2.5 h at room temperature until completely dissolved to obtain a transparent and uniform chitosan solution. Take montmorillonite (MMT) powder and weigh it at a mass ratio of CS:MMT=2:1 (w / w). Add montmorillonite to a small amount of deionized water and ultrasonically disperse for 25 min to allow it to fully exfoliate. The montmorillonite dispersion was slowly added dropwise to the chitosan solution while stirring. Stirring continued at 35°C for 3 hours to ensure uniform dispersion of the montmorillonite sheets and formation of a nanocomposite system with the chitosan molecules, resulting in a homogeneous CS-MMT nanocomposite sol. The pH of the CS-MMT nanocomposite sol was then adjusted to 6.3 with a 1 mol / L NaOH solution. After standing for 30 minutes, the sol was centrifuged to remove undispersed large particles, yielding a supernatant. This supernatant was then placed in a freeze-drying bottle and pre-frozen at -40°C for 7 hours to ensure complete freezing. The system was then placed in a freeze dryer and sublimated under a vacuum of 20 Pa, with the shelf temperature slowly increased from -40°C to 5°C until the moisture was almost completely removed, resulting in a loose and porous chitosan-montmorillonite nanocomposite for later use.

[0100] (5) Preparation of carrier material: 5 wt% polyvinyl alcohol solution and chitosan-montmorillonite nanocomposite powder were mixed at a mass ratio of 5:3 for polyvinyl alcohol solution to chitosan-montmorillonite nanocomposite powder to obtain carrier material for later use.

[0101] 1.2 Preparation of different microbial agents and compound microbial agents

[0102] a. A mixture of *Streptococcus pyogenes* R3 bacterial suspension, carrier material, and glycerol (protectant) was prepared by mixing them in a mass ratio of 1:1:0.1. The mixture was then mixed with a 1 wt% glutaraldehyde solution in a volume ratio of 20:1. The mixture was reacted at 25°C for 15 min to obtain a mixed solution. The solution was then spray-dried at an inlet temperature of 75°C, an outlet temperature of 65°C, and a feed rate of 17 mL / min to obtain *Streptococcus pyogenes* R3 microbial inoculant.

[0103] b. A mixture of Chlorella N4 bacterial suspension, carrier material, and glycerol (protectant) was prepared by mixing in a mass ratio of 1:1:0.1. The mixture was then mixed with a 1 wt% glutaraldehyde solution in a volume ratio of 20:1. The mixture was reacted at 25°C for 15 min to obtain a mixed solution. The solution was then spray-dried at an inlet temperature of 75°C, an outlet temperature of 65°C, and a feed rate of 17 mL / min to obtain Chlorella N4 microbial inoculum.

[0104] c. A composite microbial suspension was obtained by mixing *Streptococcus pyogenes* R3 suspension and *Chlorella vulgaris* N4 suspension at a volume ratio of 1:1. The composite microbial suspension, carrier material, and glycerol (protectant) were then mixed at a mass ratio of 1:1:0.1 to obtain a mixture. This mixture was then mixed with a 1 wt% glutaraldehyde solution at a volume ratio of 20:1. The mixture was reacted at 25°C for 15 min to obtain a mixed solution. The solution was then spray-dried at an inlet temperature of 75°C, an outlet temperature of 65°C, and a feed rate of 17 mL / min to obtain the composite microbial agent.

[0105] 2. Effects of different microbial agents on tobacco soil and growth traits

[0106] 2.1 Experimental Materials

[0107] Seeds: Seeds of Yunyan 87 tobacco; Microbial agents: Cladosporium R3 microbial agent, Aureobacillus N4 microbial agent, compound microbial agent.

[0108] 2.2 Experimental Methods

[0109] Using 10wt% gum arabic as a binder, microbial inoculants and tobacco seeds were mixed in a coating device to obtain coated seeds, with the coating layer controlled at 80μm.

[0110] The experiment was divided into four groups: a blank control group (CK group), a *Streptococcus pyogenes* R3 microbial inoculant group (R3), a *Citrus aureus* N4 microbial inoculant group (N4), and a compound microbial inoculant group (R3+N4). The blank control group (CK group) did not use any microbial inoculant to coat the seeds. The *Streptococcus pyogenes* R3 microbial inoculant group (R3), the *Citrus aureus* N4 microbial inoculant group (N4), and the compound microbial inoculant group (R3+N4) used their respective microbial inoculants to coat the seeds. Tobacco seeds from all treatment groups were planted under the same conditions.

[0111] 2.3 Effects of different microbial inoculants on the chemical composition of tobacco soil

[0112] The physicochemical properties of tobacco soil under different treatment groups were measured at 30, 60, and 90 days. The results are shown in Table 2. Different lowercase letters in the table indicate significant differences between different treatments at the same time. P <0.05).

[0113] Table 2. Physicochemical properties of tobacco soil from different treatment groups at different growth stages of tobacco.

[0114]

[0115] As shown in Table 2, seed coating with compound microbial agents significantly increased the contents of ammonium nitrogen, nitrate nitrogen, and available phosphorus in the soil. Different treatment groups showed different effects. At 30 days, compared with the control group, the R3+N4 group showed increases of 101.46%, 41.86%, and 51.36% in soil ammonium nitrogen, nitrate nitrogen, and available phosphorus, respectively. At 60 days, the increases were 303.39%, 64.68%, and 172.25%, respectively. At 90 days, the increases were 283.97%, 50.04%, and 73.13%, respectively, all of which were greater than the increases in the R3, N4, and control groups.

[0116] 2.4 Effects of different microbial inoculants on tobacco growth traits

[0117] 2.4.1 Effects of different complex microbial inoculants on tobacco plant height

[0118] The plant height of tobacco plants in different treatment groups was measured at 30, 60, and 90 days to compare the effects of different microbial inoculants on tobacco plant height. The results are as follows: Figure 1 As shown in the figure, different lowercase letters indicate significant differences between different treatments at the same time. P <0.05).

[0119] according to Figure 1It can be seen that after coating tobacco seeds with compound microbial agents, there was no significant difference in plant height among different treatment groups at 30 days. However, at 60 and 90 days, the plant height of R3, N4 and R3+N4 groups was significantly higher than that of CK group. The plant height of tobacco plants in R3+N4 group increased by 22.76% and 26.43% at 60 and 90 days, respectively, compared with CK group.

[0120] 2.4.2 Effects of different microbial inoculants on the maximum leaf area of ​​tobacco

[0121] Tobacco, as an economic crop primarily harvested for its leaves, relies heavily on leaf size for yield and quality. This study measured the area of ​​the largest leaves in different treatment groups at 30, 60, and 90 days of tobacco growth, comparing the effects of different microbial inoculants on the largest leaf area. The results... Figure 2 As shown in the figure, different lowercase letters indicate significant differences between different treatments at the same time. P <0.05).

[0122] according to Figure 2 It can be seen that coating seeds with compound microbial agents can significantly increase the area of ​​the largest leaf of tobacco plants. There was no significant difference in the area of ​​the largest leaf of tobacco plants among different treatment groups at 30 days, but at 60 and 90 days, the area of ​​the largest leaf of tobacco plants in the R3+N4 group was better than that in the R3, N4 and CK groups. For example, at 90 days, the increase of the R3 and N4 groups relative to the CK group was 6.86% and 0.42% respectively, while the increase of the R3+N4 group relative to the CK group was 18.43%.

[0123] 2.4.3 Effects of different microbial inoculants on the total dry weight of tobacco roots, stems and leaves

[0124] The total dry weight of roots, stems, and leaves of tobacco plants in different treatment groups was measured at 30, 60, and 90 days to compare the effects of different microbial inoculants on the total dry weight of tobacco plants. Results Figure 3 As shown in the figure, different lowercase letters indicate significant differences between different treatments at the same site. P <0.05).

[0125] according to Figure 3 It can be seen that coating seeds with compound microbial agents can significantly increase the total dry weight of tobacco plants' roots, stems and leaves; this indicates that under the phosphorus-solubilizing and nitrogen-fixing functions of compound microbial agents, tobacco growth receives sufficient nutrient supply, and the total dry weight of tobacco roots, stems and leaves is increased.

[0126] In summary, when a compound microbial agent prepared from a compound microbial suspension of *Streptococcus pyogenes* R3 and *Aureobacillus thuringiensis* N4 is used to coat tobacco seeds, it can significantly increase the content of available phosphorus, ammonium nitrogen, and nitrate nitrogen in the soil, providing sufficient phosphorus and nitrogen sources for tobacco growth and thus improving the agronomic traits of tobacco.

[0127] Example 3

[0128] 1. Effects of different ratios of bacterial suspension in compound microbial agents on soil and plants

[0129] A compound microbial inoculant was prepared using 10 wt% gum arabic as a binder. The compound microbial inoculant and tobacco seeds were mixed in a coating device to obtain coated seeds, with the coating layer controlled at 80 μm. The preparation method of the compound microbial inoculant was the same as in Example 2, except that the ratio of the two bacterial suspensions used in preparing the compound microbial inoculant was different, resulting in four treatment groups: Treatment A: the volume ratio of *Spirulina rhamnoides* R3 suspension to *Chlorella vulgaris* N4 suspension in the compound microbial inoculant was 1:2; Treatment B: the volume ratio of *Spirulina rhamnoides* R3 suspension to *Chlorella vulgaris* N4 suspension in the compound microbial inoculant was 2:1; Treatment C: the volume ratio of *Spirulina rhamnoides* R3 suspension to *Chlorella vulgaris* N4 suspension in the compound microbial inoculant was 1:1; and the blank control group: seeds were not coated. Tobacco seeds from different treatment groups were planted under the same conditions.

[0130] Sixty days after planting, tobacco plants and rhizosphere soil samples were collected to test the ammonium nitrogen, available phosphorus, tobacco plant height, and maximum leaf area in the rhizosphere soil. The growth traits of tobacco and the physicochemical properties of the tobacco soil were compared among different treatment groups. The results are shown in Table 3. Different lowercase letters in the table indicate significant differences between different treatments for the same indicator. P <0.05).

[0131] Table 3. Growth characteristics of tobacco and physicochemical properties of tobacco soil in different treatment groups

[0132]

[0133] Table 3 shows that the increases in available phosphorus in treatments A and B were 120.54% and 135.04%, respectively, both lower than that in treatment C (172.25%). A 1:1 volume ratio of Spirochete R3 suspension to Chlorella N4 suspension in the compound microbial agent resulted in a synergistic enhancement of the phosphorus-solubilizing function of both bacteria. The ratio of the two bacterial suspensions in the compound microbial agent significantly affected the soil ammonium nitrogen content. Treatment C had the highest ammonium nitrogen content (7.14 mg / kg), significantly higher than treatments A and B. There was no significant difference between treatments A and B. P>0.05), but all were significantly higher than the CK group. The increase in plant height and leaf area in treatment groups A and B was lower than that in treatment group C, further verifying that the volume ratio of Spirobrium R3 suspension to Chlorella N4 suspension in the compound microbial agent of 1:1 is the optimal ratio that takes into account phosphorus solubilization, nitrogen fixation and crop growth.

[0134] 2. Effects of different preservatives on the storage stability of coated seeds

[0135] A compound microbial agent was prepared using 10 wt% gum arabic as a binder. The compound microbial agent was mixed with tobacco seeds in a coating device to obtain coated seeds, with the coating layer controlled to be between 80 μm. The preparation method of the compound microbial agent was the same as in Example 2, except that different protective agents were used, resulting in three treatment groups: Treatment Group 1: 5 wt% glycerol + 5 wt% sucrose; Treatment Group 2: 15 wt% glycerol; Treatment Group 3: 10 wt% glycerol. Tobacco seeds with different treatments were placed under two typical seed storage conditions: room temperature storage (25℃ constant temperature, 60% relative humidity) and refrigerated storage (4℃ constant temperature, 50% relative humidity). The viable cell count of the coated seeds before and after storage was measured, and the viable cell rate was calculated. The viable cell rate of seeds coated with different protective agents under different storage conditions was compared, and the results are shown in Table 4.

[0136] Table 4. Viability of seeds coated with different preservatives under different storage conditions

[0137]

[0138] Table 4 shows that the viable cell rate of treatment group 1 after 6 months of storage at room temperature was 65.2%, lower than the 70.1% of treatment group 3. The viable cell rate of treatment group 2 after 3 months of storage at room temperature was 83.5%, slightly higher than the 82.3% of treatment group 3. However, the 15% glycerol in treatment group 2 increased the viscosity of the coating material, leading to increased particle agglomeration during spray drying and affecting coating uniformity. The viable cell retention rate of treatment group 3 after 6 months of refrigerated storage was 89.5%, close to the 88.2% of treatment group 2. Finally, considering viable cell stability, agglomeration, and cost, 10% glycerol was determined to be the optimal protective agent.

[0139] 3. Effects of different coating layer thicknesses on seed germination and plant growth

[0140] A compound microbial inoculant was prepared using 10 wt% gum arabic as a binder. The compound microbial inoculant was mixed with tobacco seeds in a coating device to obtain coated seeds. The preparation method of the compound microbial inoculant was the same as in Example 2, except that the coating layer thickness of the coated seeds was different, resulting in three treatment groups: treatment group D (40 μm), treatment group E (80 μm), and treatment group F (100 μm). Seeds from different treatment groups were subjected to germination treatment, and the germination rate was measured after 7 and 14 days. The tobacco seeds from different treatment groups were then planted under the same conditions. After 90 days, the dry weight of the tobacco leaves was measured, and the effects of different coating layer thicknesses on seed germination and plant growth were compared. The results are shown in Table 5.

[0141] Table 5. Effects of different coating layer thicknesses on seed germination and plant growth.

[0142]

[0143] Table 5 shows that the germination rate of treatment group D (40 μm) was 92.5% after 14 days, and that of treatment group E (80 μm) was 90.2%, significantly higher than that of treatment group F (81.3%). After 90 days, the order of tobacco leaf dry weight was: treatment group E > treatment group D > treatment group F. This may be because a thin coating layer led to inoculant loss, or a thick coating layer hindered water absorption and oxygen exchange in the seeds. Treatment group F experienced delayed growth during germination, affecting later accumulation. Therefore, 80 μm is the optimal coating thickness, balancing seed germination rate and inoculant retention.

[0144] In summary, the optimal preparation method for compound microbial agents is as follows:

[0145] A composite microbial suspension was prepared by mixing *Streptococcus pyogenes* R3 suspension and *Chlorella vulgaris* N4 suspension at a volume ratio of 1:1. The composite microbial suspension, carrier material, and glycerol (protectant) were then mixed at a mass ratio of 1:1:0.1 to obtain a mixture. This mixture was then mixed with a 1 wt% glutaraldehyde solution at a volume ratio of 20:1. The mixture was reacted at 25°C for 15 min to obtain a mixed solution. This solution was then spray-dried at an inlet temperature of 75°C, an outlet temperature of 65°C, and a feed rate of 17 mL / min to obtain the composite microbial agent.

[0146] The best method for seed coating treatment is:

[0147] Using 10wt% gum arabic as a binder, the compound microbial agent and tobacco seeds were mixed in a coating device to obtain coated seeds, with the coating layer thickness controlled at 80μm.

[0148] As can be seen from the above embodiments, the present invention provides a composite microbial suspension, inoculum, tobacco seed coating agent, its preparation method, and its application. The present invention utilizes polyvinyl alcohol, chitosan, and montmorillonite nanocomposite to prepare the carrier material, and finally uses the natural binder gum arabic to enhance the stability of the coating layer. Polyvinyl alcohol has good film-forming properties and water solubility, and as a carrier matrix, it can form a uniform slow-release system and is non-toxic to the bacteria. The amino groups of chitosan can form an interpenetrating network structure with the hydroxyl groups of polyvinyl alcohol through hydrogen bonds, extending the release cycle of the microbial inoculum. The montmorillonite nanocomposite can adsorb bacterial metabolites (such as acid phosphatase) through ion exchange, achieving a dual slow release of nutrients and enzymes. Gum arabic liquid, as a natural binder, has moderate viscosity, allowing the composite microbial inoculum to adhere uniformly to the seed surface, forming a coating layer of controllable thickness, with good biodegradability and no environmental pollution. This coating process greatly improves the stability and activity of the bacteria in different environments.

[0149] This invention uses a combination of *Streptococcus pyogenes* R3 and *Aureobacillus yunnanensis* N4, which have phosphorus-solubilizing and nitrogen-fixing functions, to prepare a compound microbial agent. This allows the bacteria to continuously release beneficial substances during seed germination, helping tobacco dissolve insoluble phosphorus sources in the soil, promoting nitrogen fixation and nitrogen conversion, and thus improving agronomic traits such as tobacco plant height and maximum leaf area.

[0150] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composite microbial suspension, characterized in that, Including suspensions of *Streptococcus pyogenes* R3 and *Chlorella vulgaris* N4; The volume ratio of the *Streptococcus pyogenes* R3 suspension to the *Aureobacillus chrysogenum* N4 suspension is 1~2:1~2; The *Streptococcus filiis* R3 suspension was prepared from *Streptococcus filiis* R3. The R3 classification name of the *Streptococcus* is... Herbaspirillum sp. R3 was deposited on September 20, 2023 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20231757; The Chlorella N4 bacterial suspension was prepared from Chlorella N4. The taxonomic name of the *Cyperus yunnanensis* N4 is: Chryseobacterium endophyticum N4 was deposited on March 31, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO:M 2025633.

2. The composite microbial suspension according to claim 1, characterized in that, The concentrations of the *Streptococcus pyogenes* R3 suspension and the *Chlorella vulgaris* N4 suspension were both (1~9)×10⁻⁶. 8 cfu / mL.

3. A composite microbial agent, made from the composite microbial suspension, composite carrier, and protectant as described in claim 1 or 2.

4. A method for preparing the composite microbial agent as described in claim 3, characterized in that, Includes the following steps: Step (1): Mix the composite microbial suspension, carrier material and protectant to obtain a mixture; Step (2): Mix the mixture with glutaraldehyde solution and react for 10-20 minutes to obtain a mixed solution; Step (3): Spray dry the mixed solution to obtain the composite microbial agent; The mass ratio of the composite microbial suspension, carrier material, and protectant is 1~2:1~2:0.1~0.

2.

5. The preparation method according to claim 4, characterized in that, The carrier material in step (1) includes a polyvinyl alcohol solution and a chitosan-montmorillonite nanocomposite; the mass concentration of the polyvinyl alcohol solution is 4~6wt%; the mass ratio of polyvinyl alcohol to chitosan-montmorillonite nanocomposite in the polyvinyl alcohol solution is 4~6:2~4; The protective agent in step (1) is glycerol, and the mass concentration of the glycerol is 8~12wt%.

6. The preparation method according to claim 4, characterized in that, The volume ratio of the mixture to the glutaraldehyde solution in step (2) is 15-25:1; the mass concentration of the glutaraldehyde solution is 0.5-1.5 wt%. The reaction temperature in step (2) is 20~30℃.

7. The preparation method according to claim 4, characterized in that, In step (3), the temperature of the air inlet during spray drying is 70~80℃, the temperature of the air outlet is 60~70℃, and the feed rate is 15~18mL / min.

8. A tobacco seed coating agent, characterized in that, It includes the composite microbial agent and binder as described in claim 3, wherein the binder is gum arabic liquid, and the concentration of gum arabic liquid is 5~15wt%.

9. The tobacco seed coating agent according to claim 8, characterized in that, The coating layer of the tobacco seed coating agent has a thickness of 60~100μm.

10. The application of the compound microbial agent of claim 3 or the tobacco seed coating agent of claim 8 or 9 in improving the nitrogen and phosphorus content of tobacco planting soil, as well as the growth traits of increasing tobacco plant height, tobacco leaf area, tobacco leaf dry weight, and promoting tobacco seed germination rate.

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