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

Abstract

The invention relates to the technical field of agricultural microorganisms. The invention provides a compound microorganism bacterium suspension, a bacterium agent, a tobacco seed coating agent as well as a preparation method and application of the compound microorganism bacterium suspension. The compound microorganism bacterium suspension comprises a rice seed endophytic bacterium spiraceae R3 bacterium suspension and a rice seed endophytic bacterium stenotrophomonas R5 bacterium suspension; the mass ratio of R3 to R5 is 1: (0.5-1.5); the endophytic bacterium herbapiril R3 of the rice seeds is Herbapiril sp. R3, and the preservation number of the herbapiril R3 is CCTCC (China Center For Type Culture Collection) NO: M 20231757; the rice seed endophytic bacterium stenotrophomonas R5 is Shenotrophomonas sp. R5, and the preservation number of the rice seed endophytic bacterium stenotrophomonas R5 is CCTCC (China Center for Type Culture Collection) NO: M 20231758. The compound microbial suspension is used as a seed coating agent to coat tobacco seeds, and growth of tobacco root systems can be remarkably promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganism technology, and particularly relates to a compound microbial bacteria suspension, a bacterial agent, a tobacco seed coating agent, and a preparation method and application thereof. BACKGROUND

[0002] Roots are the main way for plants to obtain water, minerals and nutrients, and the development quality thereof directly affects the growth, yield and stress resistance of plants, especially for economic crops such as tobacco, the development of roots not only determines the absorption capacity of tobacco to soil nutrients, but also determines the growth status of tobacco. However, the development of roots of current economic crops such as tobacco is often limited by many factors such as environment and soil conditions, especially in the early seedling stage, the roots cannot fully develop, which limits the growth and yield of tobacco.

[0003] Current technologies mainly focus on the overall growth regulation of tobacco, and the accurate regulation of roots is relatively lacking, and the problems of slow root growth and small root surface area still exist. Therefore, developing a technology capable of effectively promoting root development has become a key to improving the yield of tobacco. SUMMARY

[0004] The present application aims to provide a compound microbial bacteria suspension, a bacterial agent, a tobacco seed coating agent, and a preparation method and application thereof, the compound microbial bacteria suspension as a coating agent for coating tobacco seeds can significantly promote the growth of tobacco roots, can improve the root index of tobacco, and is thus beneficial to providing the required nutrients for the growth of tobacco and improving the yield of tobacco leaves.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: A compound microbial bacteria suspension, comprising a rice seed endophytic bacteria Spirosoma sp. R3 bacteria suspension and a rice seed endophytic bacteria Stenotrophomonas sp. R5 bacteria suspension; in the compound microbial bacteria suspension, the mass ratio of the rice seed endophytic bacteria Spirosoma sp. R3 and the rice seed endophytic bacteria Stenotrophomonas sp. R5 is 1:(0.5-1.5); The classification name of the rice seed endophytic bacteria Spirosoma sp. R3 is Spirosoma sp. R3, which was preserved in the China Center for Type Culture Collection on September 20, 2023, the preservation address is China. Wuhan. Wuhan University, and the preservation number is CCTCC NO: M 20231757; Herbaspirillum The classification name of the rice seed endophytic bacteria Stenotrophomonas sp. R5 is Stenotrophomonas sp. R5, which was preserved in the China Center for Type Culture Collection on September 20, 2023, the preservation address is China. Wuhan. Wuhan University, and the preservation number is CCTCC NO: M 20231758. Stenotrophomonas The classification name of the rice seed endophytic bacteria Stenotrophomonas sp. R5 is Stenotrophomonas sp. R5, which was preserved in the China Center for Type Culture Collection on September 20, 2023, the preservation address is China. Wuhan. Wuhan University, and the preservation number is CCTCC NO: M 20231758.

[0006] ​In an embodiment, the effective viable cell number of the rice seed endophytic bacteria Zoopilus R3 is (1-100) x 10 9 cfu / mL. The effective viable cell number of the rice seed endophytic bacteria Stenotrophomonas R5 is (1-100) x 10 9 cfu / mL.

[0007] Based on the same inventive concept, a composite microbial agent is also provided, which is made of the composite microbial suspension, the composite carrier and the protective agent as described above.

[0008] In an embodiment, the effective viable cell number of the rice seed endophytic bacteria Zoopilus R3 is (1-100) x 10 9 cfu / mL; and the effective viable cell number of the rice seed endophytic bacteria Stenotrophomonas R5 is (1-100) x 10 9 cfu / mL.

[0009] The application also provides a preparation method of the composite microbial agent, which comprises the following steps: (1) mixing and ultrasonicating the biochar and the inorganic clay mineral material, adding chitosan solution, and stirring to obtain a composite carrier; (2) mixing the bacterial suspension of the rice seed endophytic bacteria Zoopilus R3 and the rice seed endophytic bacteria Stenotrophomonas R5, sequentially adding the protective agent and the composite carrier, and freeze-drying to obtain the composite microbial agent.

[0010] In an embodiment, the mass ratio of the biochar, the inorganic clay mineral material and the chitosan is (4-6):(2-4):2.

[0011] In an embodiment, the mass ratio of the total volume of the bacterial suspension of the rice seed endophytic bacteria Zoopilus R3 and the bacterial suspension of the rice seed endophytic bacteria Stenotrophomonas R5 to the protective agent is 100 mL:(10-15) g.

[0012] In an embodiment, the protective agent is composed of trehalose, skimmed milk powder and proline with a mass ratio of (5-8):(3-5):2.

[0013] The application also provides an application of the composite microbial agent in preparing a tobacco seed coating agent.

[0014] The application also provides a tobacco seed coating agent containing the composite microbial suspension, wherein the effective viable cell number of the rice seed endophytic bacteria Zoopilus R3 in the composite microbial suspension is (1-100) x 10 9 cfu / mL, and the effective viable cell number of the rice seed endophytic bacteria Stenotrophomonas R5 in the composite microbial suspension is (1-100) x 109 cfu / mL.

[0015] In an embodiment, the mass ratio of the complex microbial bacteria suspension to the tobacco seeds is (0.03-0.07):1.

[0016] The application also provides the use of the complex microbial bacteria suspension, the bacterial agent or the tobacco seed coating agent in promoting the growth of tobacco.

[0017] The application also provides the use of the complex microbial bacteria suspension, the bacterial agent or the tobacco seed coating agent in promoting the growth of tobacco.

[0018] By adopting the technical scheme, the application has the following beneficial effects: The complex microbial bacteria suspension of the application can significantly promote the growth of tobacco roots by the combined action of the rice seed endophytic bacteria Sphaerotilus R3 and the rice seed endophytic bacteria Stenotrophomonas R5 which have the functions of dissolving phosphorus, fixing nitrogen and promoting growth. The roots show stronger branching ability and larger growth volume, which can improve the root index of tobacco, thereby providing the required nutrients for the growth of tobacco and improving the yield of tobacco leaves. Compared with the use of either of the two, the combined action of the two further improves the above effects and is better than the effect of using either of the two.

[0019] Further, the complex microbial agent is developed by using biochar, porous inorganic clay mineral material and chitosan as carriers, and using trehalose, proline and skimmed milk powder as protective agents. The biochar can provide rich pore structure and surface functional groups, adsorb bacterial metabolic products and release slowly. The inorganic clay mineral material can adsorb bacterial metabolic products (such as indole acetic acid IAA and acid phosphatase) by its nano-porous characteristics (specific surface area 200-300 m 2 / g), load root-promoting nutrients and prolong the action period. The amino groups of chitosan can form a network with the carboxyl groups of biochar through electrostatic interaction to form a biocompatible network, thereby enhancing the mechanical strength and slow-release performance of the coating layer. The loading capacity of the complex carrier formed by the synergistic functions of the three is greatly increased. In addition, trehalose can form a glassy matrix in freeze-drying to protect the cell membrane of the bacteria from damage by ice crystals. The casein of skimmed milk powder can be adsorbed on the surface of the bacteria to reduce protein denaturation during the drying process. Proline as an osmotic protective agent can enhance the stress resistance of the bacteria under high salt or drought conditions.

[0020] Further, by using the prepared complex microbial agent to coat the tobacco seeds, the growth of the tobacco roots can be significantly promoted, such as the roots showing stronger branching ability and larger growth volume, thereby promoting the change of the agronomic traits of tobacco and improving the yield of tobacco leaves.

[0021] BIOLOGICAL PRESERVATION INSTRUCTIONS This invention relates to the endophytic bacterium *Streptococcus pyogenes* R3 in rice seeds, classified as follows: 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.

[0022] This invention relates to the endophytic bacterium *Oligotrophomonas* R5 in rice seeds, classified as follows: Stenotrophomonas sp.R5 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 20231758. Attached Figure Description

[0023] Figure 1 Agronomic traits of tobacco treated with compound microbial suspension after 90 days of growth (different lowercase letters indicate significant differences between different treatments at the same time). P <0.05); In the CK group and R3+R5 group, the bar charts of the same trait (such as leaf length, leaf width, plant height and stem circumference) of the two groups after treatment are marked as a and b respectively, indicating that the difference is significant. The bar charts of the same trait (effective leaves) are all marked as a, indicating that the difference between the two is not significant. Figure 2 This is an experimental diagram showing the effects of compound microbial suspension on tobacco seedlings. Detailed Implementation

[0024] This invention provides a composite microbial suspension containing rice seed endophytic bacteria *Streptococcus pyogenes* R3 and rice seed endophytic bacteria *Oligotrophomonas* R5.

[0025] In this invention, the classification name of the rice seed endophytic bacterium *Streptococcus pyogenes* 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 the rice seed endophytic bacterium *Oligotrophomonas* R5 is... Stenotrophomonas sp. R5 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 20231758.

[0026] In this invention, the mass ratio of the rice seed endophytic bacteria *Streptococcus pyogenes* R3 and *Oligotrophomonas oryzae* R5 is preferably 1:(0.5-1.5), more preferably 1:(0.8-1.2), and even more preferably 1:1.

[0027] In this invention, the effective viable count of the rice seed endophytic bacterium *Streptococcus pyogenes* R3 is preferably (1-100) × 10⁻⁶. 9 cfu / mL, further preferably (5-50)×10 9 cfu / mL, more preferably 10 × 10 9 cfu / mL; the effective viable count of the rice seed endophytic bacterium Oligotrophomonas R5 is preferably (1-100)×10⁻⁶. 9 cfu / mL, further preferably (5-50)×10 9 cfu / mL, more preferably 10 × 10 9 cfu / mL.

[0028] The present invention also provides a method for preparing the aforementioned composite microbial agent, comprising the following steps: (1) After mixing biochar with inorganic clay mineral materials and sonicating, chitosan solution is added and stirred to obtain a composite carrier; wherein the inorganic clay mineral materials are attapulgite, sepiolite, etc., preferably attapulgite; and the biochar is preferably rice straw biochar.

[0029] (2) After mixing the bacterial suspensions of rice seed endophytic bacteria *Streptococcus pyogenes* R3 and rice seed endophytic bacteria *Oligotrophomonas* R5, a protectant and a composite carrier were added in sequence, and the mixture was freeze-dried to obtain a composite microbial agent.

[0030] In this invention, the preferred mass ratio of biochar, inorganic clay mineral material and chitosan is (4-6):(2-4):2, further preferably (4.5-5.5):(2.5-3.5):2, and even more preferably 5:3:2.

[0031] In this invention, the chitosan solution is preferably obtained by dissolving chitosan in acetic acid at pH 5.5; the concentration of the chitosan solution is preferably 1-3% w / v, more preferably 1.5-2.5% w / v, and even more preferably 2% w / v.

[0032] In this invention, the power of the ultrasound is preferably 200-400W, more preferably 250-350W, and even more preferably 300W; the duration of the ultrasound is preferably 30-50min, more preferably 35-45min, and even more preferably 40min.

[0033] In this invention, the mass ratio of the total volume of the bacterial suspension of *Streptococcus pyogenes* R3 and the bacterial suspension of *Oligotrophomonas oryzae* R5 to the protectant is preferably 100 mL:(10-15) g, more preferably 100 mL:(11-14) g, and even more preferably 100 mL:12 g.

[0034] In this invention, the protective agent in step (2) is composed of trehalose, skim milk powder and proline. The preferred mass ratio of trehalose, skim milk powder and proline is (5-8):(3-5):2, further preferably (5.5-7.5):(3.5-4.5):2, and even more preferably 6:4:2.

[0035] In this invention, after adding the protective agent, it is preferable to perform a pre-cooling treatment before adding the composite carrier. The temperature of the pre-cooling treatment is preferably 2-6℃, more preferably 3-5℃, and even more preferably 4℃. The time of the pre-cooling treatment is preferably 20-40 min, more preferably 25-35 min, and even more preferably 30 min.

[0036] In this invention, the volume-to-mass ratio of the mixed bacterial suspension with added protectant to the composite carrier is preferably 1 mL:(2-4) g, more preferably 1 mL:(2.5-3.5) g, and even more preferably 1 mL:3 g.

[0037] In this invention, the freeze-drying process is preferably as follows: the suspension containing bacteria obtained after mixing with the composite carrier is placed in a freeze-drying tray and placed in the pre-freezing chamber of a freeze dryer. The temperature is first lowered to -20°C at a rate of 10°C every half hour and held at that temperature for 2 hours. Then, the temperature is slowly lowered to -80°C at a rate of 5°C / min and held for 2 hours. After pre-freezing, the pressure in the freeze-drying chamber is quickly reduced to 10-20 Pa (absolute pressure) (to create a high vacuum environment). The material temperature is slowly increased from -80°C to -40°C at a rate of 2°C / h and held for 4 hours. The temperature is then increased to 20°C at a rate of 3°C / h and held for 8 hours until drying is complete.

[0038] The present invention also provides the application of the aforementioned composite microbial suspension in the preparation of tobacco seed coating agents.

[0039] This invention also provides a tobacco seed coating agent containing the aforementioned compound microbial suspension. The preferred effective viable count of *Streptococcus pyogenes* R3, an endophytic bacterium of rice seeds, in the compound microbial suspension of this invention is (1-100) × 10⁻⁶. 9 cfu / mL, further preferably (5-50)×10 9 cfu / mL, more preferably 10 × 10 9cfu / mL; the optimal effective viable count of *Oligotrophomonas R5*, an endophytic bacterium of rice seeds, in the composite microbial suspension is (1-100)×10⁻⁶. 9 cfu / mL, further preferably (5-50)×10 9 cfu / mL, more preferably 10 × 10 9 cfu / mL.

[0040] The present invention also provides the application of the compound microbial suspension, microbial agent or the tobacco seed coating agent in promoting tobacco growth or tobacco root growth.

[0041] In this invention, tobacco seeds are coated using the compound microbial suspension, microbial agent, or tobacco seed coating agent. During the coating process, the mass ratio of the compound microbial suspension to the tobacco seeds in the compound microbial suspension, microbial agent, or seed coating agent is (0.03-0.07):1, more preferably (0.04-0.06):1, and even more preferably 0.05:1.

[0042] 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.

[0043] The rice seed endophytic bacteria *Streptococcus pyogenes* R3 and *Oligotrophomonas pyogenes* R5 described in this invention are strains screened and preserved by the inventors and their team. Therefore, the rice seed endophytic bacteria *Streptococcus pyogenes* R3 and *Oligotrophomonas pyogenes* R5 used in this invention are both strains preserved in the laboratory of the inventors and their team.

[0044] Example 1: Isolation, screening, identification and preservation of *Streptococcus pyogenes* R3 and *Oligotrophomonas* R5 I. Preparation of Culture Medium 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 1 / 2LB solid 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, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0049] 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.

[0050] II. Bacterial Screening 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.

[0051] 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 6 After 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.

[0052] III. Analysis of the growth-promoting effects of endophytic bacteria 3.1 Qualitative Analysis of IAA Functions 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.

[0053] 3.2 Qualitative Analysis of Phosphorus Solubility 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.

[0054] 3.3 Qualitative Analysis of Nitrogen Fixation Function The two 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.

[0055] The study found that two strains of bacteria simultaneously possessed phosphorus-solubilizing and nitrogen-fixing functions. The two isolated and screened strains were named R3 and R5, respectively. The species of the isolated and screened bacteria were identified, and the results are shown in Table 1.

[0056] Table 1. Physiological functions and species identification of the strains

[0057] Note: "+" indicates that this function is available; "-" indicates that this function is not available.

[0058] IV. Preservation of bacterial strains 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 / min). 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).

[0059] 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.

[0060] The inoculated bacterial tubes were placed in a constant temperature incubator at 28℃ for 24 hours. After bacterial growth, they were sent to the China Center for Type Culture Collection in Wuhan, China. The taxonomic name of the rice seed endophytic bacterium *Streptococcus pyogenes* 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 the rice seed endophytic bacterium *Oligotrophomonas* R5 is... Stenotrophomonas sp. R5 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 20231758.

[0061] Example 2 1. Preparation of composite carriers Fresh rice straw was cleaned, cut into 3cm pieces, dried, and then pulverized through a 10-mesh sieve. The straw powder was placed in a nitrogen-filled muffle furnace and pyrolyzed at 700℃ for 2 hours in an oxygen-deficient environment. After cooling to room temperature, it was sieved through a 100-mesh sieve to obtain rice straw biochar (RSC). Rice straw biochar (RSC), attapulgite (ATP), and chitosan (CS) were added in a mass ratio of 5:3:2. Attapulgite (ATP) was added to the RSC suspension (10% w / v) and ultrasonically dispersed for 40 minutes (300W). Then, a chitosan (CS) solution (2% w / v, obtained by dissolving in acetic acid at pH 5.5) was added, and the mixture was magnetically stirred for 1 hour to form a gel-like composite carrier.

[0062] 2. Preparation of bacterial suspension The microbial agent used in this invention is *Streptococcus pyogenes* R3, an endophytic bacterium found in rice seeds. Herbaspirillum sp.R3) and oligotrophomonas R5 (sp.R3) and oligotrophomonas R5 Stenotrophomonas sp. R5). The taxonomic name of *Streptococcus pyogenes* R3, an endophytic bacterium in rice seeds, 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 the rice seed endophytic bacterium *Oligotrophomonas* R5 is... Stenotrophomonas sp. R5 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 20231758.

[0063] LB liquid culture medium was autoclaved at 121°C for 15 minutes and then cooled to room temperature for later use. Subsequently, endophytic bacteria *Streptococcus pyogenes* R3 and *Oligotrophomonas* R5 were inoculated into LB liquid culture medium and cultured at 28°C and 180 rpm for 24 hours. The culture was transferred to centrifuge tubes, and bacterial cells were collected by centrifugation at 6000 rpm for 10 minutes. The cells were washed twice with sterile PBS buffer to remove residual culture medium, and sterile water was used to prepare cultures with an effective viable count of 10-1. 10 CFU / mL suspensions of *Streptococcus pyogenes* R3 and *Oligotrophomonas* R5 are prepared for use.

[0064] 3. Preparation of compound microbial agents The prepared R3 and R5 bacterial suspensions were thoroughly mixed at a mass ratio of 1:1. 6% trehalose (6g trehalose per 100mL of bacterial suspension), 4% skim milk powder (4g skim milk powder per 100mL of bacterial suspension), and 2% proline (2g proline per 100mL of bacterial suspension) were added as protective agents to the resulting mixed bacterial suspension. After pre-cooling at 4°C for 30 minutes, the pre-cooled mixed bacterial suspension containing the protective agents was added to a gel-like composite carrier at a ratio of 1 mL:3 g to obtain a suspension containing bacterial cells. The suspension containing bacterial cells was placed in a freeze-drying tray and then placed in the pre-freezing chamber of a freeze dryer. The temperature was first lowered to -20°C at a rate of 10°C every half hour and held at that temperature for 2 hours. Then, the temperature was slowly lowered to -80°C at a rate of 5°C / min and held for 2 hours. After pre-freezing, the pressure in the freeze-drying chamber was rapidly reduced to 20 Pa (absolute pressure) to create a high vacuum environment. The material temperature is slowly increased from -80℃ to -40℃ at a rate of 2℃ / h and held for 4 hours. The temperature is then increased to 20℃ at a rate of 3℃ / h and held for 8 hours until drying is complete.

[0065] Example 3: Effect of different combinations of protectants on the viability of compound microbial agents Based on the preparation process of the compound microbial agent in Example 2, four additional treatment groups were set up, with only the combination of protectants changed, to explore the effect of different combinations of protectants on the viability of the compound microbial agent.

[0066] Group A: namely, compound microbial inoculant, the protectant is 6% trehalose (T) + 4% skim milk powder (M) + 2% proline (P) used in Example 2.

[0067] Group B: The preparation process of the compound microbial agent is the same as in Example 2, except that the protectant is replaced with 6% T + 4% M; Group C: The preparation process of the compound microbial agent is the same as in Example 2, except that the protectant is replaced with 6% T + 2% P; Group D: The preparation process of the compound microbial agent is the same as in Example 2, except that the protectant is replaced with 4% M + 2% P.

[0068] The effective viable count of the lyophilized bacterial agent was determined by plate counting method, and the viability rate was calculated.

[0069] Viability rate = (Number of viable bacteria after freeze-drying / Number of viable bacteria before freeze-drying) × 100% Table 2. Effects of different combinations of protectants on the viability of compound microbial agents.

[0070] Note: Different lowercase letters indicate significant differences between different treatments. P <0.05).

[0071] The results are shown in Table 2. Group A (6% T + 4% M + 2% P) had the highest viable bacterial rate (78.88%), which was significantly higher than other groups. P The concentration of trehalose (<0.05%) indicates that the three protectants work synergistically to effectively reduce cell membrane damage and protein denaturation during freeze-drying, thus maintaining cell viability. Group D had the lowest viability (49.75%), suggesting that trehalose plays a crucial role in protecting the cells from ice crystal damage.

[0072] Example 4 (I) Tobacco Seed Coating Treatment Tobacco seeds (Yunyan 87, provided by Yuxi China Tobacco Seed Co., Ltd.) were immersed in a 150 ppm gibberellin solution for 12 hours to break dormancy and promote early radicle development, with gentle stirring during the soaking process. The treated seeds were rinsed three times with sterile water to remove residual gibberellin solution, and then air-dried until the moisture content was <8%. They were then poured into the coating machine drum and idled for 2 minutes to allow friction to accumulate a negative charge on the seed surface. The composite microbial agent prepared in Example 2 was fed into the nozzle via a vibrating feeder. Corona discharge caused the powder particles to become positively charged, and under the influence of the electric field, they were uniformly adsorbed onto the negatively charged seed surface. The composite microbial agent accounted for 5% of the tobacco seed mass. After coating, the seeds continued to rotate in the drum while sterile hot air at 35°C (flow rate 2 m / s) was introduced for 20 minutes to dry the coating layer until the moisture content decreased to 7%.

[0073] (II) Root-promoting effect of compound microbial agents Four processing groups were set up: blank group (CK group), R3 group, R5 group, and R3+R5 group.

[0074] Tobacco seeds were coated with R3 bacterial suspension (R3 group); tobacco seeds were coated with R5 bacterial suspension (R5 group); tobacco seeds were coated with the compound microbial agent prepared in Example 2 (R3+R5 group); the CK group was not coated. The coating method was the same as in Example 4(a).

[0075] In the tobacco experimental field in Linli County, Changde City, Hunan Province, tobacco seeds coated with different inoculants were sown, and root indicators of tobacco were measured at different growth stages (30 days, 60 days and 90 days after transplanting).

[0076] Table 3. Effects of growth-promoting microbial functional agents on root indicators of tobacco at different growth stages.

[0077] Note: Different lowercase letters indicate significant differences between different treatments at the same time. P <0.05).

[0078] Table 3 shows that, compared with uncoated tobacco (CK group), seeds coated with R3, R5, and the compound microbial agent (R3+R5) all promoted the increase of root volume, total root length, and root surface area of ​​flue-cured tobacco to varying degrees. The root-promoting effects of single-strain treatments R3 and R5 were similar. Compared with the CK group, the root volume, total root length, and root surface area of ​​tobacco treated with the compound microbial agent (R3+R5) increased by 39.05%, 90.70%, and 31.26% at 30 days; by 59.72%, 66.33%, and 41.34% at 60 days; and by 48.06%, 78.95%, and 31.18% at 90 days.

[0079] (III) Impact on tobacco agronomic traits Based on the above Example 4 (II), tobacco agronomic traits were measured using tobacco plants 90 days after planting.

[0080] Compared to tobacco without seed coating (CK group), the compound microbial inoculant (R3+R5) significantly increased leaf length, leaf width, plant height, and stem circumference. While the number of effective leaves increased, the difference was not statistically significant. Figure 1 Among them, the increases in leaf length, leaf width, plant height and stem circumference reached 45.14%, 61.35%, 45.05% and 59.82%, respectively.

[0081] (iv) Impact on tobacco seedling stage Tobacco seeds Yunyan 87 (R3+R5 group) coated with the compound microbial agent prepared in Example 2 and uncoated tobacco seeds Yunyan 87 (CK group) were sown in seedling trays containing sterilized seedling substrate (peat moss:vermiculite:perlite = 3:2:1) and placed in a greenhouse (temperature 25±2℃, humidity 60±5%, light 12h / d). Samples were taken at the seedling stage, and the agronomic traits of each group of tobacco were compared.

[0082] Figure 2 The results showed significant differences in plant growth between tobacco seedlings coated with the compound microbial inoculant (R3+R5) and uncoated tobacco seedlings (CK group). Regarding root systems, the R3+R5 group had more developed root systems, with more numerous, longer, and denser roots, which facilitated the absorption of more water and nutrients. The CK group, on the other hand, had relatively sparse and shorter roots. In terms of leaves, the R3+R5 group had wider leaves and more robust plants overall, while the CK group had relatively narrower, thinner leaves and slightly weaker plant growth.

[0083] In conclusion, coating with compound microbial agents (R3+R5) can significantly promote the growth of tobacco seeds during the seedling stage, resulting in better performance in both root development and leaf growth.

[0084] In summary, this invention utilizes a compound microbial agent prepared from rice seed endophytic bacteria *Streptococcus pyogenes* R3 and *Oligotrophomonas* R5 as a seed coating agent to coat tobacco seeds. This significantly promotes tobacco root growth, improves root indicators, and thus provides the necessary nutrients for tobacco growth, thereby increasing tobacco leaf yield.

[0085] 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, The mixture contains a suspension of *Streptococcus pyogenes* R3 and *Oligotrophomonas spp.* R5, both endophytic bacteria of rice seeds. The mass ratio of *Streptococcus pyogenes* R3 to *Oligotrophomonas spp.* R5 in the composite microbial suspension is 1:(0.5-1.5). The classification name of the rice seed endophytic bacterium *Streptococcus pyogenes* R3 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 CCTCCNO: M 20231757; The taxonomic name of the endophytic bacterium *Oligotrophomonas* R5 in rice seeds is: Stenotrophomonas sp. R5 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 20231758.

2. The composite microbial suspension according to claim 1, characterized in that, The effective viable count of *Streptococcus pyogenes* R3, an endophytic bacterium in rice seeds, is (1-100)×10⁻⁶. 9 cfu / mL; The effective viable count of the endophytic bacterium *Oligotrophomonas* R5 in the rice seeds was (1-100)×10⁻⁶. 9 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: (1) After mixing biochar with inorganic clay mineral materials and sonicating, chitosan solution was added and stirred to obtain a composite carrier; (2) After mixing the bacterial suspension of rice seed endophytic bacteria spirulina R3 and rice seed endophytic bacteria oligotrophomonas R5, a protectant and a composite carrier were added in sequence, and the mixture was freeze-dried to obtain a composite microbial agent.

5. The preparation method according to claim 4, characterized in that, The mass ratio of biochar, inorganic clay mineral material and chitosan is (4-6):(2-4):2; The total volume of the bacterial suspension of *Streptococcus pyogenes* R3 and *Oligotrophomonas oryzae* R5 endophytic bacteria in rice seeds, and the mass ratio of the protectant, is 100 mL:(10-15) g.

6. The preparation method according to claim 4, characterized in that, The protective agent is composed of trehalose, skim milk powder, and proline in a mass ratio of (5-8):(3-5):

2.

7. The application of the composite microbial agent according to claim 3 in the preparation of tobacco seed coating agent.

8. A tobacco seed coating agent, characterized in that, Containing the compound microbial agent of claim 3, wherein the effective viable count of *Streptococcus pyogenes* R3, an endophytic bacterium of rice seeds, in the compound microbial suspension is (1-100)×10⁻⁶. 9 The effective viable count of *Oligotrophomonas R5*, an endophytic bacterium in rice seeds, was (1-100)×10⁻¹⁰ CFU / mL. 9 cfu / mL.

9. The tobacco seed coating agent according to claim 8, characterized in that, The mass ratio of the compound microbial suspension to tobacco seeds was (0.03-0.07):

1.

10. The application of the compound microbial suspension of claim 1 or 2, or the compound microbial agent of claim 3, or the tobacco seed coating agent of claim 8 or 9 in promoting tobacco growth or promoting tobacco root growth.

Images