Microbial inoculant for promoting growth in control of rice smut and preparation method and application thereof

Abstract

The present application relates to the field of agricultural microorganism technology, and particularly discloses a microbial inoculum for promoting growth in the prevention and treatment of rice smut, and a preparation method and application thereof. The present application provides an application of the grass Helicobacter R3 in the preparation of a microbial inoculum for promoting growth in the prevention and treatment of rice smut, and the classification name of the grass Helicobacter R3 is Herbaspirillum sp. R3, which was preserved in the China Center for Type Culture Collection on September 20, 2023, at an address of Wuhan University, China, Wuhan, with a preservation number of CCTCC NO: M 20231757. The microbial inoculum prepared from the grass Helicobacter R3 bacterial suspension, xanthan gum and ascorbic acid in a specific ratio, in combination with a disinfectant, can be applied to the prevention and treatment of seed diseases of rice, can significantly reduce the bacterial carrying rate of the rice smut pathogen of rice seeds, and can also alleviate the oxidative damage caused by seed disinfection and improve the seed germination rate.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, and in particular to a microbial agent that promotes growth in the control of rice grain smut, its preparation method, and its application. Background Technology

[0002] Rice smut is a disease caused by pathogens. Tilletia horrida This disease, affecting the panicle of rice, is caused by a pathogen that can be transmitted through seeds. It primarily occurs during the milk-ripe to waxy-ripe stage of rice, manifesting as black spore masses on the surface of the grains. This disease not only affects the appearance and commercial value of the rice but also reduces yield and quality, making it a significant disease in rice production. The occurrence of rice grain smut is closely related to high humidity in the field, but in rainy areas or during periods of continuous rainfall in the rice panicle-filling stage, it is difficult to effectively reduce field humidity. While irrigation management can control humidity to some extent, it is difficult to precisely regulate in field cultivation. Therefore, preventing rice grain smut from its source is crucial.

[0003] Currently used chemical agents (such as carbendazim and tricyclazole) have a certain inhibitory effect on rice grain smut, but the effect is limited. Excessive disinfection may remove beneficial microorganisms on the seed surface, causing the seeds to lose some of their natural protective barrier. Its strong oxidizing properties may damage the cell membrane and seed coat structure of the seeds, thereby affecting seed germination and reducing seed viability. Summary of the Invention

[0004] The purpose of this invention is to provide a microbial agent that can promote growth in the control of rice grain smut, as well as its preparation method and application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of *Streptococcus pyogenes* R3 in the preparation of a microbial agent that promotes growth in the control of rice grain smut. The taxonomic name of *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:M20231757.

[0006] This invention provides a microbial agent that promotes growth in the control of rice grain smut, the microbial agent comprising a suspension of *Streptococcus pyogenes* R3, xanthan gum, and ascorbic acid; The mass-to-volume ratio of xanthan gum to *Streptococcus pyogenes* R3 suspension is 2-4 g: 1000 mL; The mass-to-volume ratio of ascorbic acid to *Streptococcus pyogenes* R3 suspension is 1-3 g: 1000 mL; The *Streptococcus pyogenes* R3 suspension was prepared from *Streptococcus pyogenes* R3.

[0007] Preferably, the viable bacterial concentration of the *Streptococcus pyogenes* R3 suspension is (0.5~1.5)×10⁻⁶. 8 cfu / mL.

[0008] This invention provides a method for preparing the aforementioned microbial inoculant, comprising the following steps: (1) Inoculate *Streptococcus pyogenes* R3 into LB liquid medium and culture with shaking for 22-26 h. Centrifuge at low temperature for 8-12 min, remove the supernatant to obtain bacterial cells, and mix the bacterial cells with water to achieve a viable cell concentration of (0.5-1.5)×10⁻⁶. 8 CFU / mL was used to obtain a suspension of *Streptococcus suis* R3 bacteria; (2) Mix the suspension of Straybug R3 bacteria with xanthan gum and ascorbic acid to obtain a mixed solution, and adjust the pH value to 6.8~7.2 to obtain a microbial agent.

[0009] Preferably, the temperature of the shaking culture in step (1) is 26~30℃ and the shaking speed is 170~190rpm.

[0010] Preferably, the temperature of the low-temperature centrifugation in step (1) is 3~5℃, and the speed of the low-temperature centrifugation is 5800~6200rpm.

[0011] This invention provides the application of the microbial agent described above or the microbial agent prepared by the method described above in the control of rice grain smut in rice seeds.

[0012] This invention provides a method for controlling rice grain smut and promoting rice growth using rice seeds, comprising the following steps: (1) Soak rice seeds in disinfectant for 8-12 minutes, then remove the seeds to obtain disinfected rice seeds; (2) Soak the disinfected rice seeds in a microbial agent for 15-25 minutes, then remove the seeds and let them air dry. The microbial agent is the microbial agent described above or the microbial agent prepared by the above preparation method.

[0013] Preferably, the disinfectant is a diluted 84 disinfectant.

[0014] This invention provides the application of the method in improving the germination rate and disease resistance of rice seeds.

[0015] The present invention has the following technical effects and advantages: This invention prepares a microbial agent by combining *Streptococcus pyogenes* R3 suspension, xanthan gum, and ascorbic acid in a specific ratio. This agent is applied to control rice grain smut and promote seed growth. After sterilization with a disinfectant, it significantly reduces the inoculum rate of rice grain smut pathogens in rice seeds. The microbial agent can alleviate oxidative damage caused by seed disinfection and improve seed germination rate. This invention provides a method for controlling rice grain smut and promoting seed growth in rice seeds. By treating seeds with a disinfectant combined with a microbial agent, the method reduces the inoculum rate of rice grain smut pathogens while ensuring high seed germination rates.

[0016] Preservation Instructions The classification 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. Attached Figure Description

[0017] Figure 1 A flowchart illustrating a method for controlling rice grain smut and promoting rice growth using rice seeds, as one embodiment. Detailed Implementation

[0018] This invention provides the application of *Streptococcus pyogenes* R3 in the preparation of a microbial agent that promotes growth in the control of rice grain smut. The taxonomic name of *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:M20231757.

[0019] This invention provides a microbial agent that promotes growth in the control of rice grain smut, the microbial agent comprising a suspension of *Streptococcus pyogenes* R3, xanthan gum, and ascorbic acid; The mass-to-volume ratio of xanthan gum to *Streptococcus solani* R3 suspension is 2-4 g:1000 mL, preferably 3 g:1000 mL; The mass-to-volume ratio of ascorbic acid to *Streptococcus pyogenes* R3 suspension is 1-3 g:1000 mL, preferably 2 g:1000 mL; The *Streptococcus pyogenes* R3 suspension was prepared from *Streptococcus pyogenes* R3.

[0020] In this invention, the viable bacterial concentration of the *Streptococcus pyogenes* R3 suspension is (0.5~1.5)×10⁻⁶. 8 cfu / mL, preferably 1×10 8 cfu / mL.

[0021] The preparation method of the above-mentioned microbial inoculant includes the following steps: (1) Inoculate *Streptococcus pyogenes* R3 into LB liquid medium and culture with shaking for 22-26 h. Centrifuge at low temperature for 8-12 min, remove the supernatant to obtain bacterial cells, and mix the bacterial cells with water to achieve a viable cell concentration of (0.5-1.5)×10⁻⁶. 8 The concentration of cfu / mL was used to obtain a suspension of *Streptococcus pyogenes* R3. Low-temperature centrifugation refers to centrifugation at a temperature below room temperature, specifically 3-5℃.

[0022] The preferred shaking incubation time is 24 hours; the preferred low-temperature centrifugation time is 10 minutes; the preferred concentration is 1×10⁻⁶. 8 cfu / mL; (2) Mix the suspension of *Streptococcus pyogenes* R3 with xanthan gum and ascorbic acid to obtain a mixed solution, and adjust the pH to 6.8-7.2 to obtain a microbial agent; Xanthan gum, a natural polymer, is selected as the dispersant, with an optimal addition amount of 0.3%; ascorbic acid is selected as the antioxidant, with an optimal addition amount of 0.2%. The R3 bacterial suspension is thoroughly mixed with the dispersant and antioxidant to improve the performance, stability, and effectiveness of the inoculum. The pH of the microbial inoculum is adjusted to 6.8–7.2, preferably 7.0, using 10% HCl and 10% NaOH.

[0023] In this invention, the temperature of the shaking culture in step (1) is 26~30℃, preferably 28℃; the shaking speed is 170~190rpm, preferably 180rpm.

[0024] In this invention, the temperature of the low-temperature centrifugation in step (1) is 3~5℃, preferably 4℃; the speed of the low-temperature centrifugation is 5800~6200rpm, preferably 6000rpm.

[0025] This invention provides the application of the above-mentioned microbial inoculants or the microbial inoculants prepared by the above-described preparation method in promoting growth in rice seeds for the control of rice grain smut.

[0026] This invention provides a method for controlling rice grain smut and promoting rice growth using rice seeds, comprising the following steps: (1) Soak rice seeds in disinfectant for 8-12 minutes, then remove the seeds to obtain disinfected rice seeds; The soaking time is preferably 10 minutes; (2) Soak the disinfected rice seeds in a microbial agent for 15-25 minutes, then remove the seeds and let them air dry. The soaking time is preferably 20 minutes; The microbial agent is the microbial agent described above or the microbial agent prepared by the above preparation method.

[0027] In this invention, the disinfectant is 84 disinfectant, which has an effective chlorine content of 5.0% and is used after dilution, preferably diluted 100 times.

[0028] Preferably, the seeds are air-dried in a well-ventilated place, avoiding direct sunlight, until the seed moisture content reaches the safe storage moisture standard (12%-13%).

[0029] This invention provides the application of the method in improving the germination rate and disease resistance of rice seeds.

[0030] 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.

[0031] In this example, *Streptococcus pyogenes* R3 was an endophytic bacterium screened from rice seeds of Huang Huazhan in Zhouxi Town, Kaili City, Guizhou Province, by Professor Li Juan's research group at the College of Agriculture, Hunan Agricultural University. It has been deposited at the China Center for Type Culture Collection. *Streptococcus pyogenes* R3 was activated by absorbing the *Streptococcus pyogenes* R3 endophytic bacterium from rice seeds preserved by the research group. Herbaspirillum sp. R3) frozen sample was added to LB liquid medium (pH 7.0) and shaken in a shaker (28℃, 180rpm) for 36h to obtain activated spirulina R3 for later use; 50% carbendazim and 84 disinfectant were commercially available products with an effective chlorine content of 5.0%.

[0032] Experimental Example 1: Isolation, screening, identification, and preservation of *Streptococcus pyogenes* R3 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] III. Analysis of the growth-promoting effects of endophytic bacteria 3.1 Qualitative Analysis of IAA Functions The isolated bacteria 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.

[0041] 3.2 Qualitative Analysis of Phosphorus Solubility The isolated bacteria were inoculated onto a solid culture medium for testing the phosphate-solubilizing function of the strains. The presence of phosphate-solubilizing zones on the plates was observed over 7 days. The presence of phosphate-solubilizing zones indicated that the bacteria had phosphate-solubilizing function.

[0042] 3.3 Qualitative Analysis of Nitrogen Fixation Function The isolated 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.

[0043] The study found that some strains simultaneously possess IAA production, phosphorus solubilization, and nitrogen fixation functions. The species and physiological functions of the isolated and screened bacteria are shown in Table 1.

[0044] Table 1. Physiological functions and species identification of the strains

[0045] Note: "+" indicates that this function is available; "-" indicates that this function is not available.

[0046] IV. Preservation of bacterial strains The strain was 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 cold storage, was taken out of the refrigerator and placed in a clean bench for UV sterilization for 1 h (room temperature).

[0047] 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.

[0048] The inoculated bacterial tubes were placed in a constant temperature incubator at 28°C for 24 hours. After bacterial growth, they were sent to the China Center for Type Culture Collection in Wuhan, China. The bacteria described were *Streptococcus solani* R3, and their taxonomic name is [not provided in the original text]. 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.

[0049] Experiment Example 2 1. A method for preparing a microbial inoculant that promotes growth in the control of rice grain smut. (1) Activated *Streptococcus pyogenes* R3 was inoculated into LB liquid medium and cultured with shaking at 28℃ and 180 rpm for 24 h. Then, it was centrifuged at 4℃ and 6000 rpm for 10 min. The supernatant was removed to obtain bacterial cells. The bacterial cells were mixed with sterile water to make the bacterial concentration 1×10⁻⁶. 8 CFU / mL was used to obtain a suspension of *Streptococcus suis* R3 bacteria; (2) Xanthan gum and ascorbic acid were added to the suspension of *Streptococcus pyogenes* R3 at a mass-to-volume ratio of 3g:1000mL and 2g:1000mL respectively. The pH was adjusted to 7.0 to obtain the microbial agent.

[0050] 2. The impact of different seed treatment methods on seed biocontrol indicators Experimental materials: early rice variety "Xiangzaoxian 45" and rice grain smut pathogen ( Tilletia horrida All strains were provided by the Institute of Plant Protection, Hunan Academy of Agricultural Sciences.

[0051] Experimental method: Using rice grain smut pathogen ( Tilletia horrida The strain infected the seeds of rice variety "Xiangzaoxian 45" and the pathogen carrying rate of the seeds was detected. The seeds carrying 14.8% of the rice grain smut pathogen were used as experimental materials, and the untreated seeds were used as the control group (CK group). The treatment groups were divided into groups A, B, C, D and E.

[0052] Treatment Group A: Soak the seeds in 50% carbendazim solution (800 times dilution) for 10 minutes, remove the seeds and rinse them with clean water to remove excess fungicide residue, and let the seeds air dry in a well-ventilated place.

[0053] Treatment Group B: Soak the seeds in 50% carbendazim solution (800 times dilution) for 10 minutes, remove the seeds and rinse with clean water to remove excess fungicide residue, and obtain disinfected rice seeds. Then, soak the disinfected rice seeds in the prepared microbial agent for 20 minutes, remove the seeds, rinse with clean water to remove excess fungicide residue, and let the seeds air dry in a well-ventilated place.

[0054] Treatment Group C: Soak the seeds in a 100-fold dilution of 84 disinfectant for 10 minutes, remove the seeds and rinse them with clean water to remove excess disinfectant residue, and let the seeds air dry in a well-ventilated place.

[0055] Treatment group D: Soak the seeds in the prepared microbial inoculant for 20 minutes, take out the seeds, rinse them with clean water to remove excess inoculant residue, and let the seeds air dry in a well-ventilated place.

[0056] Treatment Group E: Soak rice seeds in disinfectant (84 disinfectant diluted 100 times) for 10 minutes, remove the seeds and rinse with clean water to remove excess disinfectant residue, and obtain disinfected rice seeds. Soak the disinfected rice seeds in the microbial agent prepared in Example 2 (1. A method for preparing a microbial agent that promotes growth in the prevention and control of rice grain smut) for 20 minutes, remove the seeds, rinse with clean water to remove excess agent residue, and air dry the seeds in a well-ventilated place until the seed moisture content reaches the safe storage moisture standard (12%~13%) before storage.

[0057] The bacterial infection rate, germination rate, and oxidative damage index of seeds in the CK group, treatment A group, treatment B group, treatment C group, treatment D group, and treatment E group were measured respectively.

[0058] The method for determining the bacterial carriage rate is as follows: The "tissue isolation method" was used for determination. One hundred treated seeds were randomly selected, surface-sterilized (immersed in 75% ethanol for 30 seconds), and the embryos were cut open and inoculated into PDA medium. The seeds were cultured at 28°C for 5 days to observe for any changes. Tilletia horrida The growth of black spore masses was analyzed, and the inoculum rate was calculated (number of inoculum-carrying seeds / total number of seeds × 100%). The method for determining germination rate is as follows: Seeds were placed in germination boxes and cultured for 7 days in an artificial climate chamber at 25℃, 12h / d light, and 70% humidity. Germination rate was calculated (number of normally germinated seeds / number of tested seeds × 100%). Oxidative damage indicators are determined by measuring the malondialdehyde (MDA) content in seeds. The lower the MDA content, the less oxidative damage. The determination method is the "thiobarbituric acid method".

[0059] The results of seed bacterial infestation rate, germination rate and oxidative damage index of different treatment groups are shown in Table 2.

[0060] Table 2. Results of seed bacterial infection rate, germination rate, and oxidative damage indices in different treatment groups.

[0061] As shown in Table 2, the bacterial infection rate of treatment group E was only 0.8%, which was significantly lower than that of the control group CK (14.8%) and treatment D (14.2%), and better than treatments A (2.8%), B (1.5%), and C (1.9%). Meanwhile, the germination rate of treatment E (95.3%) was significantly higher than that of the CK group, and better than all other groups. Furthermore, the MDA content (4.2 nmol / g) was the lowest. This indicates that the method proposed in this application for treating rice smut in rice seeds can effectively disinfect pathogens and alleviate the oxidative damage of 84 disinfectant through microbial agents, achieving a dual effect of "biological control + growth promotion".

[0062] 3. Optimization and verification of soaking time in seed treatment scheme The treatment scheme for group E in section "2. Effects of Different Seed Treatments on Seed Biocontrol Indicators" was repeated, except for the soaking time of the microbial agent. Three treatment groups were set up: group F (soaking time 10 min), group G (soaking time 20 min), and group H (soaking time 40 min). All other steps were exactly the same. Rice seeds from different treatment groups were obtained, and the bacterial infection rate and germination rate were measured. The methods for measuring the bacterial infection rate and germination rate were the same as those in section "2. Effects of Different Seed Treatments on Seed Biocontrol Indicators". The results of the bacterial infection rate and germination rate of the different treatment groups are shown in Table 3.

[0063] Table 3. Results of seed bacterial infestation rate and germination rate in different treatment groups

[0064] As shown in Table 3, the bacterial load rate of all three treatment groups was below 1.5%, and the germination rate was above 93%, with little difference between them. Among them, treatment G had the highest germination rate, while treatment H had a slightly lower bacterial load rate but no significant advantage, and extending the soaking time would increase the operating cost. Therefore, the soaking time for treatment G was optimal.

[0065] 4. Optimization and validation of the dosage of xanthan gum, a microbial inoculant. The amount of xanthan gum in the microbial inoculant used in "1. A method for preparing a microbial inoculant that promotes growth in the control of rice grain smut" was modified. Three treatment groups were set up: a control group (CK group), treatment group I, and treatment group II. The control group did not contain xanthan gum. In treatment group I, the mass-to-volume ratio of xanthan gum to *Streptococcus spp.* R3 suspension was 3 g:1000 mL. In treatment group II, the mass-to-volume ratio of xanthan gum to *Streptococcus spp.* R3 suspension was 6 g:1000 mL. All other steps were identical, and the corresponding microbial inoculants were obtained. The scheme of treatment group E in Experiment Example 2 was repeated, and the rice seeds from the control group (CK group), treatment group I, and treatment group II were soaked for 20 min, with all other steps identical, to obtain rice seeds from different treatment groups. The stratification of different treatments of microbial agents was determined after 24 hours, and the concentration of bacteria in the lower layer was measured. Then, the bacterial load and germination rate of rice seeds in different treatment groups were measured. The effects of different amounts of xanthan gum on the microbial agents and seeds were compared. The results of the effects of different amounts of xanthan gum on the microbial agents and seeds are shown in Table 4. The methods for measuring the bacterial load and germination rate are the same as those in "2. Effects of different seed treatments on seed biocontrol indicators".

[0066] Table 4. Effects of different amounts of xanthan gum on microbial inoculants and their seeds.

[0067] As shown in Table 4, the microbial inoculants obtained in the CK group showed severe stratification, with a bacterial concentration of only 0.52 × 10⁻⁶. 8 cfu·mL - ¹, the bacterial load increased to 2.3%. Treatment I showed the highest microbial germination rate, and its agent viscosity was moderate, making it easier to handle than Treatment II. Therefore, the optimal mass-to-volume ratio of xanthan gum to *Streptococcus solani* R3 suspension in the microbial agent was 3 g: 1000 mL.

[0068] As can be seen from the above embodiments, the present invention provides a microbial inoculant for promoting growth in the control of rice grain smut, its preparation method, and its application. The present invention prepares a microbial inoculant from *Streptococcus pyogenes* R3 suspension, xanthan gum, and ascorbic acid in a specific ratio. This inoculant is applied to control rice grain smut and promote growth. After sterilization with a disinfectant, it significantly reduces the inoculant rate of rice grain smut pathogens in rice seeds. The microbial inoculant can alleviate oxidative damage caused by seed disinfection and improve seed germination rate. The present invention provides a method for controlling rice grain smut in rice seeds by treating seeds with a disinfectant combined with a microbial inoculant. This method reduces the inoculant rate of rice grain smut pathogens in rice seeds while ensuring the germination rate of rice seeds.

[0069] 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. Application of *Streptococcus pyogenes* R3 in microbial agents promoting the growth of rice grain smut, wherein the classification name of *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.

2. A microbial inoculant for promoting growth in the control of rice grain smut, characterized in that, The microbial agent includes a suspension of Straybug R3 bacteria, xanthan gum, and ascorbic acid; The mass-to-volume ratio of xanthan gum to *Streptococcus solani* R3 suspension is 2-4 g: 1000 mL; The mass-to-volume ratio of ascorbic acid to *Streptococcus pyogenes* R3 suspension is 1-3 g: 1000 mL; The *Streptococcus pyogenes* R3 suspension was prepared using *Streptococcus pyogenes* R3 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable cell concentration of the *Streptococcus pyogenes* R3 suspension was (0.5~1.5)×10⁻⁶. 8 cfu / mL.

4. The method for preparing the microbial inoculant according to claim 2 or 3, characterized in that, Includes the following steps: (1) Inoculate *Streptococcus pyogenes* R3 into LB liquid medium and culture with shaking for 22-26 h. Centrifuge at low temperature for 8-12 min, remove the supernatant to obtain bacterial cells, and mix the bacterial cells with water to achieve a viable cell concentration of (0.5-1.5)×10⁻⁶. 8 CFU / mL was used to obtain a suspension of *Streptococcus suis* R3 bacteria; (2) Mix the suspension of Stray spirulina R3 with xanthan gum and ascorbic acid to obtain a mixed solution, and adjust the pH value to 6.8~7.2 to obtain a microbial agent.

5. The preparation method according to claim 4, characterized in that, The temperature of the shaking culture in step (1) is 26~30℃ and the shaking speed is 170~190rpm.

6. The preparation method according to claim 4, characterized in that, The temperature of the low-temperature centrifugation in step (1) is 3~5℃, and the speed of the low-temperature centrifugation is 5800~6200rpm.

7. The application of the microbial agent according to claim 2 or 3, or the microbial agent prepared by the preparation method according to any one of claims 4 to 6, in promoting growth in rice seeds for the control of rice grain smut.

8. A method for controlling rice grain smut and promoting rice growth using rice seeds, characterized in that, Includes the following steps: (1) Soak rice seeds in disinfectant for 8-12 minutes to obtain disinfected rice seeds; (2) Soak the disinfected rice seeds in a microbial agent for 15-25 minutes, then remove the rice seeds and let them air dry. The microbial agent is the microbial agent according to claim 2 or 3, or the microbial agent prepared by the preparation method according to any one of claims 4 to 6.

9. The method according to claim 8, characterized in that, The disinfectant is a diluted 84 disinfectant.

10. The application of the method according to claim 8 or 9 in improving the germination rate and disease resistance of rice seeds.

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