A strain of Paenibacillus polymyxa Se202205, a bacterial agent and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Paenibacillus polymyxa Se202205, a bacterial agent and an application. The Paenibacillus polymyxa Se202205 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 15, 2023, with a deposit number of CGMCC No. 28465. The Paenibacillus polymyxa Se202205 strain provided by the present invention not only has a good antagonistic effect on a variety of plant pathogenic fungi, but also has the ability to efficiently convert sodium selenite to generate nano-selenium, and has good development and application potential in selenium-enriched microbial preparations of biocontrol agents.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Paenibacillus polymyxa Se202205, a bacterial agent and an application. Background Art

[0002] Selenium is one of the trace elements necessary for the life activities of organisms. It has important functions such as improving immunity, removing free radicals in the body, delaying aging, anti-cancer, regulating the human microcirculation system, and synthesizing antioxidant system enzymes. The lack of selenium may cause disorders related to the human heart, bones, muscles and immunity. The appropriate intake of selenium supplements can significantly reduce the probability of these diseases.

[0003] As an essential nutrient, how to supplement selenium safely and effectively is a problem to be solved. In order to find the most appropriate method for human selenium supplementation, domestic and foreign nutrition societies, selenium research organizations and other institutions have done a lot of research in this regard. Finley JW et al. (2010) found a safe way to supplement selenium, which is to take selenium through plants, because plants can use selenium in the environment to convert and synthesize the selenium they need, while improving the biological activity of selenium. In recent years, a large number of studies have shown that plants can well convert and utilize selenium in the soil, and it has also been confirmed that moderate addition of selenium is beneficial to plant growth and development. However, there are not many areas in my country with high selenium content in most soils, so the content of selenium that can be absorbed through plant conversion is not high. In order to meet the human body's selenium content, it is not enough to obtain it from natural food. Therefore, the selection of selenium-enriched agricultural products, how to enrich selenium, and the application of selenium-enriched reagents have become the focus of people's attention.

[0004] At present, there are several methods to enrich plants with selenium: applying selenium-containing fertilizers to the soil, spraying selenium fertilizers on the leaves, soaking seeds in selenium-containing liquids, and biological breeding (Broadley et al, 2006). The simplest, most economical, and most easily operated method is soil fertilization or spraying foliar fertilizers. Studies have shown that spraying organic selenium fertilizers on Hami melon leaves (Zhang Yangyang et al, 2015), spraying inorganic selenium foliar fertilizers on potted broad beans (Zhang Huahua et al, 2014), wheat (Liu Qing et al, 2016), and spreading selenium mineral powder fertilizers on corn bases (Liu Qing et al, 2016) have all shown that adding selenium-rich fertilizers externally can increase the selenium content in crops. Converting inorganic selenium into organic selenium through crops is both economical and effective, and everyone can take in enough selenium through daily diet, and it can also maximize the safety of selenium supplementation. In the early days of my country, selenium mineral powder was used as a base application or selenate / selenite was sprayed on crops to achieve the purpose of producing selenium-rich agricultural products. However, studies have found that the absorption and conversion rate of inorganic selenium by most crops, especially staple crops, is very low. In addition, when selenium mineral powder is applied as the base, heavy metals are easily brought into the soil, causing secondary pollution. Inorganic selenium such as selenate / selenite is easily washed away by rainwater and seeps into the ground or flows into rivers, lakes, and form non-point source pollution.

[0005] Therefore, it is urgent to find a low-toxic selenium substitute to meet people's needs. In addition to inorganic selenium and organic selenium, there is also a nano-sized elemental selenium that has the same biological activity as organic selenium. Biological nano-selenium is transformed by microorganisms and is insoluble in water. When used for crop spraying, it is more tightly bound to the surface of the crop and is not easily washed away by rainwater. The crop conversion utilization rate is higher and the spraying operation time window is wider. Studies have found that low-toxicity, high-biologically active nano-selenium can meet people's needs, so how to prepare nano-selenium has attracted widespread attention. The biological preparation of nano-selenium is to use the effective substances produced during the growth and development of microorganisms to reduce selenium salts (sodium selenite, selenious acid, etc.) to synthesize red nano-selenium, or the effective substances in plant extracts to reduce sodium selenite to red nano-selenium. It is currently a hot spot in the field of research and preparation of nano-selenium. Nano-selenium synthesized by biological methods has high cell activity, small size, uniform particle size, mild reaction conditions, strong stability, and environmental friendliness. It has become a new trend in the preparation of nano-selenium at home and abroad.

[0006] At present, the preparation of nano-selenium by microbial method is a hot topic in the research of preparing nano-selenium. The reaction process of microbial synthesis of nano-selenium is mild, the nano-selenium particles are stable, the dispersion is high, and the toxicity is low. Nano-selenium can be prepared well without pollution to the environment. The preparation of nano-selenium by microorganisms is not restricted by seasonal factors such as environment, and no additional reducing agent or stabilizer is required, which can effectively improve the shortcomings of chemical methods. In recent years, the ability of various bacteria to reduce selenate or selenite to synthesize nano-selenium has been gradually confirmed. For example, ZL201710517574.X discloses a method and application for preparing biologically rich nano-elemental selenium. There are many types of bacteria that have been reported to produce nano-selenium at home and abroad, including Bacillus cereus CC-1 (Che Lin, 2018), Copper-Greed Bacillus (Yang Ying et al., 2020), Proteus mirabilis YC801 (Wang Yuting, 2019), Bacillus amyloliquefaciens (Li Lijun et al., 2019), Pseudomonas aeruginosa (Sourabh et al., 2017), Agrobacterium (Bajaj et al., 2012), Bacillus Sp.MSh-1 (Mojtaba et al., 2010), Acinetobacter Sp.s W30 (Wadhwani et al., 2017) and Lactobacillus plantarum (Chen Dong et al., 2020). In addition, there are some reports on the use of fungi to reduce selenite to synthesize nano-selenium, such as Saccharomyces cerevisiae (Hariharan et al., 2012), Soil Mold (Katayoun et al., 2012), Streptomyces bikini (Sholkamy et al., 2015), etc. In order to solve the above-mentioned problems,

[0007] However, due to the high cytotoxicity of selenate or selenite as the raw materials of bio-organic selenium / bio-nanoselenium, the growth of many microorganisms will be inhibited or even killed when they are cultured and transformed in a high-selenium environment; at the same time, although some microorganisms can tolerate high concentrations of selenium, they do not transform. Therefore, how to screen and improve the selenium tolerance and inorganic selenium transformation ability of microorganisms is still an urgent problem to be solved.

[0008] Paenibacillus polymyxa has a certain control effect on various plant diseases such as Verticillium wilt, chickpea wilt, rapeseed rot, black pine root rot, etc. The U.S. Environmental Protection Agency (EPA) has listed it as one of the commercially applicable microorganisms. However, there are few research reports on the selenium conversion ability of this bacterium. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a strain of Bacillus polymyxa Se202205, a bacterial agent and an application. The Bacillus polymyxa Se202205 strain provided by the present invention not only has a good antagonistic effect on a variety of plant pathogenic fungi, but also has the ability to efficiently convert sodium selenite to produce nano-selenium, and has good development and application potential in selenium-enriched microbial preparations of biocontrol agents.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] The present invention provides a strain of Paenibacillus polymyxa Se202205, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on September 15, 2023, with a deposit number of CGMCC No. 28465.

[0012] The present invention also provides the use of the polymyxa bacillus Se202205 described in the above technical solution in preventing and controlling plant diseases.

[0013] Preferably, the plant diseases include one or more of rice sheath blight, anthracnose and banana wilt.

[0014] The present invention also provides the use of the Paenibacillus polymyxa Se202205 described in the above technical solution in converting inorganic selenium into nano-selenium.

[0015] The present invention also provides a biocontrol agent, wherein the biocontrol agent contains Paenibacillus polymyxa Se202205, and the content of Paenibacillus polymyxa Se202205 is ≥1.0×10 9 cfu / g.

[0016] The invention provides a selenium-enriched bacterial agent, which is prepared by the following preparation method: inoculating the polymyxa bacillus Se202205 described in the above technical scheme into a selenium-containing culture medium and fermenting it at 30°C for 2 to 3 days to obtain the selenium-enriched bacterial agent, wherein the total content of nano-selenium and organic selenium in the selenium-enriched bacterial agent is 300-3000 mg / kg.

[0017] Preferably, the selenium-containing culture medium comprises 5-10 g / L glucose, 2-5 g / L sucrose, 2-8 g / L beef extract, 6-10 g / L peptone, 2-5 g / L sodium chloride and 0.3-3.0 g / L sodium selenite, with a pH of 6.5-7.5.

[0018] The present invention also provides the use of the selenium-enriched bacterial agent described in the above technical solution in the production of selenium-enriched rice.

[0019] Preferably, the application includes: diluting the selenium-enriched bacterial agent 300 times and spraying it during the tillering and heading stage of rice and the early stage of heading and flowering.

[0020] Preferably, the rice is sprayed twice from the late tillering stage to the heading stage with an interval of 10-15 days, and sprayed once in the early heading and flowering stage.

[0021] The beneficial effects of the present invention are:

[0022] 1. The polymyxa bacillus Se202205 provided by the present invention has an inhibitory effect on the pathogens of rice sheath blight, anthracnose and banana wilt.

[0023] 2. The polymyxa bacillus Se202205 provided by the present invention is resistant to jinggangmycin, carbendazim and tebuconazole.

[0024] 3. The Bacillus polymyxa Se202205 provided by the present invention can convert inorganic selenium into nano-selenium, and is applied to selenium-enriched rice. After the rice matures, the selenium content in the stem reaches 0.325±0.25 mg / kg, the selenium content in the husk is 0.225±0.30 mg / kg, and the selenium content in the rice reaches 0.307±0.13 mg / kg, meeting the standard for selenium-enriched rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 This is the result of phylogenetic analysis of Paenibacillus polymyxa Se202205 strain;

[0027] Figure 2 The inhibitory effect of Paenibacillus polymyxa Se202205 on the growth of various plant pathogens;

[0028] Figure 3 This is the result of converting sodium sialic acid into elemental selenium by Paenibacillus polymyxa Se202205;

[0029] Figure 4 is the transformation ability of Paenibacillus polymyxa Se202205 to sodium selenite;

[0030] Figure 5 This is an analysis chart of the resistance of strain Se202205 to commonly used chemical agents.

[0031] Biological Deposit Description

[0032] Paenibacillus polymyxa Se202205, Latin name Paenibacillus polymyxa, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 15, 2023, with the deposit number: CGMCC No.28465. The address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0033] The present invention provides a strain of Paenibacillus polymyxa Se202205, which was deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection on September 15, 2023, with a deposit number of CGMCC No. 28465. The Paenibacillus polymyxa Se202205 provided by the present invention is a pure bacterial culture isolated and screened from a mixture of rhizosphere soil and sugarcane rotten leaves in a sugarcane field in Luoxu Town, Wuming District, Nanning City, Guangxi Zhuang Autonomous Region, and has the following biological characteristics: the colony forms a milky white colony on NA culture medium, which is translucent, smooth and mucus-like; Gram-staining is positive, the bacterial cells are rod-shaped, and can produce spores under adverse conditions.

[0034] The present invention also provides the use of the polymyxa bacillus Se202205 described in the above technical solution in preventing and controlling plant diseases. In the present invention, the plant diseases preferably include one or more of rice sheath blight, anthracnose and banana wilt.

[0035] The present invention also provides the use of the Paenibacillus polymyxa Se202205 described in the above technical solution in converting inorganic selenium into nano-selenium.

[0036] The present invention also provides a biocontrol agent, wherein the biocontrol agent contains Paenibacillus polymyxa Se202205, and the content of Paenibacillus polymyxa Se202205 is ≥1.0×10 9The present invention has no special limitation on the preparation method of the biocontrol agent, and it can be obtained by conventional culture methods.

[0037] The present invention also provides a selenium-enriched bacterial agent, which is prepared by the following preparation method: the polymyxa bacillus Se202205 described in the above technical scheme is inoculated in a selenium-containing medium and fermented at 30°C for 2 to 3 days to obtain a selenium-enriched bacterial agent, wherein the total content of nano-selenium and organic selenium in the selenium-enriched bacterial agent is 300-3000 mg / kg. In the present invention, the selenium-containing medium is preferably 5-10 g / L of glucose, 2-5 g / L of sucrose, 2-8 g / L of beef extract, 6-10 g / L of peptone, 2-5 g / L of sodium chloride and 0.3-3.0 g / L of sodium selenite, pH 6.5-7.5. The present invention does not specifically limit the inoculation amount of the polymyxa bacillus Se202205, and conventional methods can be used.

[0038] The present invention also provides the use of the selenium-enriched bacterial agent described in the above technical solution in the production of selenium-enriched rice. In the present invention, the use preferably includes: diluting the selenium-enriched bacterial agent 300 times and spraying it during the tillering and heading stage of rice and the early stage of heading and flowering. In the present invention, it is preferably sprayed twice from the late tillering stage to the heading stage of rice, with an interval of 10-15d, and preferably sprayed once in the early stage of heading and flowering.

[0039] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Strain isolation, purification and identification

[0042] Strain Se202205 is a pure bacterial culture isolated and screened from a mixture of rhizosphere soil and sugarcane leaf rot in a sugarcane field in Luoxu Town, Wuming District, Nanning City, Guangxi Zhuang Autonomous Region. Its biological characteristics are as follows: the colonies form milky white colonies on NA culture medium, which are translucent, smooth and mucous; Gram staining is positive, the bacterial cells are rod-shaped, and can produce spores under adverse conditions.

[0043] Classification and Identification of the Strain The 16S rDNA of the strain was amplified by specific primers and sequence comparison analysis was performed. The results showed that the homology between strain Se202205 and Paenibacillus polymyxa was more than 99%. The 16S rDNA sequence of strain Se202205 is shown in SEQ ID No.1.

[0044]

[0045] The results of strain phylogenetic analysis are shown in Figure 1 Therefore, based on the colony and morphological characteristics, strain Se202205 was identified as Paenibacillus polymyxa.

[0046] The strain Se202205 has been applied for patent deposit and is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.28465.

[0047] Example 2

[0048] Inhibitory activity of strain Se202205 against pathogenic bacteria

[0049] The inhibitory activity of Se202205 strain against the growth of several common plant fungal pathogens, such as rice sheath blight pathogen [Rhizoctonia solani], anthracnose pathogen (Colletotrichum gloeosporioides), banana wilt pathogen Fusarium oxysporum, etc., was determined: the plate confrontation culture method was used, and a 5mm bacterial cake of the corresponding pathogenic fungi was inoculated in the center of the PDA plate, and then the activated cultured strain Se202205 was streaked with an inoculation loop about 1 cm away from the edge of the plate. The culture plate without streaking but only inoculated with pathogenic fungi was used as the control, and the plates were placed at a constant temperature of 28℃ for culture. Due to the difference in radial growth rates of the three pathogenic fungi, the antibacterial experiment against rice sheath blight pathogen was carried out after 2 days of confrontation culture, while the antibacterial experiments against anthracnose and Fusarium oxysporum were carried out after 5 days of confrontation culture. Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0050] The inhibitory effect of strain Se202205 on the growth of the above three pathogenic fungi is shown in Figure 2 ; According to the formula for inhibition rate, the results showed that the inhibition rates of the strain on the radial growth of three pathogenic fungi, Rhizoctonia solani, Colletotrichum gloeosporioides and Fusariumoxysporum, were 62.50%, 68.75% and 37.60%, respectively.

[0051] Example 3

[0052] Determination of selenium tolerance and selenium conversion ability of strain Se202205

[0053] Analytical pure sodium selenite was prepared into a selenium salt solution with a concentration of 20 mg / mL. A certain amount of selenium salt solution was added to the bacterial culture medium NA to make the selenium salt concentration of the culture medium 133.33 mg / L. The strain Se202205 to be screened was inoculated into the culture medium and the growth of the strain was observed. The results showed that the strain Se202205 could still grow normally under the stress of the selenium salt concentration and produced a large amount of red elemental selenium ( Figure 3 ).

[0054] Determine the transformation ability of strain Se202205 to sodium selenite: activate the strain and prepare a concentration of 1*10 6 cuf / mL of seed liquid was inoculated into LB culture medium containing selenium salt concentrations of 300, 500, 1000, 1500, 2000, 2500, and 3000 mg / L at an inoculum size of 1%, and LB culture medium without selenium salt was used as the control treatment; 3 replicates were set for each treatment, and the liquid volume in each bottle was 200 mL; cultured for 3 days at 30°C and a shaking speed of 180 rpm, all fermentation broths treated with selenium salt turned dark red, and red precipitates were attached to the bottom and body of the triangular flask, while the control did not change accordingly ( Figure 4 ); the fermentation broth treated with selenium was centrifuged (10000rpm, 2 minutes) for solid-liquid separation, and a large amount of red elemental selenium and bacterial cells were precipitated; the fermentation broth supernatant and precipitate were sent to a qualified analysis and testing agency for selenium content testing. The test results showed that the content of inorganic selenium in the total selenium of all fermentation systems treated with different selenium salt concentrations was less than 1%, indicating that more than 99% of the selenium salt was converted into elemental selenium and bacterial organic selenium (Table 1).

[0055] Table 1 Results of the transformation ability test of strain Se202205 on sodium selenite

[0056]

[0057] Embodiment 4:

[0058] Analysis of the drug resistance of strain Se202205 to common chemical agents

[0059] (1) Jinggangmycin for the prevention and treatment of rice sheath blight, tebuconazole for the prevention and treatment of rice false smut, and carbendazim, a commonly used agent for the prevention and treatment of plant pathogenic Fusarium oxysporum, were used as test agents to analyze the growth of strain Se202205 under culture conditions containing a certain concentration (EC50 of the agent when preventing and treating the corresponding disease). The source of the agent and basic information are as follows:

[0060] 24% Jinggangmycin A, aqueous solution, produced by Wuhan Kono Biotechnology Co., Ltd.

[0061] 50% carbendazim, suspension concentrate, produced by Anhui Guangxin Agricultural Chemical Co., Ltd.

[0062] 75% trifloxystrobin·tebuconazole, water dispersible granules, produced by Bayer GmbH, Germany.

[0063] (2) The Ec50 of Jinggangmycin against sheath blight is 0.0264ug / mL. Divided by the actual content of the agent 24%, the actual dosage should be 0.125ug / mL; the Ec50 of tebuconazole against rice false smut is 0.048ug / mL. Divided by the actual content of the agent 75%, the actual dosage should be 0.067ug / mL; the Ec50 of carbendazim against Fusarium oxysporum (taking banana wilt pathogen as an example) is 0.1835ug / mL. Divided by the actual content of the agent 50%, the actual dosage should be 0.36ug / mL.

[0064] (3) The seed solution of Se202205 strain with a concentration of 1*106cuf / mL was prepared and inoculated into 100mL LB medium at an inoculum size of 1%, and the above three chemical agents were added respectively to make the final concentration equal to the EC50 of the agents for controlling the corresponding disease pathogens. The treatment without any agent was used as the control, and 4 replicates were set for each treatment. After inoculation, each treatment was placed at 30°C and cultured at a shaking speed of 180 rpm for 36 h, and the OD600 values ​​of the culture solutions of different treatments were measured.

[0065] (4) Analysis results are shown in Figure 5 , strain Se202205 can grow normally in the culture solution containing the above-mentioned agents, and the growth of the bacteria treated with the added agents is not significantly different from that of the control treatment without the agents, and the growth of the strain in the culture solution containing Jinggangmycin is higher than that of the control. The results of this study show that when carrying out corresponding disease prevention and control, the microbial preparation prepared by strain Se202205 can be mixed with the above-mentioned agents to prevent and control the corresponding diseases, reduce the use of agents, and reduce their environmental pollution risks.

[0066] Example 5

[0067] Preparation of selenium-rich bacterial agent and its application in selenium-enriched rice production

[0068] The experiment was carried out at the scientific research and experimental base of the Guangxi Academy of Agricultural Sciences in Nanning, Guangxi from June to July 2023. The first spraying was carried out at the end of rice tillering on June 5. The rice variety was Guangyou No. 3.

[0069] Preparation method of selenium-rich bacterial agent: strain Se202205 is activated and cultured to prepare a concentration of 1*10 6cuf / mL of seed liquid was inoculated into selenium-containing culture medium (glucose 10.0 g / L, sucrose 2.0 g / L, beef extract 5.0 g / L, peptone 10.0 g / L, sodium chloride 3.0 g / L, sodium selenite 2.0 g / L, pH 7.0) at an inoculum size of 2%, and cultured and fermented for 80 h at 30°C and a shaking speed of 180 rpm to obtain a selenium-rich bacterial agent rich in nano-selenium and bacterial organic selenium.

[0070] Application of selenium-rich bacteria agent in selenium-rich rice production: dilute the above selenium-rich bacteria agent 300 times and spray it. Spray it twice from the tillering stage to the heading stage of rice, with an interval of 15 days. Spray the selenium-rich bacteria agent once at the early stage of heading and flowering of rice.

[0071] After the rice matured, samples were taken for selenium content testing: rice stalks, rice husks and rice grains were tested separately. The test results showed that after three times of spraying with selenium-enriched bacteria, the selenium content of rice stalks reached 0.325±0.25mg / kg, the selenium content of husks reached 0.225±0.30mg / kg, and the selenium content of rice grains reached 0.307±0.13mg / kg, reaching the standard of selenium-enriched rice.

[0072] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Paenibacillus polymyxa ( Paenibacillus polymyxa )Se202205, characterized in that The polymyxa bacillus Se202205 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 15, 2023, with the deposit number: CGMCC No.28465.

2. Use of the polymyxa bacillus Se202205 according to claim 1 in preventing and controlling plant diseases; The plant diseases are one or more of rice sheath blight, anthracnose and banana wilt, and the pathogen of anthracnose is Colletotrichum gloeosporioides .

3. Use of the polymyxa bacillus Se202205 described in claim 1 in converting inorganic selenium into nano-selenium.

4. A biocontrol agent, characterized in that: The biocontrol agent contains Paenibacillus polymyxa Se202205, and the content of Paenibacillus polymyxa Se202205 is ≥1.0×10 9 cfu / g, and the deposit number of the polymyxa bacillus Se202205 is: CGMCC No.28465.

5. A selenium-enriched bacterial agent, characterized in that: The selenium-enriched bacterial agent is prepared by the following preparation method: inoculating the polymyxa bacillus Se202205 described in claim 1 into a selenium-containing culture medium and fermenting at 30° C. for 2 to 3 days to obtain a selenium-enriched bacterial agent, wherein the total content of nano-selenium and organic selenium in the selenium-enriched bacterial agent is 300-3000 mg / kg.

6. The selenium-enriched bacterial agent according to claim 5, characterized in that The selenium-containing culture medium comprises: 5-10 g / L glucose, 2-5 g / L sucrose, 2-8 g / L beef extract, 6-10 g / L peptone, 2-5 g / L sodium chloride and 0.3-3.0 / L sodium selenite, with a pH of 6.5-7.

5.

7. Use of the selenium-enriched bacterial agent according to claim 6 in producing selenium-enriched rice.

8. The use according to claim 7, characterized in that: The application comprises: diluting the selenium-enriched bacterial agent 300 times and spraying it during the tillering and heading stage of rice and the early stage of heading and flowering.

9. The use according to claim 8, characterized in that: Spray twice from the late tillering stage to the heading stage of rice, with an interval of 10-15 days, and spray once before the heading and flowering stage.

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