Composting bacillus A1 and application thereof in preparation of preparation for improving resistance of rice sheath blight disease

Abstract

The invention discloses application of compost bacillus A1 in preparation of a preparation for improving resistance to rice sheath blight disease, and relates to the technical field of microbial pesticides and biological control of plant diseases, the compost bacillus A1 is obtained through separation, and the preservation number of the compost bacillus A1 is CGMCC No.35058. The biocontrol agent prepared from the compost bacillus A1 obtained through separation has a good inhibition effect on rhizoctonia solani. As the compound belongs to a biological agent, a series of problems caused by the use of chemical pesticides are completely avoided, the pollution-free production of the rice is facilitated, the use amount of other chemical pesticides can be not used or reduced, the planting cost can be reduced, and the economic development of the rice is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticides and biological control of plant diseases, and relates to Bacillus compostii A1, specifically to the application of Bacillus compostii A1 in the preparation of agents to improve resistance to rice sheath blight. Background Technology

[0002] Rice sheath blight is a fungal disease caused by Rhizoctonia solani AG1-IA, resulting in an average annual global yield reduction of 10–30%. The disease primarily affects the leaf sheath and leaves. In the early stages, dark green, water-soaked, oval lesions form on the leaf sheath near the water surface, gradually expanding into cloud-like patterns. Under high humidity, white mycelium and dark brown sclerotia develop.

[0003] Currently, the control of rice sheath blight combines agricultural measures with chemical control. Agricultural control includes sclerotium retrieval, appropriate planting density, and water and fertilizer management. Chemical control relies primarily on chemical agents, mainly triazoles and succinate dehydrogenase inhibitors, which easily induce drug resistance, pesticide residues, and ecological risks. Therefore, biological control technology for rice sheath blight has become a research hotspot in recent years. Biological control refers to the use of organisms or their metabolites to control harmful plant and animal populations or reduce their harmfulness. Compared with traditional control methods, the main advantages of biological control technology are its minimal environmental impact and high specificity. The challenge and key point lies in finding highly efficient, stable, and harmless functional microorganisms.

[0004] Bacillus compostii is a Bacillus species that rapidly colonizes in humus environments. It can produce antimicrobial lipopeptides and plant hormones. However, current technologies are mostly focused on promoting composting or soil improvement. There are no reports on its use for foliar biocontrol of rice sheath blight and on clarifying its immune activation mechanism. Summary of the Invention

[0005] To address the aforementioned deficiencies in existing technologies, this invention aims to provide Bacillus compostii A1 and its application in the preparation of agents to enhance resistance to rice sheath blight. This strain possesses characteristics such as high efficiency and stability, solving the problems of low foliar colonization rate and unstable efficacy of existing biocontrol bacteria, and significantly inhibiting the spread of rice sheath blight lesions.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a strain of Bacillus stercoris A1 resistant to rice sheath blight, with accession number CGMCC No.35058.

[0008] The present invention also provides the application of the above-mentioned Bacillus compostii A1 in resistance to rice sheath blight.

[0009] The present invention also provides a biocontrol agent for resisting rice sheath blight, the biocontrol agent comprising an active ingredient and an auxiliary ingredient, wherein the active ingredient comprises the above-mentioned Bacillus compostii A1 or its fermentation broth.

[0010] Preferably, the auxiliary components are a carrier, a dispersant, a protectant, and a synergist, wherein the dispersant is Tween 20 and the mass fraction of Tween 20 is 0.01% to 0.05%; the active ingredients also include seaweed oligosaccharides.

[0011] Preferably, the formulation of the biocontrol agent is a wettable powder or a microcapsule suspension.

[0012] Preferably, the total concentration of viable bacteria in the biocontrol agent is 1×10⁻⁶. 7 -1×10 9 CFU / mL.

[0013] The present invention also provides a method for preparing a fermentation broth containing bacteria resistant to rice sheath blight, comprising: culturing the above-mentioned Bacillus compostii A1 to obtain the fermentation broth.

[0014] Preferably, the method includes: inoculating Bacillus composting strain A1 into LB liquid medium and culturing at 28-35°C to obtain a bacterial fermentation broth with an OD600 value of (0.7-1.2).

[0015] The present invention also provides a bacterial fermentation broth prepared by the above-described preparation method.

[0016] The present invention also provides a method for preventing rice sheath blight, comprising: spraying the rice leaves with the above-mentioned microbial fermentation liquid at the four-leaf stage, and / or tillering stage, and / or booting stage, wherein the spray volume of the microbial fermentation liquid is (4~6) ml / plant.

[0017] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0018] This invention utilizes the isolated Bacillus compostii A1 to prepare a biocontrol agent targeting rice sheath blight. This biocontrol agent is a biological agent, completely avoiding the problems associated with the use of chemical pesticides, thus promoting pollution-free rice production. It eliminates or reduces the need for other chemical pesticides, which not only reduces planting costs but also benefits the economic development of rice cultivation. Furthermore, the biocontrol agent prepared from this strain exhibits excellent inhibitory effects against rice sheath blight pathogens. Attached Figure Description

[0019] Figure 1 The results show the antagonistic effect of strain A1 against *Rhizoctonia solani* (A is the control group, B is the experimental group).

[0020] Figure 2 The relative lesion area of ​​rice sheath blight after spraying with a biocontrol agent containing strain A1 (p < 0.01).

[0021] Figure 3 The relative expression level of OsNPR1 after spraying a biocontrol agent containing strain A1 (CK is the control group, A1 is strain A1; internal control is OsUBQ).

[0022] Figure 4 The relative expression level of OsPR2 after spraying with a biocontrol agent containing strain A1; the internal control was OsUBQ).

[0023] Figure 5 The relative expression level of OsMAPK6 after spraying with a biocontrol agent containing strain A1 (internal reference was OsUBQ). Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, the reagents and materials used in the following examples are all conventional commercially available products;

[0026] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0027] Where there is no conflict, the embodiments and features in the embodiments of this invention can be combined with each other. The invention will now be described in detail with reference to the embodiments.

[0028] The following is the method for preparing the culture medium used in the examples:

[0029] LB solid medium (LB plate): 5 g yeast extract, 10 g tryptone, 10 g NaCl, 12 g agar, distilled water to a final volume of 1000 mL, pH 7.0 - 7.2.

[0030] LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, distilled water to a final volume of 1000 mL, pH 7.0 - 7.2. PDA solid medium (PDA plates): 200 g potato, 20 g sucrose, 15-18 g agar powder, 1000 mL deionized water.

[0031] Example 1 Isolation and Identification of Strain A1

[0032] I. Isolation of strain A1

[0033] 1. Sampling

[0034] Five-point sampling was used to sample the rhizosphere soil (soil attached to the root surface after pulling up rice plants and shaking off large clumps of soil), tissue at the boundary between diseased and healthy rice, and floating matter on the surface of rice paddy water in rice fields infected with sheath blight (the rice was sourced from normally planted rice plants in Taojiang, Yiyang, Hunan). 50 g of soil or 5 g of tissue was collected from each point and mixed into one sample (approximately 200 g of soil or 20 g of tissue), which was then placed in a sterile sampling bag and stored at 4°C (for no more than 48 h).

[0035] 2. Sample pretreatment:

[0036] Soil samples: Take 5 g of soil sample, add 45 mL of sterile water, shake for 30 min, let stand for 10 min, take 10 mL of supernatant, and treat in an 80°C water bath for 10 min (to kill non-spore-forming bacteria).

[0037] Tissue samples: cut into small pieces with sterile scissors, add 10 mL of sterile water and grind, take 5 mL of supernatant and heat-treat as above.

[0038] 3. Isolation and Culture

[0039] Take 100 µL of the heat-treated sample suspension, spread it on an LB plate, and incubate it upside down at 37°C for 24-48 h to obtain a single colony of strain A1.

[0040] 4. Cultivation characteristics

[0041] Further investigation into the culture characteristics of strain A1 revealed that its growth temperature range is 4-50°C, with an optimal growth temperature of 30-37°C. Growth slows above 45°C and ceases at 55°C. The strain adapts to a pH range of 5.0-9.0, with an optimal pH of 6.5-7.5. Growth rates decrease by more than 50% at pH values ​​below 5.5 or above 8.5. This strain exhibits some salt tolerance, growing in LB medium containing 0-8% NaCl, with an optimal salt concentration of 0.5%. Growth is significantly inhibited at salt concentrations >6%.

[0042] This strain is aerobic, strictly aerobic, and grows only at the surface of the liquid in deep liquid media. When punctured in a soft agar column containing 1% agar, it grows only on the surface of the medium.

[0043] II. Identification of strain A1

[0044] The 16S rRNA sequence of strain A1 was amplified and sequenced using primers (F: 5'-CCTACGGGAGGCAGCAG-3'; R: 5'-ATTACCGCGGCTGCTGG-3'). Sequence alignment was performed using NCBI, and the isolated strain A1 was finally identified as Bacillus stercoris, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 3, 2025, with accession number CGMCC No. 35058.

[0045] Example 2 Preparation of biocontrol agents for strain A1

[0046] This embodiment involves culturing strain A1 obtained from screening in Example 1 and preparing it into a biocontrol agent, including the following:

[0047] Single colonies of Bacillus compostii A1 were picked and inoculated into LB liquid medium. After incubation at 32°C and 180 rpm for 16 h, the culture was centrifuged for 5 min, washed twice with PBS, and the OD600 was adjusted to 0.8 (≈1×10⁻⁶). 8 The fermentation broth, obtained by adding CFU / mL, is a cell suspension containing Bacillus compostii A1. By adding carriers, dispersants, protectants, and synergists, a biocontrol agent containing strain A1 can be prepared.

[0048] As another preferred embodiment, the dispersant is Tween, and the amount added is 0.01%~0.05% by volume.

[0049] As another preferred embodiment, the dispersant is Tween, and the amount added is 0.02% by volume.

[0050] As another preferred embodiment, the concentration of Bacillus compostingus A1 in the fermentation broth is 1×10⁻⁶. 7 -1×10 9 CFU / mL.

[0051] As another preferred embodiment, the formulation of the biocontrol agent is a wettable powder or a microcapsule suspension;

[0052] As another preferred embodiment, the active ingredients of the biocontrol agent include strain A1 and seaweed oligosaccharides.

[0053] Example 3 Antagonism Experiment

[0054] The plate confrontation culture method was used, with a 5 mm mycelial cake (mycelial side down) of *Rhizoctonia solani* inoculated in the center of a PDA plate. The experimental group was inoculated with single colonies of strain A1 isolated in Example 1 around the perimeter of the plate. The plates were incubated in the dark at 28°C, and mycelial growth was observed daily. The control group was not inoculated with strain A1 isolated in Example 1. The results showed that the inhibition zone width in the experimental group was significantly greater than that in the control group (see...). Figure 1 This indicates that strain A1 has a strong antagonistic effect on the growth of *Rhizoctonia solani*.

[0055] Example 4: Pot Experiment

[0056] Normally planted rice plants were selected from Taojiang County, Yiyang City, Hunan Province. At the four-leaf stage, 5 mL / plant of the biocontrol agent prepared in Example 2 was sprayed (experimental group), while the control group was sprayed with water. After 24 hours, the plants were inoculated with *Rhizoctonia solani* fungal cakes (Ø5 mm), and the lesion area was measured after 72 hours. Figure 1 As shown, compared with the control group, foliar spraying of a biocontrol agent containing strain A1 reduced the relative lesion area of ​​rice sheath blight by ≥55% (see...). Figure 2 This indicates that strain A1 can inhibit the occurrence of rice sheath blight to a certain extent.

[0057] As another preferred embodiment, the biological agent can also be used by seed coating (1×10⁻⁶). 6 CFU / seed or rhizosphere drip irrigation.

[0058] Example 5 Gene Expression

[0059] The biological agent prepared in Example 2 was applied to rice leaves using a spray inoculation method, serving as the experimental group, while the control group was sprayed with water. Samples were taken 0-24 h after inoculation, and qPCR was used for detection. The results showed that at 12 h after inoculation, the relative expression levels of OsNPR1, OsPR2, and OsMAPK6 in the experimental group were 4.2, 5.1, and 3.7 times that of the control group, respectively (p < 0.01) (see...). Figure 3 , Figure 4 and Figure 5 The above results indicate that strain A1 can significantly upregulate the expression levels of immune marker genes OsNPR1, OsPR2, and OsMAPK6.

[0060] This demonstrates that strain A1 activates the OsMAPK6 cascade, promotes the accumulation of OsNPR1 nuclei, initiates the expression of pathogenesis-related proteins such as OsPR2, and induces systemic acquired resistance (SAR), thereby limiting the spread of R. solani.

[0061] It should be noted that subsequent field trials involved spraying once each at the tillering and heading stages. Based on the data from the above examples, it was confirmed that the biocontrol agent containing strain A1, applied to rice leaves, exhibited significant resistance to rice sheath blight. The calculated field dosage was 5 L / 667 m³. 2 This strain is naturally isolated, easily colonized, has a half-life of <7 days, and poses no risk of residue.

[0062] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A strain of Bacillus stercoris A1 resistant to rice sheath blight, characterized in that, Its accession number is CGMCC No.35058.

2. The application of Bacillus compostingus A1 as described in claim 1 in resistance to rice sheath blight.

3. A biocontrol agent for rice sheath blight, characterized in that, The biocontrol agent includes an active ingredient and an auxiliary ingredient, wherein the active ingredient comprises Bacillus compostii A1 or its fermentation broth as described in claim 1.

4. The biocontrol agent as described in claim 3, characterized in that, The auxiliary components include a carrier, a dispersant, a protectant, and a synergist. The dispersant is Tween 20, and the amount of Tween 20 added is 0.01% to 0.05% by volume. The active ingredients also include seaweed oligosaccharides.

5. The biocontrol agent as described in claim 3, characterized in that, The formulation of the biocontrol agent is a wettable powder or a microcapsule suspension.

6. The biocontrol agent as described in claim 3, characterized in that, The total concentration of viable bacteria in the biocontrol agent is 1×10⁻⁶. 7 -1×10 9 CFU / mL.

7. A method for preparing a fermentation broth containing a microbial strain resistant to rice sheath blight, characterized in that, include: The fermentation broth is obtained by culturing the Bacillus compostingus A1 as described in claim 1.

8. The preparation method according to claim 7, characterized in that, include: Bacillus compostii strain A1 was inoculated into LB liquid medium and cultured at 28-35℃ to obtain a fermentation broth with an OD600 value of (0.7-1.2).

9. The fermentation broth prepared by the method described in claim 7 or 8.

10. A method for preventing rice sheath blight, characterized in that, include: During the four-leaf stage, and / or tillering stage, and / or booting stage of rice, the fermentation liquid of the strain as described in claim 9 is sprayed onto the rice leaves, wherein the spray volume of the fermentation liquid is (4 ~ 6) ml / plant.

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