Streptomyces murinus and application thereof

Abstract

This invention discloses a strain of Streptomyces gravidarum and its application, belonging to the field of biotechnology. The preservation number of the Streptomyces gravidarum is: CCTCC NO: M 20251912. The Streptomyces gravidarum can be used to control rice sheath blight.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a strain of Streptomyces gravidarum and its applications. Background Technology

[0002] Streptomyces boulardii ( Streptomyces murinus As an important member of the *Streptomyces* genus, *Streptomyces* has gradually gained attention in the field of biological control in recent years. Studies have shown that *Streptomyces* var. *glaucus* possesses abundant metabolites and exhibits significant antagonistic activity against various plant pathogens. For example, previous studies have reported that *Streptomyces* var. *glaucus* is effective against *Fusarium oxysporum* watermelon-specific strains. Fusarium oxysporumf sp. hiveum), Fusarium solani ( Fusarium solani ) and Strange Long-beaked Shells ( Ceratocystis paradoxa It exhibits good inhibitory effects on plant pathogens such as *Streptomyces oryzae*. Its mechanism of action may include the production of antimicrobial substances such as antibiotics, chitinases, and β-1,3-glucanases, as well as inhibiting the growth and reproduction of pathogens through competition for nutrients and space. However, research on the control of rice sheath blight by *Streptomyces oryzae* is still in its early stages, with few related reports. In-depth research on the control efficacy, mechanism of action, and application technology of *Streptomyces oryzae* against rice sheath blight is expected to provide new ideas and methods for the biological control of rice sheath blight, develop efficient, safe, and environmentally friendly biological control products, fill the research gap in this field, and is of great significance for promoting the green and sustainable development of the rice industry. Summary of the Invention

[0003] In order to overcome the problems existing in the prior art, the present invention proposes a strain of Streptomyces gravidus and its application.

[0004] To achieve the above objectives, the first aspect of the present invention provides a strain of Streptomyces gravidarum (… Streptomyces murinus Its accession number is: CCTCC NO: M 20251912.

[0005] A second aspect of the present invention provides a microbial agent, wherein the active ingredient of the microbial agent is the Streptomyces gravidarum described in claim 1.

[0006] The third aspect of this invention provides the application of the *Streptomyces gravidus* of the first aspect and the inoculant of the second aspect in the control of rice sheath blight.

[0007] The fourth aspect of the present invention provides a method for preventing and controlling rice sheath blight, the method comprising applying the inoculant described in the first aspect and / or the second aspect to rice cultivation soil.

[0008] Through the above technical solution, the present invention can achieve at least the following beneficial effects: the *Streptomyces oryzae* of the present invention can effectively prevent and control rice sheath blight. Attached Figure Description

[0009] Figure 1 This is a morphological diagram of *Streptomyces griseus* from this invention on PDA medium; Figure 2 These are morphological images of the hyphae and spores of *Streptomyces griseus* in this invention under an optical microscope; Figure 3 This is the phylogenetic tree of Streptomyces griseus in this invention; Figure 4 This is a diagram of the plate confrontation test between Streptomyces boulardii P-11F and Rhizoctonia solani in this invention; Figure 5 This invention relates to the inoculation experiment of Streptomyces gravidarum P-11F and Rhizoctonia solani detached leaf samples. Figure 6 This invention relates to a pot experiment of Streptomyces boulardii P-11F in rice and a bar chart of the disease index.

[0010] The *Streptomyces gravidarum* provided by this invention ( Streptomyces murinus (Classified and named as: Streptomyces griseus P-11F) Streptomyces murinus P-11F was deposited on August 27, 2025, at the China Center for Type Culture Collection (CCTCC), located at 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China (Wuhan University), with accession number CCTCC NO: M 20251912. Detailed Implementation

[0011] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0012] In this invention, *Streptomyces oryzae* P-11F and *Streptomyces oryzae* CCTCC NO: M 20251912 are the same strain, have the same meaning, and their names (number P-11F) can be used interchangeably.

[0013] During the research process, the inventors of this invention isolated a strain of Streptomyces griseus, named Streptomyces griseus P-11F, which was deposited on August 27, 2025, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with accession number CCTCC NO: M 20251912.

[0014] Based on this, the first aspect of the present invention provides a strain of Streptomyces gravidus, whose preservation number is CCTCC NO: M20251912.

[0015] Further research has revealed that *Streptomyces gravidus* can be used to treat rice sheath blight.

[0016] Based on this, the second aspect of the present invention provides the application of the above-mentioned Streptomyces gravidus in the prevention and control of rice sheath blight.

[0017] In this invention, there are no particular restrictions on the specific dosage of Streptomyces gravidus, as long as it can achieve the prevention and control of rice sheath blight.

[0018] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate understanding by those skilled in the art.

[0019] 1. Isolation of strains The inventors obtained a biocontrol Streptomyces strain resistant to rice sheath blight by sampling, isolating, and purifying soil samples from the experimental field (25°8′15″N, 102°45′5″E) behind Yunnan Agricultural University in Kunming, Yunnan Province. The strain was named Streptomyces p-11F.

[0020] 2. Identification of strains 2.1 Inoculate the strain onto PDA medium, as shown in the attached diagram. Figure 1 As shown, Colony morphology: Colonies are round or irregular in shape, with a relatively rough surface, wrinkles, and protrusions, possibly due to abundant spore production. The colonies are generally light brown (early incubation) or grayish-brown (late incubation), gradually darkening in color with prolonged incubation, consistent with the "grayish" in the name of *Streptomyces oryzae*. Vigorous growth on the culture medium indicates good growth capacity on that medium. The culture medium turns yellow, possibly due to the diffusion of substances produced by the strain's metabolism into the medium.

[0021] 2.2 Morphological observation of mycelia and spores of the strain under an optical microscope As attached Figure 2 As shown, when *Streptomyces griseus* grows in a culture medium, it first forms substrate hyphae that penetrate deep into the medium. Under a 40x objective lens, these hyphae are colorless to light gray, approximately 0.5-0.8 μm in diameter, and are characterized by multi-branching and lack of septa. They can extend extensively through branching, penetrating deep into the culture medium to absorb nutrients such as carbon and nitrogen sources. The substrate hyphae are the main site of the strain's nutrient metabolism, and their vigorous growth provides the material basis for the subsequent formation of aerial hyphae and spores. When culture conditions are suitable (e.g., sufficient nutrients and good aeration), the substrate hyphae grow upwards and extend to the surface of the culture medium, forming aerial hyphae. The aerial hyphae are darker in color than the substrate hyphae, ranging from grayish-white to mouse gray, and are slightly thicker in diameter (approximately 0.8-1.2 μm). They also have branching ability and appear as a fluffy or fibrous covering on the colony surface.

[0022] After the spore hyphae mature, they break to form individual spores. The spores of *Streptomyces griseus* are elliptical or cylindrical, approximately (0.8-1.2) μm × (1.5-2.0) μm in size. Scanning electron microscopy reveals that the spore surface is mostly smooth or slightly wrinkled, with a few strains possibly exhibiting wart-like protrusions (this varies slightly depending on culture conditions or strain subtype). The spore color is consistent with the aerial hyphae, ranging from grayish-brown to light brown, and when accumulated, they form a distinct powdery colony appearance.

[0023] 2.3 Molecular Identification The actinomycete strain P-11F was identified by molecular biology using 16S rDNA sequencing.

[0024] The sequencing results showed a sequence length of 1030 pb. The sequencing results were compared with those on NCBI, and a phylogenetic tree was constructed using MEGA 8.0. The results are as follows. Figure 3 As shown in the phylogenetic tree, strain P-11F is most closely related to Streptomyces gravidarum, with a sequence similarity of 99.02%. Therefore, it is concluded that strain P-11F is Streptomyces gravidarum.

[0025]

[0026] 3. A contrastive study of Streptomyces griseus P-11F and Rhizoctonia solani on agar plates. In a plate confrontation test between *Streptomyces griseus* P-11F and *Rhizoctonia solani*, the average inhibition rate was calculated to be as high as 85.4%. The inhibition rate was calculated using the formula: Inhibition rate (%) = R − r / R × 100% R: Control growth radius of the pathogen in the absence of antagonistic microorganisms (i.e., the average distance from the inoculation point of the pathogen to the edge of the colony when cultured alone, in cm). r: The treatment growth radius of the pathogen under the action of antagonistic microorganisms (i.e., the average distance from the inoculation point of the pathogen to the edge of the colony when cultured in confrontation with antagonistic microorganisms).

[0027] As attached Figure 4 As shown, compared with the control, the growth of Rhizoctonia solani in the confrontation group was significantly inhibited: colony expansion was restricted: the colony diameter of Rhizoctonia solani was significantly smaller than that of the control, and the hyphal density was reduced; inhibition zone formation: a clear "inhibition blank zone" appeared between the inoculation areas of Streptomyces griseus and Rhizoctonia solani, indicating that Streptomyces griseus effectively blocked the hyphal extension of Rhizoctonia solani; growth competitive advantage: the colony morphology of Streptomyces griseus was intact and not disturbed by Rhizoctonia solani, reflecting its dominant position in nutrient or space competition.

[0028] 4. In vitro leaf inoculation experiment To identify Streptomyces boulardii ( Streptomyces murinus P-11F) against Rhizoctonia solani (P-11F) Rhizoctonia solani This experiment investigated the biocontrol potential of YM-Q-7 (the pathogen of rice sheath blight). Functional verification was conducted using in vitro rice leaf inoculation, and its antagonistic effect was analyzed through phenotypic observation. Specific results are as follows: (1) Experimental design and treatment setup The experiment was conducted with four treatment groups, and the biocontrol effect was evaluated by comparing leaf disease phenotypes: YM-Q-7 group: Inoculated only with Rhizoctonia solani as a pathogenic control; CK group: Inoculated with sterile water as a blank control; P-11F group: Inoculated only with Streptomyces griseus to assess the safety of biocontrol bacteria on rice; P-11F+YM-Q-7 group: Simultaneous inoculation with Streptomyces griseus and Rhizoctonia solani to verify the antagonistic effect of biocontrol bacteria.

[0029] (2) Phenotypic observation and results analysis The interaction effects of each treatment can be visually assessed by observing the disease symptoms (wilt, browning, and degree of necrosis) on detached leaves: a. Pathogenicity verification (YM-Q-7 group) Leaves inoculated only with Rhizoctonia solani ( Figure 5 The first one on the left shows severe wilting, browning, and even necrosis, indicating that Rhizoctonia solani has strong pathogenicity to rice and can efficiently induce typical symptoms of rice sheath blight, thus verifying the effectiveness of the experimental system.

[0030] b. Blank control group (CK group) Leaves inoculated with sterile water ( Figure 5 The second leaf from the left is bright green and free of disease spots, indicating that the water treatment did not damage the leaves and ruled out the interference of abiotic factors on the experimental results.

[0031] c. Safety of biocontrol bacteria (P-11F group) Leaves inoculated only with Streptomyces griseus ( Figure 5 (Third from left) No obvious lesions, proving that *Streptomyces oryzae* is non-toxic to rice leaves when used alone, thus providing a safe basis for its use as a biocontrol agent.

[0032] d. Verification of biocontrol efficacy (P-11F+YM-Q-7 group) Leaves simultaneously inoculated with Streptomyces griseus and Rhizoctonia solani ( Figure 5 Although there was localized yellowing, the lesion area and degree of withering were significantly lower than those in the YM-Q-7 group, indicating that Streptomyces gravidus effectively inhibited the infection or spread of Rhizoctonia solani.

[0033] (3) Conclusion In vitro inoculation experiments on rice showed that *Streptomyces griseus* (P-11F) has a significant biocontrol effect against *Rhizoctonia solani* (YM-Q-7), effectively reducing the severity of rice sheath blight. This strain possesses both "non-toxicity" and "strong antagonistic" characteristics, providing a high-quality candidate resource for the biological control of rice sheath blight. Further validation of its application potential can be achieved through fermentation optimization and field trials.

[0034] 5. Rice pot verification Experimental steps: Rice variety tested: Lijiang Xintuan Black Rice; Select uniform plastic pots (30cm in diameter at the top and 25cm in height), fill each pot with 3.5kg of water, and adjust the soil moisture content to 70% of field capacity. Sow 10 sterilized and pre-germinated rice seeds in each pot and place them in an artificial climate chamber (temperature 25-28℃, light 12h / d, relative humidity 75%) for cultivation. After emergence, thin the seedlings to 5 plants per pot and cultivate them until the tillering stage (seedling age 30-35 days, plant height 30-35cm) for further treatment. Sheath blight fungus ( Rhizoctonia solani): Use highly pathogenic strains (such as AG1-IA physiological race) to transfer from PDA slant medium (200g potato juice + 20g glucose + 18g agar + 1000mL water) to fresh PDA plates, incubate at 28℃ for 7 days, and collect sclerotia (or prepare mycelial cakes, 5mm in diameter) for later use. Streptomyces boulardii ( Streptomyces murinus The strain was inoculated into PDA medium at 28°C, and the spores were washed with sterile water to adjust the concentration to 10. 8 CFU / mL concentration is available for reference.

[0035] Four treatment groups were set up, with three replicates per group and five pots per replicate (60 pots in total). The specific grouping is shown in Table 1 below: Table 1 Statistical Table of Each Treatment Method

[0036] (6) Observe the moisture content and disease incidence every day after inoculation.

[0037] Incidence rate calculation method: Disease incidence rate (%) = (Number of diseased plants / Total number of plants surveyed) × 100%; Incidence rate: Through artificial inoculation, the control and treatment groups showed 100% incidence of disease. Incidence rate ( Figure 6 (First from the left).

[0038] Disease severity index grading criteria: Disease severity grading criteria (refer to the rice sheath blight grading method): Grade 0: Disease-free throughout the entire plant Grade 1: The third leaf sheath (counting from the bottom up) and the leaves below are affected; Grade 2: Disease occurs on the second leaf sheath; Grade 3: Disease occurs on the first leaf sheath; Level 4: Disease occurs on the flag leaf sheath or rice panicle; Disease index calculation method: Disease index = [Σ(number of diseased plants at each level × level value) / (total number of plants surveyed × highest level value)] × 100; Prevention and control efficacy (%) = [(Disease index of blank control group - Disease index of treatment group) / Disease index of blank control group] × 100%; Disease index: When treated with "R. solani" alone (red bar), the disease index was approximately 84.46, indicating severe disease; while when treated with the combination of "p-11F + R. solani" (green bar), the disease index dropped to approximately 46.34, indicating a significant reduction in disease severity. This demonstrates that "p-11F" has an inhibitory effect on rice sheath blight and can alleviate the severity of the disease. Figure 6 (Second from the left). The prevention and control effect reached 45.13%.

[0039] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A strain of Streptomyces griseus ( Streptomyces murinus Its accession number is: CCTCC NO: M 20251912.

2. A microbial agent, wherein the active ingredient of the microbial agent is *Streptomyces gravidarum* as described in claim 1.

3. The application of the *Streptomyces gravidus* as described in claim 1 or the fungal agent as described in claim 2 in the control of rice sheath blight.

4. A method for controlling rice sheath blight, characterized in that: The method includes applying the *Streptomyces gravidarum* of claim 1 and / or the microbial agent of claim 2 to rice cultivation soil.

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