Bacillus altitudinis strain JR-1 and application thereof in preventing and treating rice blast

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Bacillus oryzae* JR-1 and its application in the control of rice blast. The *Bacillus oryzae* JR-1 provided by this invention has the preservation number CCTCC NO: M 20252406. It was isolated from the rhizosphere soil of a high-quality dryland rice variety, Dianheyou 615, in a healthy plot adjacent to a rice blast-affected area. It possesses the ability to solubilize phosphorus, protease, cellulase, and laccase, effectively inhibiting rice blast pathogens, especially the mycelial growth of highly pathogenic rice blast pathogens, with an inhibition rate of 78.69%. Furthermore, in vitro leaf inoculation and root drenching experiments show that the fermentation broth of *Bacillus oryzae* JR-1 provided by this invention can effectively inhibit the occurrence of rice blast, exhibiting good preventive and curative effects against rice blast, especially in the Dianheyou 615 rice variety.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus oryzae JR-1 and its application in the prevention and control of rice blast. Background Technology

[0002] Rice, as a staple food for humankind, is crucial for global development and population growth. High-quality dryland rice is a type of premium rice variety grown on dry land that exhibits yield and quality comparable to paddy rice. It possesses characteristics such as strong soil-penetrating ability, well-developed root systems, vigorous tillering, good drought resistance, high yield, and rice quality meeting national high-quality rice standards. Changes in dryland habitats have led to significant shifts in the predominance of diseases affecting high-quality dryland rice; however, fungal pathogens... Magnaporthe oryzae Rice blast caused by the disease remains a devastating disease in rice production, causing 10% to 20% of yield loss each year. In severely affected areas, yield losses can reach 40% to 50%, or even result in total crop failure.

[0003] Currently, the main methods for controlling rice blast are breeding resistant varieties and chemical control. Because rice blast is a fungal disease, it mutates and differentiates rapidly, exhibiting high diversity with new physiological races constantly emerging. Conventional breeding methods have long breeding cycles, and the resistant varieties produced often exhibit limited resistance and lose resistance quickly, making it difficult to achieve broad-spectrum and long-lasting resistance, hindering the development of the breeding industry. While chemical control has indeed shown significant effectiveness in field trials, the advent and use of chemical pesticides have, to some extent, solved the problem of rice blast control. However, with increasing public awareness of food safety, their excessive use has led to new problems such as increased pathogen resistance, worsened environmental pollution, and rice residues. Chemical control methods have introduced plant resistance and food safety issues, while traditional agricultural control methods struggle to achieve the desired control effects. In this context, utilizing beneficial microorganisms to control the disease has become a new direction for prevention and control. Microorganisms are diverse, mainly including bacteria, fungi, actinomycetes, and some viruses. Due to their simple structure, rapid reproduction, and strong colonization ability, bacteria are increasingly valued by plant pathologists for their ability to control plant diseases. They are also the most promising and valuable biocontrol agents.

[0004] Previous studies have reported that Bacillus bacteria, such as Bacillus subtilis, are the most effective antagonistic bacteria against rice blast fungus when isolated and screened from various microenvironments. Bacillus subtilis The lipopeptide compounds synthesized and secreted by Bs916 are key factors in rice's resistance to various diseases, including rice blast; Bacillus megaterium (B. megaterium) Bacillus megaterium The inoculant developed by Bacillus licheniformis (Bacillus licheniformis) has shown good field control efficacy against rice blast and has been widely applied; Bacillus licheniformisBS-3 achieves highly effective inhibition of rice blast fungus by producing a 31 kDa antifungal polypeptide. Currently, research on *Bacillus pyriformis* (BG) is underway. Bacillus altitudinis Reports on the use of *Bacillus oryzae* as a biocontrol bacterium to inhibit rice blast fungus are scarce, and existing *Bacillus oryzae* strains have limited inhibitory effects on the fungus. For example, existing technology CN119020193A discloses a strain of *Bacillus oryzae* B7, which has some biocontrol activity against rice blast fungus, but the effect is limited and needs to be combined with *Pseudomonas* (…). Pseudomonas otitidis ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Use in combination with T17. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus oryzae JR-1 and its application in the prevention and control of rice blast. It has a significant effect on inhibiting rice blast fungus, especially the highly pathogenic rice blast fungus, and can significantly inhibit the occurrence of rice blast and prevent and control rice blast.

[0006] This invention provides a strain of Bacillus hygroscopicus JR-1, which is classified as follows: Bacillus altitudinis JR-1 was deposited on November 3, 2025, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 20252406.

[0007] This invention provides a microbial agent comprising Bacillus hygroscopicus JR-1 as described in the above technical solution.

[0008] Preferably, the bacterial agent is a liquid preparation of Bacillus hygroscopicus JR-1.

[0009] Preferably, the effective viable count of Bacillus hygroscopicus JR-1 in the liquid formulation is 1×10⁻⁶. 7 ~1×10 8 CFU / mL.

[0010] The present invention provides a method for preparing the bacterial agent described in the above technical solution, wherein Bacillus hygroscopicus JR-1 is inoculated into a fermentation medium for fermentation culture, and the fermentation broth is collected to obtain the bacterial agent.

[0011] Preferably, the fermentation medium includes LB liquid medium; the fermentation temperature is 28~32℃ and the time is 24~72h.

[0012] This invention provides the application of Bacillus oryzae JR-1 as described in the above-described technical solution, or the inoculum agent as described in the above-described technical solution, or the inoculum agent obtained by the preparation method described in the above-described technical solution, in inhibiting rice blast fungus and / or controlling rice blast.

[0013] Preferably, the rice blast fungus includes rice blast fungus YL22H7; the rice blast disease includes leaf blast.

[0014] Preferably, the rice blast fungus and the rice variety corresponding to the rice blast are both Dianheyou 615.

[0015] This invention provides a method for preventing and controlling rice blast, comprising the following steps: applying the Bacillus oryzae JR-1 described in the above technical solution or the inoculant described in the above technical solution or the inoculant obtained by the preparation method described in the above technical solution to rice crops.

[0016] Beneficial effects: The *Bacillus oryzae* JR-1 provided by this invention, with preservation number CCTCC NO: M 20252406, was isolated from the rhizosphere soil of high-quality dryland rice variety Dianheyou 615 in a healthy plot adjacent to a rice blast outbreak area. It possesses the ability to solubilize phosphorus, protease, cellulase, and laccase, effectively inhibiting rice blast pathogens, especially the mycelial growth of highly pathogenic rice blast pathogens, with an inhibition rate of 78.69%. Furthermore, in vitro leaf inoculation and root drenching experiments showed that the fermentation broth of *Bacillus oryzae* JR-1 provided by this invention can effectively inhibit the occurrence of rice blast, exhibiting good preventive and curative effects against rice blast, especially against the Dianheyou 615 rice variety.

[0017] Biological Preservation Information Highland Bacillus JR-1, classified as Highland Bacillus Bacillus altitudinis JR-1 was deposited on November 3, 2025, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 20252406. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 Image showing the colony morphology of Bacillus hygroscopicus JR-1 on a plate; Figure 2 Phylogenetic tree of Bacillus hygroscopicus JR-1; Figure 3 The results of biological function verification of Bacillus hygroscopicus JR-1 are shown; from left to right, they are the functional verification results of nitrogen fixation, phosphorus solubilization, protease solubilization, cellulase solubilization, laccase solubilization, siderophore production, and silicate solubilization. Figure 4 The results of the flat plate confrontation experiment in Example 3; Figure 5The results of colony diameter analysis for rice blast fungus in the control and treatment groups of Example 3 are as follows; Figure 6 The lesion condition of detached leaves in the control and treatment groups of Example 4; Figure 7 The above are the statistical results of the lesion area of ​​the detached leaves of the control group and the treatment group in Example 4; Figure 8 The lesion condition on the leaves of the control group and treatment group in the root drenching experiment of Example 5; Figure 9 The results show the statistical findings of lesion area on leaves in the control and treatment groups during the root drenching experiment in Example 5. Figure 10 The lesion condition on the leaves of the control group and treatment group in the root drenching experiment of Example 6; Figure 11 The results show the statistical findings of lesion area on leaves in the control and treatment groups during the root drenching experiment in Example 6. in, express P <0.05, express P <0.001. Detailed Implementation

[0020] This invention provides a strain of Bacillus hygroscopicus JR-1, with accession number CCTCC NO: M 20252406.

[0021] This invention isolated the aforementioned *Bacillus oryzae* JR-1 from the rhizosphere soil of *Dianheyou 615*, a high-quality dryland rice variety, from a healthy plot adjacent to a rice blast-affected area. The colony morphology on agar plates was as follows: white colonies with a rough, uneven surface, exhibiting distinct wrinkles or a granular texture, relatively dry consistency, and mostly raised shapes. Some colonies had irregular edges and showed an irregular expansion pattern. It possesses the ability to solubilize phosphorus, protease, cellulase, and laccase, effectively inhibiting *Bacillus oryzae*, especially the mycelial growth of highly pathogenic *Bacillus oryzae*, achieving an inhibition rate of 78.69%. Furthermore, in vitro leaf inoculation and root drenching experiments showed that the fermentation broth of *Bacillus oryzae* JR-1 provided by this invention effectively inhibited the occurrence of rice blast, demonstrating good preventative and curative effects against rice blast, particularly against the *Dianheyou 615* rice variety.

[0022] This invention provides a microbial agent comprising Bacillus hygroscopicus JR-1 as described in the above technical solution.

[0023] In one embodiment, the bacterial agent of the present invention is a liquid preparation of Bacillus glacialis JR-1. In another embodiment, the effective viable count of Bacillus glacialis JR-1 in the liquid preparation of the present invention is 1 × 10⁻⁶. 7 ~1×108 CFU / mL; as another embodiment, the effective viable count of Bacillus hygroscopicus JR-1 in the liquid formulation of the present invention is 1×10⁻⁶. 8 CFU / mL.

[0024] The present invention provides a method for preparing the bacterial agent described in the above technical solution, wherein Bacillus hygroscopicus JR-1 is inoculated into a fermentation medium for fermentation culture, and the fermentation broth is collected to obtain the bacterial agent.

[0025] In one embodiment, the fermentation medium of the present invention includes LB liquid medium. In one embodiment, the fermentation temperature of the present invention is 28-32°C; in another embodiment, the fermentation temperature of the present invention is 28°C. In one embodiment, the fermentation time of the present invention is 24-72 hours; in another embodiment, the fermentation time of the present invention is 36-48 hours.

[0026] This invention provides the application of Bacillus oryzae JR-1 as described in the above-described technical solution, or the inoculum agent as described in the above-described technical solution, or the inoculum agent obtained by the preparation method described in the above-described technical solution, in inhibiting rice blast fungus and / or controlling rice blast.

[0027] In one embodiment, the rice blast fungus of the present invention includes rice blast fungus YL22H7. In one embodiment, the rice blast disease of the present invention includes leaf blast. In one embodiment, the rice blast fungus and the rice variety corresponding to the rice blast disease of the present invention are both Dianheyou 615.

[0028] This invention provides a method for preventing and controlling rice blast, comprising the following steps: applying the Bacillus oryzae JR-1 described in the above technical solution or the inoculant described in the above technical solution or the inoculant obtained by the preparation method described in the above technical solution to rice crops.

[0029] In one embodiment, the application method of the Bacillus cereus JR-1 or the inoculant of the present invention includes root irrigation. In one embodiment, the application amount of the inoculant of the present invention is 40-50 mL / 9 plants; in another embodiment, the application amount of the inoculant of the present invention is 50 mL / 9 plants. In one embodiment, the rice crop of the present invention is a rice plant at the 3-leaf-1-heart stage. In one embodiment, the rice of the present invention is Dianheyou 615.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of Bacillus subtilis JR-1 provided by the present invention and its application in the control of rice blast, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Isolation and identification of strains Bacteria were isolated from the rhizosphere soil of high-quality dryland rice (Dianheyou 615) in healthy plots adjacent to rice blast-affected plots using the dilution plating method. The specific procedure was as follows: Soil samples were weighed and placed in sterile water at a ratio of 1 g:10 mL, and incubated with shaking at 28°C for 30 min to prepare 10... -1 Soil suspension; diluted sequentially in 10-fold gradients to 10... -6 Finally, a diluted soil suspension was obtained. 100 μL of the suspension was pipetted onto LB medium and incubated upside down in a 28°C incubator for 24 h. Purification was performed using the streak plate method to obtain pure culture strain JR-1. Colonies were white, with a rough, uneven surface, exhibiting obvious wrinkles or a granular texture, and were relatively dry. The colonies were mostly raised, with some colonies having irregular edges and showing an irregular expansion pattern. Figure 1 Finally, the purified strain was identified using 16S rRNA. The results showed that the full-length 16S rRNA sequence of strain JR-1 was 1404 bp, similar to that of *Bacillus hygroscopicus*. Bacillus altitudinis TA 8.2 and Bacillus hygroscopicus Bacillus altitudinis The sequence similarity of T8.28 was 100%, similar to Bacillus salsa. Bacillus safensis The sequence similarity of KLV1 was 99.86%, similar to that of Bacillus safranin. Bacillus safensis The sequence similarity of T4 was 99.79%, and it was similar to Bacillus hygroscopicus. Bacillus altitudinis The strain is in a phylogenetic branch ( Figure 2 Therefore, strain JR-1 was identified as Bacillus hygroscopicus. Bacillus altitudinis And to preserve them biologically.

[0032] Example 2 Biological function verification of Bacillus hygroscopicus JR-1 Bacillus hygroscopicus JR-1, activated for 24 hours, was inoculated onto nitrogen-fixing, phosphorus-solubilizing, protease-solubilizing, cellulase-solubilizing, laccase-solubilizing, siderophore-producing, and silicate-solubilizing media, respectively. After incubation at 28°C upside down for 72 hours, the presence of clear zones around the colonies was observed. The formation of clear zones indicated the presence of the corresponding functions. The results showed that Bacillus hygroscopicus JR-1 produced clear zones on the phosphorus-solubilizing, protease-solubilizing, cellulase-solubilizing, and laccase-solubilizing media, indicating that the Bacillus hygroscopicus JR-1 obtained in Example 1 possesses phosphorus-solubilizing, protease-solubilizing, cellulase-solubilizing, and laccase-solubilizing functions, but lacks nitrogen-fixing, silicate-solubilizing, and siderophore-producing functions. Figure 3 ).

[0033] Example 3 Inhibition rate of Bacillus oryzae JR-1 against rice blast fungus 1. Preparation of fermentation broth: Select the Bacillus hygroscopicus JR-1 obtained in Example 1 and inoculate it into LB liquid medium. Culture at 28°C and 180 r / min for 72 h with shaking to obtain the fermentation broth of Bacillus hygroscopicus JR-1.

[0034] 2. Rice blast pathogen: Rice blast fungus YL22H7, isolated from susceptible plants of Dianheyou 615, exhibits the strongest pathogenicity and is published in Pathogenicity and Genetic Variations in... Magnaporthe oryzae Isolates from One Rice Variety Planting in Paddy and Upland Fields (https: / / doi.org / 10.3390 / agronomy13051246).

[0035] 3. Using a sterile punch with a diameter of 5 mm, inoculate rice blast fungus cakes into the center of PDA medium plates. Spot-inoculate Bacillus oryzae JR-1 fermentation broth around the tested pathogen, 2 cm away. Spot-inoculate an equal volume of liquid LB medium as a control group (CK). Incubate at 28℃. When the pathogen in the control group has completely covered the culture dish, use the cross-cross method to determine the colony diameter of the tested pathogen in both the control group and the Bacillus oryzae JR-1 fermentation broth treatment group. Calculate the inhibition rate of the strains using a formula. The results are shown in Table 1 and [Table data missing]. Figures 4-5 As shown.

[0036] Table 1. Results of the inhibition rate of Bacillus oryzae JR-1 against rice blast fungus.

[0037] According to Table 1 and Figures 4-5 It can be seen that the Bacillus thuringiensis JR-1 provided by the present invention can effectively inhibit the growth of mycelium of the highly pathogenic pathogen (rice blast fungus YL22H7) of high-quality dryland rice - Dianheyou 615, and the inhibition rate reaches 78.69%.

[0038] Example 4 detached leaf testing Treatment group (JR-1) selected leaves from healthy, high-quality dryland rice plants of the variety Dianheyou 615, cut 5cm long leaf segments, and immersed them in 1×10 8 The leaves were cultured in CFU / mL Bacillus oryzae JR-1 fermentation broth at 150 rpm for 1 h with shaking. Sterile water was used as a negative control (CK). The treated leaves were placed in sterile petri dishes, and the leaf surface was gently pricked with a sterile toothpick, one prick per leaf. Each leaf was then inoculated with 10 μL of a spore suspension of rice blast fungus YL22H7 (1×10⁻⁶). 5(spores / mL) were applied to the puncture sites. The inoculated leaves were placed in an artificial climate chamber and cultured at 28℃ for 48 h (12 h light / day, 12 h darkness / day, light intensity 4000 Lux). The relative area of ​​rice blast lesions at the inoculation sites was investigated. The results showed that the *Bacillus oryzae* JR-1 provided by this invention can effectively inhibit the occurrence of rice blast in plants. Figures 6-7 ).

[0039] Example 5 The preventive effect of Bacillus oryzae JR-1 against rice blast A pot experiment was conducted with a planting density of 9 plants per pot. Once the dry-grown rice plants (Dianheyou 615) reached the 3-leaf, 1-heart stage, the irrigation concentration per pot was 1×10⁻⁶. 8 50 mL of CFU / mL Bacillus oryzae JR-1 fermentation broth was used, with three replicates per treatment. Sterile water was used as a negative control. After 24 h of treatment, the spores of rice blast fungus YL22H7 (1×10⁻⁶) were inoculated. 5 Inoculation was performed by spraying at a uniform angle (spores / mL), followed by incubation at 22℃ in the dark with humidity for the first 24 hours. Approximately 7 days after inoculation, once the disease had fully developed, the relative lesion area was calculated on a leaf-by-leaf basis. The results showed that, in disease prevention treatment, compared to the negative control (CK), the treatment group (JR-1) showed a significant reduction in the relative lesion area of ​​rice leaves after treatment with this strain, with a reduction of up to 38%. Figures 8-9 ).

[0040] Example 6 The therapeutic effect of Bacillus hygroscopicus JR-1 on rice blast A pot experiment was conducted with a planting density of 9 plants per pot. Once the dry-grown rice plants (Dianheyou 615) reached the 3-leaf-1-heart stage, each pot was first inoculated with a spore suspension (1×10⁻⁶) of rice blast fungus YL22H7. 5 Inoculation was performed using a uniform angle spray, followed by 24 hours of dark, humidified culture. The treatment group (JR-1) was then watered with a concentration of 1×10⁻⁶. 8 Fermentation broth of *Bacillus cereus* JR-1 (CFU / mL) was used in 50 mL batches, with three replicates per treatment. The negative control (CK) was treated with sterile water, also with three replicates per treatment. Incubation was carried out at 22℃. After full disease development, the relative area of ​​lesions was calculated on a leaf-by-leaf basis. Results showed that, compared to the negative control (CK), the relative lesion area of ​​rice leaves treated with the strain (JR-1) was significantly reduced, with a decrease of 53%. Figures 10-11 ).

[0041] As can be seen from the above, the Bacillus oryzae JR-1 provided by this invention has the ability to solubilize phosphorus, protease, cellulase and laccase, and can effectively inhibit the growth of mycelium of highly pathogenic fungus of high-quality dryland rice - Dianheyou 615, and inhibit the occurrence of rice blast disease in the plant.

[0042] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus hygroscopicus JR-1, characterized in that, The classification name of the Bacillus hygroscopicus JR-1 is... Bacillus altitudinis It was deposited on November 3, 2025, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University, with accession number CCTCC NO: M 20252406.

2. A microbial agent, characterized in that, It includes Bacillus hygroscopicus JR-1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a liquid preparation of Bacillus hygroscopicus JR-1.

4. The microbial agent according to claim 3, characterized in that, The effective viable count of Bacillus geysersis Jr-1 in the liquid formulation is 1×10⁻⁶. 7 ~1×10 8 CFU / mL.

5. The method for preparing the microbial agent according to any one of claims 2 to 4, characterized in that, Bacillus hygroscopicus JR-1 was inoculated into a fermentation medium for fermentation culture, and the fermentation broth was collected to obtain the bacterial agent.

6. The preparation method according to claim 5, characterized in that, The fermentation medium includes LB liquid medium; the fermentation temperature is 28~32℃ and the time is 24~72h.

7. The Bacillus oryzae JR-1 of claim 1, or the inoculum agent of any one of claims 2-4, or the inoculum agent obtained by the preparation method of claim 5 or 6, in inhibiting rice blast fungus (… Magnaporthe oryzae Applications in the prevention and control of rice blast disease.

8. The application according to claim 7, characterized in that, The rice blast disease mentioned includes leaf blast.

9. The application according to claim 7, characterized in that, The rice variety mentioned is Dianheyou 615.

10. A method for preventing and controlling rice blast, characterized in that, The process includes the following steps: applying the Bacillus oryzae JR-1 of claim 1, or the inoculant of any one of claims 2 to 4, or the inoculant obtained by the preparation method of claim 5 or 6 to rice crops; The rice blast disease mentioned includes leaf blast; The rice variety mentioned is Dianheyou 615.

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