Application of bacillus velezensis E1 in improvement of bacterial blight resistance of rice
By activating the rice immune pathway with Bacillus belye E1 to inhibit bacterial blight, the problem of unstable colonization of existing biocontrol bacteria was solved, achieving efficient and stable control of rice bacterial blight and providing a new approach for biological pesticides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YUELU MOUNTAIN LAB
- Filing Date
- 2026-01-20
- Publication Date
- 2026-05-12
AI Technical Summary
Existing Bacillus-based biocontrol bacteria have poor colonization and stability in rice fields, resulting in unstable control efficacy against rice bacterial blight and making large-scale commercial application difficult. Furthermore, chemical control strategies face problems such as resistance development and environmental pollution.
By using Bacillus belye strain E1, the growth of rice bacterial blight pathogen Xoo was inhibited through chemotactic colonization and activation of the OsMAPK6-OsWRKY13-OsOPR10 immune pathway, thereby improving rice resistance.
It significantly inhibits the proliferation of bacterial blight pathogens, activates the host immune pathway, provides stable control effects, replaces or reduces the use of chemical pesticides, and provides a basis for the development of biological pesticides.
Smart Images
Figure CN122012315A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological control technology and relates to the control of rice bacterial blight pathogens, specifically the application of Bacillus belye E1 in improving rice resistance to bacterial blight. Background Technology
[0002] Rice bacterial blight is a vascular bacterial disease caused by Xanthomonas oryzae epv. oryzae (Xoo), and is widely recognized as one of the most destructive diseases in rice-growing areas of Asia and the world. This disease primarily infects rice leaves, causing them to wither and the photosynthetic area to decrease sharply. In severe cases, it can lead to the death of the rice plant, typically resulting in an annual yield loss of 10%-30%. In epidemic years, some fields may even experience total crop failure, posing a sustained threat to food security.
[0003] For a long time, the control of rice bacterial blight has mainly relied on chemical pesticides, such as copper-based agents like Bordeaux mixture and antibiotics like streptomycin. However, this strategy of relying on chemical control faces increasingly serious challenges. First, the pathogen Xoo is highly susceptible to developing resistance under continuous selective pressure, leading to a significant decrease in pesticide efficacy. Second, the overuse of antibiotics and heavy metal copper can cause pesticide residue problems, threatening not only food safety but also polluting the paddy field ecosystem and disrupting soil microecological balance and biodiversity.
[0004] Given the limitations of chemical control, biological control using beneficial microorganisms is considered a sustainable alternative or supplementary strategy due to its environmental friendliness and low likelihood of resistance development. Among numerous biocontrol microorganisms, Bacillus spp. is considered an ideal biocontrol resource because it can produce heat-resistant spores to resist adverse environments, secrete various antimicrobial substances (such as antimicrobial lipopeptides and enzymes), and has the ability to induce systemic resistance (ISR) in plants. However, existing Bacillus biocontrol bacteria still have significant shortcomings in actual field applications. The core problem is that the colonization ability and stability of the strains on crop surfaces are greatly constrained by complex field environmental factors (such as temperature, humidity, ultraviolet radiation, and indigenous microbial communities), resulting in unstable control efficacy and hindering large-scale commercial application.
[0005] Bacillus velezensis, an important species in the Bacillus genus, has been widely reported to possess broad-spectrum antagonistic activity against various plant pathogens, such as Magnaporthe oryzae (causing rice blast) and Rhizoctonia solani (causing rice sheath blight). However, current research and patent resources are largely focused on these diseases, and there is still a lack of patented Bacillus velezensis strains with clear efficacy against another major rice disease—bacterial blight. More importantly, there is a lack of in-depth and clear research reports on how such biocontrol bacteria act on the rice-Xoo interaction system, their potential molecular mechanisms of action, and especially whether and how they can activate rice's own immune defense pathways to resist bacterial blight.
[0006] Therefore, isolating and screening a patented strain of Bacillus belyss that exhibits stable and highly effective inhibition of rice bacterial blight, and elucidating its biocontrol mechanism, is of significant theoretical and practical importance for developing novel biological pesticides, reducing the use of chemical pesticides, and promoting the development of green control technologies for rice diseases. This invention is based on this urgent need.
[0007] In summary, this invention proposes to improve the resistance of rice to bacterial blight by using strain E1 (CGMCC No. 35057) through chemotactic colonization and activation of the OsMAPK6-OsWRKY13-OsOPR10 immune pathway to inhibit Xoo. Summary of the Invention
[0008] The purpose of this invention is to provide the application of Bacillus vesiculus E1 in improving the resistance of rice bacterial blight, and to apply Bacillus vesiculus E1 bacterial suspension to the prevention and control of rice bacterial blight, thus providing a new approach and means for the prevention and control of rice bacterial blight.
[0009] To achieve the above objectives, the technical solution of the present invention is as follows:
[0010] This invention provides a Bacillus velezensis strain E1, which was isolated from healthy rice leaves from Taojiang, Yiyang, Hunan Province. It was identified as Bacillus velezensis by 16S rRNA, gyrB, and rpoB multi-gene sequencing and is deposited at the China Microbial Culture Collection Center (CGMCC) under accession number CGMCC No. 35057 on July 3, 2025.
[0011] The present invention also provides the application of the above-mentioned Bacillus berberis E1 in improving the resistance to bacterial blight in rice.
[0012] Preferably, the Bacillus berberis E1 is used as a bacterial suspension working solution.
[0013] Preferably, the bacterial suspension working solution is prepared by: activating the bacterial strain on a slant, then adjusting OD600 to 0.8, and adding 0.02% Tween-20 by volume to obtain the bacterial suspension working solution.
[0014] Preferably, the concentration of the bacterial suspension used is 1×10⁻⁶. 8 CFU / mL.
[0015] Preferably, the Bacillus berberis E1 is used for the prevention and control of diseases during the four-leaf stage of rice.
[0016] Preferably, the Bacillus belye E1 is used to activate the OsMAPK6-OsWRKY13-OsOPR10 immune pathway and inhibit bacterial blight pathogens.
[0017] The beneficial effects of this invention are:
[0018] This invention provides a highly efficient, stable, and preserved biocontrol bacterium that significantly inhibits the proliferation of Xanthomonas oryzae pv. Oryzae (Xoo), solving the problems of low colonization rate and unstable efficacy of existing biocontrol bacteriums. It clarifies a new use of strain E1 to inhibit the growth of Xanthomonas oryzae pv. Oryzae (Xoo) and activate the host immune pathway through foliar spraying, which can replace or reduce chemical fungicides and provide a theoretical basis for the development of fungicides. Attached Figure Description
[0019] Figure 1 This refers to the relative growth of rice bacterial blight pathogens after spraying E1 bacterial suspension in this invention.
[0020] Figure 2 This refers to the relative expression level of OsMAPK6 after spraying E1 bacterial suspension in this invention.
[0021] Figure 3 This refers to the relative expression level of OsWRKY13 after spraying E1 bacterial suspension in this invention.
[0022] Figure 4 This refers to the relative expression level of OsOPR10 after spraying E1 bacterial suspension in this invention. Detailed Implementation
[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0026] Example 1: Preparation of working solution of Bacillus belyssus E1 suspension
[0027] Slant activation: Bacillus berberis E1 (CGMCC No. 35057) glycerol strain preserved at -80℃ was streaked onto LB agar plates and incubated at 32℃ for 24 hours until single colonies were formed.
[0028] Liquid culture: Pick a single plump colony and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium. Place the flask in a shaker at 32℃ and 180 rpm for 16 hours to obtain the seed culture.
[0029] Cell collection and washing: Centrifuge the seed culture at 4°C and 8000 rpm for 5 minutes and discard the supernatant. Gently resuspend the cell pellet in sterile phosphate-buffered saline (PBS, pH 7.2) and centrifuge again to wash. Repeat this process twice to remove culture medium residue.
[0030] Working solution preparation: Finally, resuspend the bacterial cells in an appropriate amount of PBS, and adjust the absorbance (OD600) of the bacterial suspension at 600 nm to 0.8 using a UV spectrophotometer. Verification using the plate count method showed that this OD value corresponds to a viable bacterial concentration of approximately 1 × 10⁻⁶. 8 CFU / mL. Then, add 0.02% (v / v) of Tween-20 surfactant to the bacterial suspension and mix thoroughly to obtain the desired bacterial suspension working solution. This working solution should be prepared and used immediately.
[0031] Example 2: Effect of Bacillus belye E1 suspension on bacterial leaf blight in rice in pot cultivation
[0032] Methods: Healthy and uniform four-leaf stage rice seedlings were selected as experimental materials.
[0033] Two groups were set up: (1) E1 treatment group: using the E1 bacterial suspension working solution prepared in Example 1 (1×10 8 (2) Control group (CK): Spray with an equal volume of PBS solution containing 0.02% Tween-20. After 24 hours of treatment, artificially inoculate with bacterial blight suspension (concentration 1×10⁻⁶ CFU / mL) on each rice plant; 8 (CFU / mL). Samples were taken 14 days after inoculation.
[0034] Relative quantification of pathogens: The residual amount of Xoo in rice leaves was detected using real-time quantitative PCR (qPCR). Total DNA was extracted from the leaves and amplified using specific primers targeting the Xoo 16S rDNA gene and the rice internal reference gene OsUBQ. The absolute copy numbers of Xoo and rice genes were calculated using the standard curve method. The relative growth rate of bacterial blight pathogen was calculated using the following formula: Relative growth rate = (Xoo copy number / OsUBQ copy number) × 100%.
[0035] Results and Analysis: qPCR results showed that, compared with the control group, the relative growth of bacterial blight pathogens in rice leaves treated with E1 bacterial suspension was significantly reduced. Based on the calculation of the relative growth, the relative control efficacy of the E1 treatment group reached 65.4%. Figure 1 This result visually demonstrates the excellent control effect of strain E1 against bacterial blight on live rice.
[0036] Example 3: Gene Expression
[0037] Samples were taken 24 hours after inoculation with bacterial blight fungus suspension, and qPCR results were obtained. Figure 2 , Figure 3 , Figure 4 At 24 h, the expression of OsMAPK6 in group E1 was 3.3 times that of the control group, the expression of OsWRKY13 was 5.5 times that of the control group, and the expression of OsOPR10 was 3.6 times that of the control group (P < 0.01).
[0038] Database data reveals that OsMAPK6, OsWRKY13, and OsOPR10 are three genes playing crucial roles in rice stress resistance, respectively regulating responses to low-temperature stress and pathogen infection. The expression and regulatory effects of OsMAPK6 are context-dependent. Overexpression of OsWRKY13 reprograms signaling pathways, enabling rice to more effectively resist bacterial blight and rice blast infection, with resistance remaining stable throughout the growth cycle. Overexpression of OsOPR10 enhances rice's PTI response (such as reactive oxygen species burst), significantly increasing resistance to rice blast and bacterial blight. The verification results of this invention demonstrate that Bacillus repens E1 has a good control effect on bacterial blight and significantly enhances the expression of OsMAPK6, OsWRKY13, and OsOPR10 genes, thereby improving rice resistance to bacterial blight and providing a new approach and method for its control.
[0039] The above-described embodiments 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 type of Bacillus belesiensis E1, characterized in that, The Bacillus velezensis strain E1 was isolated from healthy rice leaves from Taojiang, Yiyang, Hunan Province. It was identified as Bacillus velezensis by 16S rRNA, gyrB, and rpoB multi-gene sequencing and is deposited at the China Microbial Culture Collection Center, accession number CGMCC No. 35057, on 2025-07-03.
2. The application of Bacillus berberis E1 as described in claim 1 in improving resistance to bacterial blight in rice.
3. The application according to claim 2, characterized in that, The Bacillus berberis E1 was used as a bacterial suspension working solution.
4. The application according to claim 3, characterized in that, The bacterial suspension working solution is prepared by: activating the bacterial strain by slant culture, then adjusting OD600 to 0.8, and adding 0.02% Tween-20 by volume to obtain the bacterial suspension working solution.
5. The application according to claim 3, characterized in that, The concentration of the bacterial suspension used was 1×10⁻⁶. 8 CFU / mL.
6. The application according to claim 2, characterized in that, The Bacillus berberis E1 strain is used for the prevention and control of diseases during the four-leaf stage of rice.
7. The application according to claim 2, characterized in that, The Bacillus belye E1 was used to activate the rice OsMAPK6-OsWRKY13-OsOPR10 immune pathway and inhibit the growth of bacterial blight pathogen.