Microbial agent for preventing and treating rice diseases

Abstract

The application belongs to the technical field of microbial agents, and particularly relates to a microbial agent for preventing and treating rice diseases. Achromobacter denitrificans The application also discloses application of a strain GPB1-5 in prevention and treatment of rice diseases, and the strain GPB1-5 is classified as Acidovorax facilis, and is preserved in China Center for Type Culture Collection, with a preservation number of CCTCC No: M2020344. The strain GPB1-5 can effectively inhibit growth of pathogenic bacteria of rice diseases, and has a potential to be developed into a microbial agent. In addition, the strain GPB1-5 has a synergistic effect when being compounded with aureobasidin, berberine or mycostatin, and can improve the prevention and treatment effect on rice diseases, and has an important significance for research and development of the microbial agent for preventing and treating rice diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a microbial inoculant for the prevention and control of rice diseases. Background Technology

[0002] Most bacterial diseases of rice are quarantine diseases, characterized by rapid outbreaks and severe damage. Common bacterial diseases of rice include bacterial leaf blight, bacterial leaf streak, bacterial grain blight, and bacterial basal rot. Among them, bacterial leaf streak, also known as fine streak or stripe disease, is caused by Xanthomonas oryzae var. oryzae (the pathogenic strain of Xanthomonas oryzae). Xanthomonas oryzae pv. oryzicola This disease, caused by [unspecified pathogen], is an important quarantine bacterial disease in rice production. It can occur throughout the entire growth period of rice, mainly affecting the leaves, but also the leaf sheaths. After infection, it causes leaf scorch, increased unfilled grains, and a decrease in thousand-grain weight, generally resulting in a yield reduction of 15-25%, and in severe cases, up to 40-60%.

[0003] For a long time, the main method for controlling plant diseases (including rice diseases) has been chemical control. Chemical pesticides have played a crucial role in plant disease control, but the long-term, high-dose, and irrational use of chemical pesticides has led to serious pesticide residue problems and environmental pollution. Simultaneously, pathogens have developed strong resistance under the selective pressure of long-term, high-dose chemical pesticides, resulting in a gradual decline in the effectiveness of chemical pesticides. To achieve the goals of plant disease control and sustainable development for humans and the environment, people have begun to explore environmentally friendly biopesticides, and microbial inoculants are one type of biopesticide.

[0004] The applicant's previous research found that strain GPB1-5 can achieve selenium enrichment in vegetables, increase the water-soluble selenium content in selenium-enriched soil, and passivate the heavy metal cadmium in the soil. Recently, regarding its effects on plant pathogens, the inventors also discovered that strain GPB1-5 can effectively inhibit the growth of rice pathogens, showing potential for development into a microbial agent, which is of great significance for the control of rice diseases. No related reports have been found yet.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a microbial agent for the prevention and control of rice diseases, so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Application of strain GPB1-5 in the control of rice diseases. The taxonomic name of strain GPB1-5 is Denitrifying Achromobacterium (GBB1-5). Achromobacter denitrificans (), deposited at the China Center for Type Culture Collection, accession number CCTCC No: M2020344.

[0008] This invention also provides a microbial inoculant, comprising GPB1-5 bacterial powder; the taxonomic name of the GPB1-5 strain is *Achromobacterium denitrifyingus* (GBB1-5). Achromobacter denitrificans (), deposited at the China Center for Type Culture Collection, accession number CCTCC No: M2020344; Furthermore, the effective viable count of the GPB1-5 bacterial powder is 5 × 10⁻⁶. 8 cfu / g.

[0009] Furthermore, the microbial agent is a compound of strain GPB1-5 bacterial powder and a second active ingredient; wherein the second active ingredient is chlortetracycline, berberine, or kasugamycin.

[0010] Furthermore, the mass ratio of the bacterial powder of strain GPB1-5 to chlortetracycline is 1-8:8-1.

[0011] Furthermore, the mass ratio of the strain GPB1-5 bacterial powder to berberine is 1-4:20-1.

[0012] Furthermore, the mass ratio of the strain GPB1-5 bacterial powder to streptomycin is 1-7:30-1.

[0013] The present invention also provides the application of the aforementioned microbial agent in the prevention and control of rice diseases.

[0014] Furthermore, the rice diseases mentioned include bacterial diseases of rice.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The strain GPB1-5 of the present invention can effectively inhibit the growth of rice pathogens and has the potential to be developed into a microbial agent.

[0016] (2) When the bacterial powder of strain GPB1-5 of the present invention is combined with chlortetracycline, berberine or kasugamycin, it has a synergistic effect and can improve the control effect on rice diseases. It is of great significance for the research and development of microbial agents for the control of rice diseases. Detailed Implementation

[0017] The following is a clear and complete description of the technical solution of this invention patent. Obviously, the described embodiments are some, but not all, of the embodiments of this invention.

[0018] 1. Materials and Methods 1.1 Test pathogens Xanthomonas streak pathogenic type ( Xanthomonas oryzae pv. oryzicola ), isolated from rice bacterial leaf streak disease samples from the rice experimental field of the Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences.

[0019] 1.2 Culture medium NA medium: 3g beef extract, 5g peptone, 1g yeast extract, 10g sucrose, 17g agar, 1L distilled water, pH adjusted to 7.0; NB medium: 3g beef extract, 5g peptone, 1g yeast powder, 10g sucrose, 1L distilled water, pH adjusted to 7.0; LB medium: 10g tryptone, 5g yeast extract; 10g sodium chloride, 1L distilled water, pH adjusted to 7.0; Fermentation medium: 5g tryptone, 2g yeast extract, 1.5g potassium nitrate, 0.5g magnesium sulfate, 0.1g calcium chloride, 1L distilled water, pH 7.0.

[0020] 1.3 Test reagents GPB1-5 bacterial powder: After activating strain GPB1-5, pick a single colony and inoculate it into LB liquid medium. Incubate at 30°C and 180 rpm on a shaker until the logarithmic growth phase. Add fresh LB medium to adjust the viable count to 10⁻⁶. 9 The seed culture was obtained at cfu / mL. A 10% inoculum was added to a fermenter containing fermentation medium. Fermentation was carried out at 30°C, 200 rpm, and aeration to maintain dissolved oxygen at 0.8 mg / L for 36 hours. The fermentation broth was centrifuged at 5000×g and 4°C for 15 minutes, and the cell pellet was collected. The cell pellet was resuspended in sterile physiological saline pre-cooled to 4°C, and centrifuged again at 5000×g and 4°C for 15 minutes, and the cell pellet was collected. The cell pellet was then slowly resuspended in a cryoprotectant (by mass percentage: trehalose 12%, skim milk 6%, magnesium chloride 0.15%, ascorbic acid 0.08%, mannitol 4%, and the remainder sterile water). The pellet was then freeze-dried under vacuum (-40°C, 15 Pa vacuum for 12 hours; then heated to 25°C at a rate of 5°C / h and dried under 8 Pa vacuum) until the moisture content was below 5%. The pellet was then pulverized through a 100-mesh sieve, and soluble starch was added to adjust the viable count to 5×10⁻⁶. 8 cfu / g indicates the bacterial powder of strain GPB1-5.

[0021] 94% chlortetracycline technical grade (Shanghai Nongle Biological Products Co., Ltd.), 75% berberine technical grade (Inner Mongolia Qingyuanbao Biotechnology Co., Ltd.), 98% kasugamycin technical grade (Wuhan Xinxinjiali Biotechnology Co., Ltd.) GPB1-5 bacterial powder was diluted with 0.1% Tween-80 aqueous solution to prepare single-agent stock solutions at 100% by weight. Aureomycin, berberine, and streptomycin were dissolved and then diluted with 0.1% Tween-80 aqueous solution to prepare single-agent stock solutions. Multiple formulations were set up, and each single agent and each formulation mixture was further diluted with 0.1% Tween-80 aqueous solution to create five mass concentration gradients to determine the virulence of the single agents and mixtures against the tested pathogens.

[0022] It should be noted that previous compatibility tests showed that chlortetracycline, berberine, and streptomycin had good compatibility with strain GPB1-5, so these three were selected as the compounding factors for strain GPB1-5.

[0023] 1.4 Test Methods Following the filter paper diffusion method described by Wei Haijuan et al. (2011), the test pathogens were picked and cultured in 100 mL of NB medium at 30 °C with shaking at 180 r / min for 24 h. The bacterial suspension concentration was then adjusted to 1.0 × 10⁻⁶ using a hemocytometer. 8 CFU / mL. Pour 10 mL of NA medium cooled to approximately 50°C into a 9 cm diameter petri dish, shake well, cool, and prepare a plate. Spread 50 μL of bacterial suspension evenly onto the cooled NA plate. Punch 5 mm diameter filter paper discs, sterilize them, and immerse them in the solution for 1 hour. Remove and drain excess solution. Place one filter paper disc in the center of the bacterial plate. Use a treatment soaked in 0.1% Tween-80 aqueous solution as a blank control. Each treatment is repeated three times, with three plates per replicate. After incubation at 30°C for 48 hours, measure the diameter of the inhibition zone using the cross-sectional method and calculate the inhibition rate.

[0024]

[0025] 1.5 Data Analysis The drug concentration was converted to a logarithmic value, and the corresponding inhibition rate was converted to a probability value. A regression equation for virulence and EC was calculated with the logarithmic concentration as the independent variable and the inhibition rate probability value as the dependent variable. 50 Values ​​and correlation coefficients. The co-toxicity coefficient (CTC value) of the mixture was calculated using the Sun Yunpei method, and the synergistic effect of the agents was evaluated. The results are shown in Tables 1-4.

[0026] Table 1. Results of virulence assay of single agents against the pathogen of bacterial leaf streak of rice.

[0027] Table 1 shows that the bacterial powder of strain GPB1-5 can inhibit the growth of the pathogen causing bacterial leaf streak in rice, and its EC50 content is [missing information]. 50The concentration was 42.809 mg / L, indicating its potential for development into a microbial inoculant, which is of great significance for the control of rice diseases. In addition, the EC50 values ​​of chlortetracycline, berberine, and streptomycin against the pathogen of rice bacterial leaf streak were determined. 50 The values ​​were 5.195 mg / L, 1.437 mg / L and 7.239 mg / L, respectively.

[0028] Table 2. Virulence test results of GPB1-5 bacterial powder combined with chlortetracycline against the pathogen of rice bacterial leaf streak.

[0029] As shown in Table 2, within a mass ratio of 1-8:8-1, the combination of GPB1-5 bacterial powder and aureomycin exhibits a co-toxicity coefficient greater than 120 in inhibiting the growth of rice bacterial leaf streak pathogen, demonstrating a synergistic effect and improving the control of rice diseases.

[0030] Table 3. Virulence test results of GPB1-5 bacterial powder combined with berberine on the pathogen of rice bacterial leaf streak.

[0031] As shown in Table 3, within a mass ratio of 1-4:20-1, the co-toxicity coefficient of the combination of strain GPB1-5 powder and berberine in inhibiting the growth of rice bacterial leaf streak pathogen is greater than 120, showing a synergistic effect and improving the control effect on rice diseases.

[0032] Table 4. Virulence test results of GPB1-5 bacterial powder combined with streptomycin on the pathogen of bacterial leaf streak of rice.

[0033] As shown in Table 4, within a mass ratio of 1-7:30-1, the combination of GPB1-5 bacterial powder and streptomycin showed a co-toxicity coefficient of greater than 120 in inhibiting the growth of rice bacterial leaf streak pathogen, indicating a synergistic effect and improving the control of rice diseases.

[0034] In summary, the strain GPB1-5 of this invention can effectively inhibit the growth of rice pathogens and has the potential to be developed into a microbial agent. Furthermore, the GPB1-5 bacterial powder exhibits a synergistic effect when combined with chlortetracycline, berberine, or streptomycin, which can improve the control effect against rice diseases. This is of great significance for the research and development of microbial agents for the control of rice diseases.

Claims

1. The application of a strain GPB1-5 in the control of rice diseases, characterized in that, The strain GPB1-5 is taxonomically named Denitrifying Achromobacterium (GPB1-5). Achromobacter denitrificans (), deposited at the China Center for Type Culture Collection, accession number CCTCC No: M2020344.

2. A microbial inoculant, characterized in that, The microbial agent includes GPB1-5 bacterial powder; the taxonomic name of the strain GPB1-5 is *Achromobacter denitrifying bacteria* (GPB1-5). Achromobacter denitrificans (), deposited at the China Center for Type Culture Collection, accession number CCTCC No: M2020344.

3. The microbial agent according to claim 2, characterized in that, The effective viable count of the GPB1-5 bacterial powder was 5 × 10⁻⁶. 8 cfu / g.

4. The microbial agent according to claim 2, characterized in that, The microbial agent is a compound of strain GPB1-5 bacterial powder and a second active ingredient; wherein the second active ingredient is chlortetracycline, berberine or kasugamycin.

5. The microbial agent according to claim 4, characterized in that, The mass ratio of the strain GPB1-5 bacterial powder to chlortetracycline is 1-8:8-1.

6. The microbial agent according to claim 4, characterized in that, The mass ratio of the strain GPB1-5 bacterial powder to berberine is 1-4:20-1.

7. The microbial agent according to claim 4, characterized in that, The mass ratio of the strain GPB1-5 bacterial powder to the streptomycin compound is 1-7:30-1.

8. The application of the microbial agent according to any one of claims 2-7 in the prevention and control of rice diseases.

9. The application according to claim 8, characterized in that, The rice diseases mentioned include bacterial diseases of rice.

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