Biocontrol inoculant of bacillus megaterium Sneb753 for inducing rice to resist false smut and banded sclerotial blight and application of biocontrol inoculant

Abstract

The invention relates to a biocontrol inoculant of bacillus megaterium Sneb753 for inducing rice to resist false smut and banded sclerotial blight and application of the biocontrol inoculant, and belongs to the technical field of microbial pesticides. The bacterial strain involved in the invention is bacillus megaterium Sneb753, which has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 16, 2024, and the preservation number is CGMCC No.33086. The invention also relates to a preparation method of the bacillus megaterium Sneb753. The bacillus megaterium Sneb753 can be prepared into a biocontrol inoculant which is used for inducing rice to generate rice false smut and rice sheath blight disease resistance through seed treatment or spraying, the rice false smut and the rice sheath blight disease of the rice can be effectively prevented and treated, and the growth of the rice can be promoted by treating the rice through fermentation liquor of the strain or an extracted metabolite; the bactericidal composition has a good application prospect in the aspects of preventing and treating rice false smut and banded sclerotial blight of rice.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial pesticides, and particularly relates to a biocontrol agent of Bacillus megaterium Sneb753 for inducing rice resistance to rice false smut and sheath blight, and application thereof. Background Art

[0002] Rice false smut is a fungal disease of rice caused by Ustilaginoidea virens, which occurs in rice-producing areas all over the world. In severe cases, the incidence rate can reach 30%, causing a 20% to 30% reduction in yield, which is manifested as an increase in the rate of blighted rice and a decrease in 1000-grain weight. More seriously, the six fungal toxins it produces, such as fusarium rust and smut, are highly toxic to mammals, causing organ lesions and affecting reproductive function. Rice sheath blight is a disease caused by Rhizoctonia solani, which generally causes 10% to 20% losses. It is particularly serious in densely planted and high-yield fields, with a loss rate of more than 50%, which is manifested as leaf death, reduced fruit set rate, and reduced 1000-grain weight.

[0003] The prevention and control methods for the two diseases are mainly chemical prevention and breeding of disease-resistant varieties. The use of chemical fungicides can easily cause pathogens to develop drug resistance, pollute the environment, and endanger the ecological balance; there are still many problems to be studied and solved in the combined application of biological agents and chemical agents. If seed treatment can effectively control rice false smut and rice sheath blight, it will be a good method with high efficiency, low cost and no pollution with great application prospects. Therefore, it is of great significance to use bacterial fermentation liquid to induce rice to prevent and control rice false smut and rice sheath blight. Summary of the invention

[0004] In view of the above shortcomings, the purpose of the present invention is to provide a biocontrol agent of Bacillus megaterium Sneb753 that induces rice resistance to rice false smut and rice sheath blight and its application. Taking the induced immunity to rice false smut and rice sheath blight as the standard, the bacterial fermentation broth with the ability to induce infection of two pathogenic fungi, U. virens and R. solani, is sought from the strain storage of the Northern Nematode Research Institute of Shenyang Agricultural University, in order to find new resources for the research and development of new biological control of two rice fungal diseases.

[0005] The technical solution adopted by the invention is: a bacterium Bacillus megaterium Sneb753, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration in Beijing, with a deposit number of CGMCC No.33086, a deposit date of December 16, 2024, and a Latin name of Bacillus megaterium.

[0006] A biological control bacterial agent comprising the Bacillus megaterium Sneb753.

[0007] Application of biological control agents in controlling rice false smut and rice sheath blight.

[0008] A method for preparing a metabolite fermentation liquid, the technical key points of which are that the test tube strain of Bacillus megaterium Sneb753 is activated, inoculated into a liquid fermentation medium for fermentation culture, and the metabolite fermentation liquid is obtained after fermentation culture for 3 days.

[0009] Furthermore, the method for preparing the metabolite fermentation broth is characterized in that the formula of the liquid fermentation medium per liter is: the formula of the fermentation medium is: 10-15g of tryptone, 1-5g of yeast extract, 5-10g of sodium chloride, 17-20g of agar, and distilled water is adjusted to 1L, and sterilized at 121°C for 30min.

[0010] A metabolite fermentation liquid, the technical key point of which is that it is prepared according to a metabolite fermentation liquid preparation method.

[0011] Application of metabolite fermentation liquid in preventing and controlling rice false smut and rice sheath blight.

[0012] A seed coating agent containing microbial metabolites, the technical key point of which is that it contains the fermentation liquid of the metabolites.

[0013] The seed coating or seed dressing is used in inducing rice to produce resistance to rice false smut and rice sheath blight.

[0014] The beneficial effects of the present invention are as follows: the biocontrol agent of Bacillus megaterium Sneb753 for inducing rice resistance to false smut and sheath blight and its application can be prepared into the biocontrol agent, metabolite fermentation liquid and seed coating for preventing and controlling false smut and sheath blight, can effectively prevent and control the infection of false smut (U. virens) and sheath blight (R. solani), has a significant effect, can promote plant growth, and has a good application prospect in preventing and controlling false smut and sheath blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a colony morphology diagram of Bacillus megaterium Sneb753 in the present invention;

[0017] Figure 2This is a phylogenetic tree of the 16S rDNA sequence of Bacillus megaterium Sneb753 compared with its closely related species;

[0018] Figure 3 The growth of rice seeds coated with the fermentation liquid of Bacillus megaterium Sneb753 in a potted experiment 40 days after sowing;

[0019] Figure 4 The growth of rice seeds coated with the fermentation liquid of Bacillus megaterium Sneb753 in a potted experiment 60 days after sowing;

[0020] Figure 5 The growth of rice seeds coated with the fermentation liquid of Bacillus megaterium Sneb753 was shown in Figure 120 (the Sneb196 group was a negative control that inhibited growth and rice resistance). DETAILED DESCRIPTION

[0021] To make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the following is a brief description of the present invention in conjunction with the attached Figure 1-Figure 5 The present invention is further described in detail with reference to the accompanying drawings and specific embodiments.

[0022] Embodiment 1:

[0023] The present embodiment provides a Bacillus megaterium Sneb753 that has induced resistance to rice and can prevent and control rice false smut and rice sheath blight. The colony morphology, gene structure characteristics, and potted plant and field test effects of the Bacillus megaterium Sneb753 of the present invention are described below in combination with relevant experimental data.

[0024] 1. Colony morphology and microbiological characteristics

[0025] The colony morphology of Bacillus megaterium Sneb753 is as follows Figure 1 shown.

[0026] After culturing for 24 hours in the dark on LB solid medium, the diameter of the colonies on the nutrient-rich medium can reach 3.0 μm or larger; the surface is moist and flat, light yellow, opaque and sticky.

[0027] The culture conditions of the Bacillus megaterium Sneb753 are as follows:

[0028] Lysing broth agar medium (LB) solid medium (1L) formula: tryptone 10g, yeast extract 5g, sodium chloride 10g, agar 17-20g, distilled water 1L, sterilized at 121℃ for 30min. Natural pH.

[0029] Slant culture method: 28 。 C for 24 h.

[0030] 2. Physiological and biochemical characteristics

[0031] Bacillus megaterium Sneb753 is Gram-positive early and is an aerobic bacterium. Other characteristics are shown in Table 1 below:

[0032] Table 1 Physiological and biochemical characteristics of strain Sneb753

[0033]

[0034] 3. Gene structure characteristics

[0035] Figure 2 shown.

[0036] Embodiment 2:

[0037] This embodiment also provides a method for preparing metabolites for preventing and controlling rice false smut and rice sheath blight using Bacillus megaterium Sneb753.

[0038] The method is specifically as follows: after activating the test tube strain of Bacillus megaterium Sneb753, inoculate it into a 250ml triangular flask (each flask contains 150ml LB liquid fermentation medium) for culturing, the culturing conditions are: 28°C, 150r / min shaking fermentation for 3 days, and obtain metabolites with induced resistance to rice false smut and rice sheath blight. The formula of each liter of the LB solid culture medium (1L) is: the formula of the fermentation medium is: 10g of tryptone, 1g of yeast extract, 5g of sodium chloride, 17g of agar, 1L of distilled water, and sterilized at 121°C for 30min.

[0039] After the test tube strain of Bacillus megaterium Sneb753 was activated, it was inoculated into a 250 mL triangular flask (each bottle contained 150 mL LB liquid fermentation medium) for culture. The culture conditions were as follows: 28-32°C, 150-160 r / min shaking fermentation for 3 days, the bacterial solution was centrifuged and the supernatant was removed, resuspended with sterile water, and the OD value was adjusted to 1.0 with a spectrophotometer. The concentration of the bacterial suspension was 1X10 8 cfu / mL, and metabolites with induced resistance to rice false smut and rice sheath blight were obtained.

[0040] Embodiment 3:

[0041] The difference between this embodiment and embodiment 2 is that after the test tube strain of Bacillus megaterium Sneb753 is activated, it is inoculated into a 250 ml triangular flask (each bottle contains 150 ml LB liquid fermentation medium) for culture, and the culture conditions are: 32°C, 160r / min shaking fermentation for 3 days, and metabolites with induced resistance to rice false smut and rice sheath blight are obtained. The formula of the LB solid culture medium (1L) per liter: the formula of the fermentation medium is: 15g tryptone, 5g yeast extract, 10g sodium chloride, 20g agar, 1L distilled water, and sterilized at 121°C for 30min.

[0042] Embodiment 4:

[0043] Experiment on preparing metabolites of Bacillus megaterium Sneb753 to prevent and control rice false smut and rice sheath blight.

[0044] 1. Preparation of test preparations

[0045] The fermentation liquid of metabolites obtained from the prevention and control of rice false smut and rice sheath blight prepared in the above “IV” is set aside.

[0046] 2. Tested rice varieties and pathogenic fungi

[0047] The rice variety tested was Nipponbare.

[0048] The tested pathogen strains were provided by the College of Plant Protection, Shenyang Agricultural University.

[0049] 3. Effect of indoor strain Sneb753 on rice growth

[0050] Use 1.0% sodium hypochlorite solution to disinfect rice seeds for 3-5 minutes, then rinse with sterile water for 3-5 times, repeat 3 times; use the reserved bacterial solution to treat the seeds at a ratio of 1:20-50 (ml / g) of fermentation solution volume to rice seed weight, treat in the dark at 28℃ for 24-36 hours, and air dry. Take a small sealed bag and write the corresponding number, add 10-15ml of sterile water, sprinkle the treated seeds in, soak under light conditions for 24-36 hours, until the seeds absorb water and swell, use the sterile water treatment group as the control, and set up 3 biological replicates for each treatment.

[0051] Direct seeding of indoor potted plants: Mix the sterilized soil and the substrate for seedling cultivation in a volume ratio of 1:1 and set aside. Prepare a 15L bucket, put 10L of the reserved mixed soil in each bucket, and add an appropriate amount of water until the soil is fully hydrated (water can seep out by pressing hard). Dig a small hole with a diameter of about 2.5cm and a depth of about 2.5cm in the center of each bucket, slowly and evenly put an appropriate amount of soaked rice seeds in each hole, and gently cover the hole with 2cm of mixed soil with dry soil. Starting from germination, the plant height, aboveground fresh weight and underground fresh weight of rice were investigated at 20d, 40d and 60d respectively.

[0052] The data results for 20 days are shown in Table 2:

[0053] Table 2 Statistical survey on potted effect of indoor Bacillus megaterium Sneb753 at 20 days

[0054]

[0055] The growth status of the plants at 40 days is as follows Figure 3 ; The data results are shown in Table 3:

[0056] Table 3 Statistical survey on potted effect of indoor Bacillus megaterium Sneb753 at 40 days

[0057]

[0058] The growth status of the plants at 60 days is as follows Figure 4 ; The data results are shown in Table 4:

[0059] Table 4 Statistical survey on potted effect of indoor Bacillus megaterium Sneb753 at 60 days

[0060]

[0061] From the statistical survey results of potted plant effects in Tables 2 to 4, it can be seen that the plant height, aboveground fresh weight and underground fresh weight of rice plants after the seed coating treatment of strain Sneb753 were significantly higher than those of the blank control at 20d, 40d and 60d. The results show that Sneb753 can promote the growth of rice plants in the early stage.

[0062] 4. Field efficacy test of strain Sneb753 against rice false smut and rice sheath blight

[0063] In the rice experimental field of the College of Agriculture, Lunan Campus, Shenyang Agricultural University, at 41°49′N, 123°34′E, field experiments were conducted on May 5, 2023 and April 27, 2024, respectively, for preliminary screening of biocontrol bacteria and re-verification of growth promotion and control effects. The experiments were designed in randomized blocks. In both experiments, rice seeds treated with sterile water were used as blank controls. A total of 1,470 treatments were used in the preliminary screening, with 1 group for each treatment and 3 biological replicates for each group. After investigation and analysis, it was found that seeds coated with fermentation liquid of 12 bacterial strains such as Sneb753 can stimulate rice resistance to rice false smut and rice sheath blight; these 12 bacterial strains were used to obtain fermentation liquid for seed treatment again during re-verification, with 8 groups for each treatment and 3 biological replicates for each group. Field plot design: Field direct seeding was performed in the reserved moist field with a row spacing of 15 cm and a column spacing of 20 cm. The hole diameter was about 2.5 cm and the depth was about 2.5 cm. Slowly and evenly place 40 to 45 soaked rice seeds in each hole, gently smooth the hole, wait for germination, and set up protection rows around the plot. Field management is carried out in the conventional way of rice fields, and the inoculation of rice false smut and rice sheath blight will be completed on August 5, 2023 and July 31, 2024, respectively. According to the standards of rice false smut disease index grading (grade 0: healthy, no bent fruits; grade 1: 1 bent fruit per panicle; grade 2: 2 bent fruits per panicle; grade 3: 3-5 bent fruits per panicle; grade 4: 6-9 bent fruits per panicle; grade 5: 10 or more bent fruits per panicle) and rice sheath blight disease index grading (grade 0: healthy, the whole plant is disease-free; grade 1: the fourth leaf blade and all the following leaf sheaths and leaves are diseased; grade 3: the third leaf blade and all the following leaf sheaths and leaves are diseased; grade 5: the second leaf blade and all the following leaf sheaths and leaves are diseased; grade 7: the flag leaf and the following leaf sheaths are diseased; grade 9: the whole plant is diseased, or even dead), 5 rice plants were randomly selected from each treatment at maturity, and the disease indexes of rice false smut and rice sheath blight were investigated and analyzed.

[0064] The data results for 120d in 2023 are shown in Table 5:

[0065] Table 5 Statistical survey on the field effect of 120d Bacillus megaterium Sneb753 in 2023

[0066]

[0067] The data results for 120d in 2024 are shown in Table 6:

[0068] Table 6 Statistical survey on the field effect of 120d Bacillus megaterium Sneb753 in 2024

[0069]

[0070] The field effect test of Sneb753 on controlling rice false smut and rice sheath blight is shown in Table 7:

[0071] Table 7 Disease index of rice false smut and rice sheath blight in each fermentation liquid treatment group at 120 days

[0072]

[0073] The results in Tables 5 and 6 show that in the two-year field trial, the plant height, aboveground fresh weight, underground fresh weight and 100-grain weight of rice plants after Sneb753 fermentation liquid seed coating treatment were significantly higher than those of the blank control at 120 days. The results in Table 7 show that in terms of the incidence of rice false smut and rice sheath blight in the field, the disease indexes of rice false smut and rice sheath blight treated with strain Sneb753 seeds were 1.67 and 9.72, respectively, which were reduced by 88.64% and 66.68% compared with the blank control. Plant status Figure 5 The test results show that the strain Sneb753 can induce resistance in rice and reduce the infection of rice false smut and rice sheath blight.

[0074] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A bacterium Bacillus megaterium Sneb753, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, Beijing, with a deposit number of CGMCC No.33086, a deposit date of December 16, 2024, and a Latin name of Bacillus megaterium .

2. A biological control agent comprising the Bacillus megaterium Sneb753 according to claim 1.

3. The biological control agent according to claim 2 is used to control rice false smut ( Ustilaginoidea virens ) and rice sheath blight ( Rhizoctonia solani ) in applications.

4. A method for preparing a metabolite fermentation broth, characterized in that: The test tube strain of Bacillus megaterium Sneb753 according to claim 1 is activated and then inoculated into a liquid fermentation medium for fermentation culture, and a fermentation liquid of metabolites is obtained after the fermentation culture.

5. The method for preparing a metabolite fermentation broth according to claim 4, characterized in that: The formula of the liquid fermentation medium per liter is: the formula of the fermentation medium is: 10-15g of tryptone, 1-5g of yeast extract, 5-10g of sodium chloride, 17-20g of agar, distilled water to 1L, and sterilized at 121°C for 30 min.

6. A metabolite fermentation broth, characterized in that: Prepared according to the method of claim 4.

7. Use of the metabolite fermentation liquid as claimed in claim 6 in preventing and controlling rice false smut and rice sheath blight.

8. A seed coating agent containing microbial metabolites, characterized in that: A fermentation broth comprising the metabolite product according to claim 6.

9. Use of the seed coating or seed dressing as claimed in claim 8 in inducing resistance of rice to rice false smut and rice sheath blight.

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