A biocontrol agent for controlling tobacco angular leaf spot, its preparation method and application

Abstract

This invention relates to the field of tobacco disease control methods, specifically disclosing a biocontrol agent for controlling tobacco angular leaf spot, its preparation method, and its application. The biocontrol agent is composed of a mixture of *Streptomyces flavus* fermentation broth and *Snipoma fasciatus* fermentation broth. The active ingredient in the *Streptomyces flavus* fermentation broth is *Streptomyces flavus* 168 (CGMCC No. 15355); the active ingredient in the *Snipoma fasciatus* fermentation broth is *Snipoma fasciatus* Sneb2006 (CGMCC No. 28087). The biocontrol agent of this invention is ideal for controlling tobacco angular leaf spot, exhibiting multiple effects including induction, prevention, and treatment, with significant synergistic effects. It is safe for humans and animals, environmentally friendly, highly resistant, and possesses stable and long-lasting antibacterial activity, showing promising potential applications.

Description

Technical Field

[0001] This invention relates to the field of tobacco disease control methods, specifically to a biocontrol agent for controlling tobacco angular leaf spot, its preparation method, and its application. Background Technology

[0002] The pathogen of tobacco angular leaf spot is *Pseudomonas syringa* epv. tabaci, belonging to the genus *Pseudomonas*. It is a common bacterial leaf spot disease in all tobacco-growing areas and often occurs in combination with wildfire disease. Except for a few tobacco-growing areas in Sichuan and Shandong provinces, the damage is generally mild. Tobacco bacterial angular leaf spot mostly occurs in the later stages of growth, mainly affecting leaves, capsules, sepals, and stems. Infected leaves develop polygonal to irregular dark brown spots with distinct edges and no obvious yellow halo. The color of the spots varies considerably, ranging from dark brown to brown at the edges and yellowish-brown or milky white in the center. Multiple concentric rings are often observed in the spots. The lower leaves of the tobacco plant tend to have more spots. Under humid conditions, pus oozes from the spots, forming a mucous film. When dry, the spots rupture and fall off. Leaves with many spots turn completely yellow and wither, losing their harvesting and curing value.

[0003] Tobacco angular leaf spot pathogens can overwinter on seeds or in diseased plant debris remaining in the soil, becoming the primary source of infection for the following year. Planting diseased seedlings is one of the main causes of disease outbreaks in the field. The pathogen can enter through wounds and natural pores. After infection in the field, the pathogen is mainly spread by wind and rain, causing reinfection. The activities of insects, humans, and livestock can also play a role in spreading the pathogen. Under suitable conditions, the incubation period of the pathogen is only 4 days. In southern tobacco-growing areas, the disease begins to occur in May-June, and in northern tobacco-growing areas in July-August, with major outbreaks often occurring after storms. High humidity is required for pathogen invasion, and rain splashing is necessary for spread. Extensive waterlogging of leaves, creating a water-soaked appearance, facilitates rapid disease expansion, a phenomenon often observed after storms; this is called "rain-induced waterlogging." During the day, stomata are open, making leaves prone to waterlogging. A high-nitrogen, low-potassium nutrient system also increases leaf waterlogging and reduces the tobacco plant's resistance, thus significantly impacting disease development. Overcrowding in tobacco planting leads to poor ventilation and light penetration, and excessive moisture in the field. During storms, leaves easily rub against each other, creating numerous wounds that facilitate the invasion and spread of pathogens. Pruning too early or too late also hinders the plant's normal nutrient utilization and exacerbates disease. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a biocontrol agent for preventing and controlling tobacco angular leaf spot, its preparation method, and its application, aiming to solve the problem of tobacco angular leaf spot easily developing on tobacco plants after storms.

[0005] The technical solution of the present invention is as follows:

[0006] Firstly, a biocontrol agent for preventing and controlling tobacco angular leaf spot disease, wherein the biocontrol agent is composed of a mixture of Streptomyces flavus fermentation broth and Streptomyces adhering to the surface fermentation broth;

[0007] The ratio of the fermentation broth of Streptomyces flavus to the fermentation broth of Streptomyces obliterans is 8-10:6-8.

[0008] The active ingredient in the Streptomyces flavus fermentation broth is Streptomyces flavus 168, with the preservation number CGMCC No. 15355;

[0009] The active ingredient in the *Sneb. viscous* fermentation broth is *Sneb. viscous* 2006, with the preservation number CGMCC No. 28087.

[0010] Furthermore, the viable count of Streptomyces flavus 168 in the biocontrol agent is not less than 1 x 10^6. 8 cfu / mL; viable count of *Sneb. 2006* cfu / mL; not less than 5 × 10⁻⁶. 8 cfu / mL.

[0011] The second aspect is a method for preparing a biocontrol agent for preventing and controlling tobacco angular leaf spot, comprising:

[0012] Step 1: Prepare the fermentation broth of Streptomyces flavus and Streptomyces adhesiveus separately;

[0013] Step 2: Mix the fermentation broth of Streptomyces flavus and the fermentation broth of Streptomyces adhering to the surface at a mass ratio of 8-10:6-8 to obtain the biocontrol agent.

[0014] Furthermore, the preparation of the fermentation broth of Streptomyces flavus includes the following steps:

[0015] S1: Activate and culture Streptomyces flavus in a solid culture medium. After 24 hours of culture at 28°C, inoculate the seed culture into liquid Gao's medium.

[0016] S2: The seed culture prepared in S1 was inoculated into a new liquid culture medium at a 5% inoculation rate and cultured at 28℃ and 140r / min for 48h to obtain the fermentation broth.

[0017] Furthermore, in S1 and S2, the culture medium used to activate the strains was Gao's medium.

[0018] Furthermore, the preparation of the fermentation broth of *Strombus aegyptium* includes the following steps:

[0019] S1: The *Strombus aegyptiacus* was inoculated into a solid culture medium for activation and culture at 32°C for 24 hours. After that, it was inoculated into a liquid culture medium to obtain the seed culture.

[0020] S2: The seed liquid prepared in S1 was inoculated into a new liquid culture medium at an inoculation rate of 10%, and cultured at 32℃ and 150r / min for 48h to obtain the fermentation broth.

[0021] Furthermore, in S1 and S2, the culture medium used to activate the strains was NA medium.

[0022] Thirdly, the application of biocontrol agents, characterized in that the biocontrol agents are used to prevent tobacco angular leaf spot, and the tobacco angular leaf spot is caused by infection with the pathogenic species of Pseudomonas syringae. When using them, they can be applied from transplanting to maturity. The effect is best when used before and at the early stage of disease. The dosage is 100 mL / mu each time.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The biocontrol agent prepared by this invention can inhibit the growth of Pseudomonas syringae pathogenic species in tobacco, providing technical support and theoretical basis for the prevention and control of tobacco angular leaf spot;

[0025] 2. The biocontrol agent prepared by this invention can not only prevent tobacco angular leaf spot caused by *Pseudomonas syringae* pathogenic species, but also induce disease resistance in tobacco, enhancing its control effect against tobacco angular leaf spot. The two bacteria can enhance each other's inhibitory effect against *Pseudomonas syringae* pathogenic species.

[0026] 3. The effective components of the biocontrol agent prepared by this invention are Streptomyces flavus 168 and Streptomyces obliterans Sneb2006. Currently, no strains of these strains have been found to be used in the prevention and control of tobacco angular leaf spot, but this invention has discovered a new use for them in the prevention and control of tobacco angular leaf spot.

[0027] The biocontrol agent is composed of a mixture of fermentation broths of *Streptomyces flavus* and *Syngonium fasciatus*. The active ingredient in the *Streptomyces flavus* fermentation broth is *Streptomyces flavus* 168, with preservation number CGMCC No. 15355; the active ingredient in the *Syngonium fasciatus* fermentation broth is *Syngonium fasciatus* Sneb2006, with preservation number CGMCC No. 28087. This biocontrol agent can inhibit the growth of *Pseudomonas syringae* pathogenic species in tobacco, providing technical support and theoretical basis for the prevention and control of tobacco angular leaf spot disease caused by infection with *Pseudomonas syringae* pathogenic species in tobacco. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0029] The biocontrol agent for controlling tobacco angular leaf spot according to the present invention is composed of a mixture of *Streptomyces flavus* fermentation broth and *S. aegyptium* fermentation broth; the mass ratio of *Streptomyces flavus* fermentation broth to *S. aegyptium* fermentation broth is 8-10:6-8; the active ingredient of the *Streptomyces flavus* fermentation broth is *Streptomyces flavus* 168, with preservation number CGMCC No. 15355; the active ingredient of the *S. aegyptium* fermentation broth is *S. aegyptium* Sneb2006, with preservation number CGMCC No. 28087.

[0030] Example 1

[0031] This embodiment provides a method for preparing a biocontrol agent for preventing and controlling tobacco angular leaf spot.

[0032] I. Preparation of bacterial strains

[0033] 1. Test strains

[0034] Streptomyces microflavus var. liaoningensis, strain 168, was isolated, purified, and deposited in soil on February 12, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 15355.

[0035] Sneb2006 Ensifer adhaerens was deposited on August 2, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28087.

[0036] Gao's medium: 20g soluble starch, 0.5g NaCl (sodium chloride), 1g KNO3 (potassium nitrate), 0.5g K2HPO4 (potassium dihydrogen phosphate), 0.5g MgSO4·7H2O (magnesium sulfate heptahydrate), 0.01g FeSO4·7H2O (ferrous sulfate heptahydrate), 15-20g agar (agar is omitted for liquid medium), 1000mL water, pH 7.4-7.6, sterilized at 121℃ for 20min.

[0037] NA medium: 3g beef extract, 10g peptone, 5g NaCl, 18g agar (agar is omitted for liquid medium), 1000mL water, pH 7.4, sterilized at 121℃ for 20min.

[0038] 2. Preparation of biocontrol agents

[0039] The preparation of fermentation broth for Streptomyces flavus includes the following steps:

[0040] S1: Activate and culture Streptomyces flavus in a solid culture medium. After 24 hours of culture at 28°C, inoculate the seed culture into liquid Gao's medium.

[0041] S2: The seed culture prepared in S1 was inoculated into a new liquid culture medium at a 5% inoculation rate and cultured at 28℃ and 140r / min for 48h to obtain the fermentation broth.

[0042] The preparation of fermentation broth for *Strombus amygdalinus* includes the following steps:

[0043] S1: The *Strombus aegyptiacus* was inoculated into a solid culture medium for activation and culture at 32°C for 24 hours. After that, it was inoculated into a liquid culture medium to obtain the seed culture.

[0044] S2: The seed liquid prepared in S1 was inoculated into a new liquid culture medium at an inoculation rate of 10%, and cultured at 32℃ and 150r / min for 48h to obtain the fermentation broth.

[0045] The viable count of Streptomyces flavus 168 is not less than 1 x 10. 8 cfu / mL; viable count of *Sneb. 2006* cfu / mL; not less than 5 × 10⁻⁶. 8 cfu / mL.

[0046] The Streptomyces flavus fermentation broth and the Streptomyces adhesiveus fermentation broth were mixed evenly at a mass ratio of 10:6.

[0047] Example 2

[0048] This embodiment provides an investigation into the effectiveness of a biocontrol agent in controlling the pathogen causing tobacco angular leaf spot.

[0049] Test reagents: biocontrol agent from Example 1, Streptomyces flavus fermentation broth, Streptomyces adhesiveus fermentation broth, thiabendazole copper (positive control), and sterile water (blank control).

[0050] Experimental method: The inhibition zone method was used to detect the inhibitory effect of biocontrol agents on Pseudomonas syringae.

[0051] After sterilizing the NA solid medium, wait until its temperature drops below 42℃, then add the pre-activated *Pseudomonas syringae* bacterial suspension to the medium, mix thoroughly, and pour into agar plates for later use. Place a sterilized Oxford cup (6 mm inner diameter) in the agar plate, add 100 μL of the test reagent and sterile water, and incubate for 30 h. Measure the size of the inhibition zone. Calculate the inhibition rate using the following formula.

[0052] Relative inhibition rate = (diameter of treatment inhibition zone - diameter of control zone) / diameter of treatment inhibition zone × 100%

[0053] Experimental results:

[0054] Table 1. Antibacterial effect of the biocontrol agent prepared in this invention against *Pseudomonas syringae*.

[0055] Test reagents Diameter of the inhibition zone (cm) Relative antibacterial rate (%) Biocontrol agents 3.3 72.73 Streptomyces flavus fermentation broth 2.7 66.67 Adhesive sword fungus fermentation broth 3.0 70.00 Thiamethoxam 2.8 67.86 sterile water 0.9 -

[0056] As shown in Table 1, the antibacterial rate of the biocontrol agent in this invention reaches 72.73%, which is higher than that of applying Streptomyces flavus and Streptomyces fasciatus fermentation broth alone. Meanwhile, the antibacterial rate of thiabendazole copper is 67.86%, indicating that the biocontrol agent has a very good antibacterial effect on Pseudomonas syringae.

[0057] Example 3

[0058] This embodiment provides a field efficacy test of a biocontrol agent for controlling tobacco angular leaf spot.

[0059] Test reagents: biocontrol agent from Example 1, fludioxonil (positive control), and sterile water (blank control).

[0060] Experimental method: The tobacco variety selected was Liaoyan No. 15, and the amount of tobacco used was 10m³. 2 As one group, 20 groups were sprayed with the treatment solution before the tobacco disease appeared, with a spraying rate of 100 mL / mu, and 4 replicates, designated as treatment A; another 20 groups were sprayed with the treatment solution at the initial stage of disease, with a spraying rate of 100 mL / mu, and 4 replicates, designated as treatment B. The control efficacy was calculated based on the disease index after 20 days.

[0061] Disease grading standards:

[0062] Grade 0: All leaves are disease-free;

[0063] Grade 1: The area of ​​lesions accounts for less than 1% of the leaf area;

[0064] Grade 3: Lesions cover 1% to 5% of the leaf area;

[0065] Level 5: Lesions cover 5% to 10% of the leaf area;

[0066] Level 7: Lesions cover 10%–20% of the leaf area;

[0067] Level 9: The lesion area accounts for more than 20% of the leaf area.

[0068]

[0069]

[0070] Experimental results:

[0071] In this experiment, the biocontrol agent showed a preventive and therapeutic effect of 80.70% against tobacco angular leaf spot, which was much higher than the effect of the positive control thiabendazole copper. This indicates that the fermentation broth of Streptomyces flavus and Streptomyces adoxoides has a high control effect on tobacco angular leaf spot pathogens. At the same time, after the drug was applied at the early stage of the disease, the biocontrol agent also had a good control effect of 75.73%, which was higher than the control effect of thiabendazole copper.

[0072] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A biocontrol agent for preventing and controlling tobacco angular leaf spot, characterized in that, The biocontrol agent is composed of a mixture of Streptomyces flavus fermentation broth and Streptomyces adhesiveus fermentation broth. The fermentation broth of Streptomyces flavus and the fermentation broth of Streptomyces adhesiveus are used in a mass ratio of 8~10:6~8. The active ingredient in the Streptomyces flavus fermentation broth is Streptomyces flavus 168, with the preservation number CGMCC No. 15355; The active ingredient in the fermentation broth of *Sneb. 2006* is *Sneb. 2006*, with the preservation number CGMCC No. 28087. The viable count of Streptomyces flavus 168 in the biocontrol agent is not less than 1 × 10⁻⁶. 8 cfu / mL; viable count of *Sneb. 2006* cfu / mL; not less than 5 × 10⁻⁶. 8 cfu / mL.

2. A method for preparing a biocontrol agent for controlling tobacco angular leaf spot as described in claim 1, characterized in that, include: Step 1: Prepare the fermentation broth of Streptomyces flavus and Streptomyces adhesiveus separately; Step 2: Mix the fermentation broth of Streptomyces flavus and the fermentation broth of Streptomyces adhering to the surface at a mass ratio of 8~10:6~8 to obtain the biocontrol agent.

3. The method for preparing the biocontrol agent for controlling tobacco angular leaf spot according to claim 2, characterized in that, The preparation of fermentation broth for Streptomyces flavus includes the following steps: S1: Activate and culture Streptomyces flavus in a solid culture medium. After 24 hours of culture at 28°C, inoculate the seed culture into liquid Gao's medium. S2: The seed culture prepared in S1 was inoculated into a new liquid culture medium at a 5% inoculation rate and cultured at 28℃ and 140 r / min for 48 h to obtain the fermentation broth.

4. The method for preparing the biocontrol agent for controlling tobacco angular leaf spot according to claim 3, characterized in that, In S1 and S2, the culture medium used to activate the strains was Gao's medium.

5. The method for preparing the biocontrol agent for controlling tobacco angular leaf spot according to claim 2, characterized in that, The preparation of fermentation broth for *Strombus amygdalinus* includes the following steps: S1: The *Strombus aegyptiacus* was inoculated into a solid culture medium for activation and culture at 32°C for 24 hours. After that, it was inoculated into a liquid culture medium to obtain the seed culture. S2: The seed culture prepared in S1 was inoculated into a new liquid culture medium at an inoculation rate of 10%, and cultured at 32℃ and 150r / min for 48h to obtain the fermentation broth.

6. The method for preparing the biocontrol agent for controlling tobacco angular leaf spot according to claim 5, characterized in that, In S1 and S2, the culture medium used to activate the strains was NA medium.

7. The application of the biocontrol agent for controlling tobacco angular leaf spot as described in claim 1, characterized in that, The biocontrol agent is used to control tobacco angular leaf spot, which is caused by infection with the tobacco pathogenic species *Pseudomonas syringae*.

8. The application of the biocontrol agent for controlling tobacco angular leaf spot according to claim 7, characterized in that, It can be applied from transplanting to maturity. The best effect is achieved when applied before and at the early stage of disease. The dosage is 100 mL / acre each time.

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