Biocontrol microbial inoculum for preventing and treating angular leaf spot of tobacco as well as preparation method and application of biocontrol microbial inoculum

Abstract

The invention relates to the field of tobacco disease control methods, and particularly discloses a biocontrol microbial inoculum for controlling tobacco angular leaf spot and a preparation method and application thereof.The biocontrol microbial inoculum is formed by mixing streptomyces microflavus fermentation liquor and ensifer adhesion fermentation liquor, the active ingredient of the streptomyces microflavus fermentation liquor is streptomyces microflavus 168, and the preservation number of the streptomyces microflavus fermentation liquor is CGMCC No.15355; the active ingredient of the ensifer mucilaginosus fermentation liquor is ensifer mucilaginosus Sneb2006, and the preservation number of the ensifer mucilaginosus Sneb2006 is The biocontrol inoculant provided by the invention is very ideal for preventing and treating angular leaf spot of tobacco, has multiple effects of induction, prevention and treatment, and has a remarkable synergistic effect; the compound is safe to human and livestock, environment-friendly, high in stress resistance and stable and lasting in bacteriostatic activity, and has a potential application prospect.

Description

Technical Field

[0001] The invention relates to the field of tobacco disease prevention and control methods, and in particular to a biocontrol agent for preventing and controlling tobacco angular spot disease, and a preparation method and application thereof. Background Art

[0002] The pathogen of tobacco angular leaf spot is Pseudomonas syringaepv.tabaci, which belongs to the genus Pseudomonas. It is one of the common bacterial leaf spot diseases in tobacco-growing areas, and often occurs in combination with wildfire. Except for individual tobacco-growing areas in Sichuan and Shandong provinces, the damage is generally mild. Bacterial angular leaf spot of tobacco often occurs in the late growth period, mainly harming leaves, capsules, sepals, stems, etc. When the leaves are infected, polygonal to irregular black-brown lesions are produced on the leaves, with obvious edges and no obvious yellow halo around them. The color of the lesions also varies greatly, dark brown or brown on the edges and yellow-brown or milky white in the center, and the lesions often have multiple ring patterns. There are often more lesions on the leaves at the bottom of the tobacco plant. Under humid conditions, pus appears on the lesions and a film is formed. When dry, the lesions break and fall off. Leaves with more lesions turn yellow and lose their value for roasting.

[0003] Tobacco angular leaf spot pathogens can overwinter on seeds or on diseased residues remaining in the soil, becoming the primary source of infection the following year. Planting diseased seedlings is one of the main causes of field disease. The pathogen can invade through wounds and natural openings. After the disease occurs 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. The disease begins to occur in the southern tobacco areas in May-June and in the northern tobacco areas in July-August, and the pandemic often occurs after a storm. The pathogen needs high humidity to invade people, and it needs rain splashing to spread. Any large area of ​​leaves filled with water to form waterlogging is conducive to the rapid spread of the disease, and this situation occurs after a storm. This phenomenon is called "wind and rain water filling". The pores are open during the day, and the leaves are easily filled with water. The high nitrogen and low potassium nutritional status can also increase the degree of water filling of the leaves and lead to a decrease in the disease resistance of tobacco plants, which has an important impact on the development of the disease. The density of tobacco planting is too high, the ventilation and light transmission are poor, and the field is too humid. In case of stormy weather, the leaves are easy to rub against each other, causing a large number of wounds, which is conducive to the invasion and spread of pathogens. Topping too early or too late is not conducive to the normal utilization of nutrients in the plant and will aggravate the degree of disease. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a biocontrol agent for preventing and treating tobacco angular spot disease, and a preparation method and application thereof, aiming to solve the problem that tobacco angular spot disease is easily produced on tobacco plants after a storm.

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

[0006] In the first aspect, a biocontrol agent for preventing and treating tobacco angular spot disease is prepared by mixing a fermentation liquid of Streptomyces leucoderma and a fermentation liquid of Xanthomonas australis;

[0007] The dosage ratio of the fermentation liquid of Streptomyces tenuifolius to the fermentation liquid of Xanthophyllales adhering to is 8-10:6-8;

[0008] The active ingredient of the Streptomyces leucophylla fermentation broth is Streptomyces leucophylla 168, and its deposit number is CGMCC No.15355;

[0009] The active ingredient of the fermentation liquid of the sticky sword fungus is the sticky sword fungus Sneb2006, and the preservation number is CGMCC No.28087.

[0010] Furthermore, the number of viable bacteria of Streptomyces leucoderma 168 in the biocontrol agent is not less than 1 x 10 8 cfu / mL; the number of viable bacteria of Sneb2006 is not less than 5×10 8 cfu / mL.

[0011] In a second aspect, a method for preparing a biocontrol agent for preventing and treating tobacco angular spot disease comprises:

[0012] Step 1, respectively preparing a fermentation broth of Streptomyces tenuifolius and a fermentation broth of Xanthophyllales adhering to the streptomyces;

[0013] Step 2: uniformly mix the fermentation liquid of Streptomyces tenuifolius and the fermentation liquid of Xanthophyllales adhering to a mass ratio of 8-10:6-8 to obtain a biocontrol agent.

[0014] Furthermore, the preparation of the fermentation broth of Streptomyces tenuifolius comprises the following steps:

[0015] S1: The Streptomyces tenuifolius was activated and cultured in a solid culture medium at 28°C for 24 hours before being inoculated into a liquid Gould's medium to obtain a seed solution;

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

[0017] Furthermore, in S1 and S2, the culture medium used for activating the strain is Gottlieb's medium.

[0018] Furthermore, the preparation of the fermentation liquid of the sticky sword fungus comprises the following steps:

[0019] S1: Inoculate the sticky sword fungus into a solid culture medium for activation culture at 32°C, and after 24 hours, inoculate into a liquid culture medium to obtain a seed solution;

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

[0021] Furthermore, in S1 and S2, the culture medium used for activating the strain is NA medium.

[0022] The third aspect is the application of the biocontrol agent, which is characterized in that the biocontrol agent is used to prevent tobacco angular spot disease, and the tobacco angular spot disease is caused by infection by Pseudomonas syringae pv. tobacco. When in use, it can be applied from transplanting to maturity, and the effect is best when used before and in the early stage of the disease, and the dosage is 100 mL / mu each time.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The biocontrol agent prepared by the present invention can inhibit the growth of tobacco pathogenic variants of Pseudomonas syringae, providing technical support and theoretical basis for the prevention and treatment of tobacco angular spot disease;

[0025] 2. The biocontrol agent prepared by the present invention can not only prevent tobacco angular spot disease caused by Pseudomonas syringae pv. tobacco, but also induce tobacco to produce disease resistance, thereby enhancing the prevention and treatment of tobacco angular spot disease. The two bacteria can enhance each other's antibacterial effect on Pseudomonas syringae pv. tobacco.

[0026] 3. The active ingredients of the biocontrol agent prepared by the present invention are Streptomyces tenuifolius 168 and Sneb2006. Currently, the above strains have not been found to be useful for the prevention and treatment of tobacco angular spot disease, but the present invention has discovered a new use for the prevention and treatment of tobacco angular spot disease.

[0027] The biocontrol agent is prepared by mixing the fermentation broth of Streptomyces septifolium and Xylotrichum truncatum, wherein the active ingredient of the fermentation broth of Streptomyces septifolium is Streptomyces septifolium 168, with a preservation number of CGMCC No. 15355; the active ingredient of the fermentation broth of Xylotrichum truncatum is Xylotrichum truncatum Sneb2006, with a preservation number of CGMCC No. 28087. The biocontrol agent can inhibit the growth of tobacco pathogenic variants of Pseudomonas syringae, and provides technical support and theoretical basis for the prevention and treatment of tobacco angular spot disease caused by infection of tobacco pathogenic variants of Pseudomonas syringae. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0029] The biocontrol agent for preventing and treating tobacco angular spot disease of the present invention is prepared by mixing a fermentation liquid of Streptomyces leucodermi and a fermentation liquid of Xanthophyta glutinosus; the mass dosage ratio of the fermentation liquid of Streptomyces leucodermi and the fermentation liquid of Xanthophyta glutinosus is 8-10:6-8; the active ingredient of the fermentation liquid of Streptomyces leucodermi is Streptomyces leucodermi 168, and its preservation number is CGMCC No. 15355; the active ingredient of the fermentation liquid of Xanthophyta glutinosus is Sneb 2006, and its preservation number is CGMCC No. 28087.

[0030] Example 1

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

[0032] 1. Strain Preparation

[0033] 1. Test strains

[0034] 168 Streptomyces microflavus var.liaoningensis, isolated and purified from soil on February 12, 2018 and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration. The address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain deposit number is: CGMCC No.15355;

[0035] Sneb2006 Ensifer adhaerens was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 2, 2023. The depository address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain deposit number is CGMCC No. 28087.

[0036] Gao's medium: soluble starch 20g, NaCl (sodium chloride) 0.5g, KNO3 (potassium nitrate) 1g, K2HPO4 (potassium dihydrogen phosphate) 0.5g, MgSO4·7H2O (magnesium sulfate heptahydrate) 0.5g, FeSO4·7H2O (ferrous sulfate heptahydrate) 0.01g, agar 15-20g (agar is not added for liquid culture medium), water 1000mL, pH 7.4-7.6, sterilization at 121℃ for 20min.

[0037] NA medium: 3 g beef extract, 10 g peptone, 5 g NaCl, 18 g agar (no agar for liquid culture medium), 1000 mL water, pH 7.4, sterilization at 121°C for 20 min.

[0038] 2. Preparation of biocontrol agents

[0039] The preparation of the fermentation broth of Streptomyces tenuiflavus comprises the following steps:

[0040] S1: The Streptomyces tenuifolius was activated and cultured in a solid culture medium at 28°C for 24 hours before being inoculated into a liquid Gould's medium to obtain a seed solution;

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

[0042] The preparation of the fermentation liquid of the sticky sword fungus comprises the following steps:

[0043] S1: Inoculate the sticky sword fungus into a solid culture medium for activation culture at 32°C, and after 24 hours, inoculate into a liquid culture medium to obtain a seed solution;

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

[0045] The viable count of Streptomyces tenuifolius 168 is not less than 1 x 10 8 cfu / mL; the number of viable bacteria of Sneb2006 is not less than 5×10 8 cfu / mL.

[0046] The fermentation liquid of Streptomyces tenuifolius and the fermentation liquid of Xanthoceras adhering were uniformly mixed at a mass ratio of 10:6.

[0047] Example 2

[0048] This example provides a survey on the effectiveness of a biocontrol agent in controlling tobacco angular leaf spot pathogens.

[0049] Test agents: the biocontrol agent in Example 1, fermentation broth of Streptomyces gracilis, fermentation broth of Xanthophyllales, thiophanate-methyl (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°C, pour the pre-activated Pseudomonas syringae liquid into the medium, mix evenly and pour into the plate for later use. Place a sterilized Oxford cup (inner diameter 6mm) in the plate, inject 100μL of the test agent and sterile water into the cup, and measure the size of the inhibition zone after culturing for 30h. Calculate the inhibition rate using the following formula.

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

[0053] Experimental results:

[0054] Table 1 Antibacterial effect of the biocontrol agent prepared by the present invention on Pseudomonas syringae

[0055] Test drug Diameter of inhibition zone (cm) Relative antibacterial rate (%) Biocontrol agents 3.3 72.73 Streptomyces leucoflavus fermentation broth 2.7 66.67 Fermentation broth of Xanthoceras adherans 3.0 70.00 Thiophanate 2.8 67.86 Sterile water 0.9 -

[0056] As can be seen from Table 1, the antibacterial rate of the biocontrol agent of the present invention reaches 72.73%, which is higher than the control effect of applying the fermentation broth of Streptomyces tenuifolius and Xanthophyllales adhering alone. At the same time, the antibacterial rate of thiophanate-methyl is 67.86%, indicating that the antibacterial effect of the biocontrol agent on Pseudomonas syringae is very considerable.

[0057] Example 3

[0058] This example provides a biocontrol agent for determining the efficacy of controlling tobacco angular leaf spot in the field.

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

[0060] Test method: Select the tobacco variety Liaoyan No. 15, every 10m 2 As a group, 20 groups were sprayed with the treatment solution before the tobacco disease occurred, with a spraying amount of 100 mL / mu and 4 replicates, which was set as treatment A; another 20 groups were sprayed with the treatment solution at the early stage of the disease, with a spraying amount of 100 mL / mu and 4 replicates, which was set as treatment B. The control effect was calculated based on the disease index 20 days later.

[0061] Disease classification standards:

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

[0063] Level 1: The lesion area accounts for less than 1% of the leaf area;

[0064] Level 3: The lesion area accounts for 1% to 5% of the leaf area;

[0065] Level 5: The lesion area accounts for 5% to 10% of the leaf area;

[0066] Level 7: The lesion area accounts for 10% to 20% of the leaf area;

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

[0068]

[0069]

[0070] Test results:

[0071] In this experiment, the preventive and therapeutic effect of the biocontrol agent on tobacco angular leaf spot was as high as 80.70%, which was much higher than the control effect of the positive control thiophanate-copper, indicating that the fermentation broth of Streptomyces tenuifolius and the fermentation broth of Xanthophyllales citrate had a high preventive and therapeutic effect on tobacco angular leaf spot pathogen; at the same time, after using the drug in the early stage of the disease, the biocontrol agent also had a good preventive effect, reaching 75.73%, which was higher than the preventive effect of thiophanate-copper.

[0072] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0073] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A biocontrol agent for preventing and treating tobacco angular spot disease, characterized in that: The biocontrol agent is prepared by mixing the fermentation liquid of Streptomyces leucoderma and the fermentation liquid of Xanthomonas aconitifolius. The mass usage ratio of the fermentation broth of Streptomyces tenuifolius and the fermentation broth of Xanthophyllales adhering to is 8-10:6-8; The active ingredient of the Streptomyces leucophylla fermentation broth is Streptomyces leucophylla 168, and its deposit number is CGMCC No. 15355; The active ingredient of the fermentation liquid of the sticky sword fungus is the sticky sword fungus Sneb2006, and the preservation number is CGMCC No.28087.

2. The biocontrol agent for preventing and treating tobacco angular spot disease according to claim 1, characterized in that: The number of viable bacteria of Streptomyces leucophylla 168 in the biocontrol agent is not less than 1 x 10 8 cfu / mL; the number of viable bacteria of Sneb2006 is not less than 5×10 8 cfu / mL.

3. A method for preparing the biocontrol agent for preventing and treating tobacco angular spot disease according to claim 1, characterized in that: include: Step 1, respectively preparing a fermentation broth of Streptomyces tenuifolius and a fermentation broth of Xanthophyllales adhering to the streptomyces; Step 2: Evenly mix the fermentation broth of Streptomyces tenuifolius and the fermentation broth of Xanthophyllales adhering to a mass ratio of 8-10:6-8 to obtain a biocontrol agent.

4. The method for preparing the biocontrol agent for preventing and treating tobacco angular spot disease according to claim 3, characterized in that: The preparation of the fermentation broth of Streptomyces tenuiflavus comprises the following steps: S1: The Streptomyces tenuifolius was activated and cultured in a solid culture medium at 28°C for 24 hours before being inoculated into a liquid Gould's medium to obtain a seed solution; S2: The seed solution prepared in S1 was inoculated into a new liquid culture medium at a 5% inoculation rate, and cultured at 28°C and 140 r / min for 48 h to obtain a fermentation liquid.

5. The method for preparing the biocontrol agent for preventing and treating tobacco angular spot disease according to claim 4, characterized in that: In S1 and S2, the culture medium used for activating the strains was Gottlieb's medium.

6. The method for preparing the biocontrol agent for preventing and treating tobacco angular spot disease according to claim 3, characterized in that: The preparation of the fermentation liquid of the sticky sword fungus comprises the following steps: S1: Inoculate the sticky sword fungus into a solid culture medium for activation culture at 32°C, and after 24 hours, inoculate into a liquid culture medium to obtain a seed solution; S2: The seed solution prepared in S1 was inoculated into a new liquid culture medium at an inoculation rate of 10%, and cultured at 32° C. and 150 r / min for 48 h to obtain a fermentation liquid.

7. The method for preparing the biocontrol agent for preventing and treating tobacco angular spot disease according to claim 6, characterized in that: In S1 and S2, the medium used for activating the strains was NA medium.

8. Use of the biocontrol agent for preventing and treating tobacco angular spot disease according to claim 1, characterized in that: The biocontrol agent is used for preventing and treating tobacco angular spot disease, and the tobacco angular spot disease is caused by infection of Pseudomonas syringae pv. tobacco.

9. The use of the biocontrol agent for preventing and treating tobacco angular spot disease according to claim 8, characterized in that: It can be applied from transplanting to maturity. It is best to use it before and in the early stage of disease. The dosage is 100mL / mu each time.

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