Biological control agent for tobacco bacterial wilt

Abstract

The invention provides a biological control agent for tobacco bacterial wilt, and belongs to the technical field of tobacco disease control methods. The biological control agent is prepared by uniformly mixing Penicillium reevesii and Angelica talaromyces fermentation broth according to a mass ratio of 2: 3. The biological control agent has a strong inhibition effect on tobacco bacterial wilt, the biological control agent is applied 10 days after transplanting, the biological control agent is applied again every 15 days, and the tobacco bacterial wilt can be effectively controlled. The medicament disclosed by the invention is non-toxic, free of pathogenicity, safe to human and livestock, environment-friendly and excellent and lasting in disease-resistant effect, and has a potential application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco disease prevention and control methods, and in particular relates to a biological control agent for tobacco bacterial wilt. Background Art

[0002] Tobacco is a species of the genus Nicotiana in the Solanaceae family. It is the main tobacco crop used by humans to produce cigarettes. When Christopher Columbus first discovered the New World in 1493, the Indians of Hispaniola introduced tobacco to him. Tobacco is a native plant of tropical and subtropical America, but it is commercially grown all over the world. It is also grown as a horticultural plant or weed. Tobacco growth requires a temperature of 20 to 30 degrees Celsius, an air humidity of 80 to 85%, and a low concentration of nitrogen in the soil. Tobacco is a robust annual small-branched herb with huge leaves up to 2.5 meters tall. The whole body is covered with short, sticky glandular hairs, which secrete yellow sticky substances containing nicotine. In some tobacco-producing areas in my country, bacterial wilt is common, and some fields even have no harvest. The epidemic of bacterial wilt is becoming more and more serious, so the prevention and control of the disease should be taken seriously. In the early stage of the disease, 1 to 2 leaves can often be seen withering and drooping at noon on a sunny day, and they can recover at night. Observe the stem. There are often chlorotic stripes on the stem on the side of the wilting leaves. The root system on the side of the rotten stem rots into dark brown to black. If the wilting leaves are picked, the vascular bundles of the petioles are blackened. The infected leaves are still green and hanging on the stems at the beginning, so it is called "bacterial wilt". Since the diseased leaves often wilt on one side in the early stage, it is commonly known as "half-side madness". When the weather is hot, the leaves of the diseased plants are irregularly burned, the leaves become dry, and the edges fall off. The stems of the diseased plants are upright, and dead leaves hang on the stems. When the soil is humid, the roots become soft and sticky, causing the whole plant to die. The cross-section of the tobacco stem with stripes shows that the vascular tissue has turned brown or brown. The pith begins to rot and becomes honeycomb-like, and a cavity forms at the base of the tobacco stem. There is a sticky milky white to light brown shiny mucus at the vascular incision; sticky filaments can also be seen. To identify this disease, you can place the diseased vascular bundle piece in a container filled with light salt water. After a few minutes, you can see a milky white mist. Currently, most of the registered pesticides for bacterial wilt are chemical agents, such as thiophanate-methyl and trichloroisocyanuric acid. However, long-term use of chemical agents to prevent and control the disease can easily cause pathogens to develop drug resistance. At the same time, long-term use also causes harm to the environment. Therefore, it is urgent to find new and efficient prevention and control methods. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a biological control agent for tobacco bacterial wilt, aiming to solve the problem that most of the registered agents for tobacco bacterial wilt in the prior art are chemical agents, such as thiophanate-methyl, trichloroisocyanuric acid, etc., but long-term use of chemical agents to prevent and control diseases can easily make pathogens resistant, and long-term use also causes harm to the environment.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a biological microbial agent for preventing and treating tobacco bacterial wilt, comprising a fermentation liquid of Penicillium reiterii and a fermentation liquid of Talaromyces angelicae, wherein the mass ratio thereof is 1-3:2-4.

[0005] Preferably, the mass ratio of Penicillium rettii to Talaromyces angelicae is 2:3.

[0006] Preferably, the preparation of the biological control agent comprises the following steps: (1) Penicillium reiterii and Talaromyces angelicae were inoculated into LB solid culture medium for activation; (2) The activated Penicillium reiterii and Talaromyces angelicae were inoculated into LB liquid culture medium respectively, and cultured at 32°C and 180 r / min for 24 h to obtain seed solution; (3) The seed liquid was transferred to new LB liquid culture medium respectively, and cultured at 37°C and 180 r / min for 48 h to obtain Penicillium reiterii fermentation liquid and Talaromyces angelicae fermentation liquid; (4) The fermentation liquid of Penicillium reiterii and the fermentation liquid of Talaromyces angelicae are mixed in a mass ratio of 1-3:2-4 to obtain a biological control agent for controlling tobacco bacterial wilt.

[0007] Preferably, the number of viable bacteria in the fermentation broth of Penicillium rettii is greater than 5×10 8 cfu / mL; the number of viable bacteria in the angelica tularensis fermentation liquid is greater than 1×10 8 cfu / mL.

[0008] The invention also provides a method for using the pharmaceutical composition, wherein the biological agent is applied 10 days after the tobacco is transplanted, and then applied again after an interval of 15 days, with each application amount being 80 ml / mu.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a biological control agent for tobacco bacterial wilt. The agent prepared by the present invention can achieve effective control of tobacco bacterial wilt by application in tobacco fields, which is greatly improved compared with conventional control and can reach 83.48%, and provides a technical idea for disease control in the tobacco planting process; the present invention can reduce the use of chemical pesticides and the risk of drug resistance, protect the ecological environment, meet the requirements of green plant protection and ecological civilization construction, is safe and harmless to the environment, and has good development prospects and application potential. DETAILED DESCRIPTION

[0010] The invention provides a biological control agent for tobacco bacterial wilt, comprising a fermentation liquid of Penicillium reinhardtii and a fermentation liquid of Talaromyces angelicae, wherein the mass ratio of the fermentation liquid of Penicillium reinhardtii and Talaromyces angelicae is 1-3:2-4, wherein the mass ratio of the fermentation liquid of Penicillium reinhardtii and Talaromyces angelicae is preferably 2:3, so as to better exert the control effect of the agent.

[0011] Penicillium reiterii used in the present invention: Penicillium reiterii I stole Penicillium , isolated from the rhizosphere soil of healthy tobacco in Dandong, Liaoning Province, and deposited in the General Microbiology Center of China Microbiological Culture Collection on September 25, 2024. The address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No.41503.

[0012] The angelica tularensis used in the present invention is: Talaromyces angelicae Talaromyces angelicae , isolated from the rhizosphere soil of healthy tobacco in Dandong, Liaoning Province, and deposited in the General Microbiology Center of China Culture Collection on September 12, 2024. The address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41506.

[0013] In this embodiment, the preparation of the biological control agent includes the following steps: (1) Penicillium reiterii and Talaromyces angelicae were inoculated into LB solid culture medium for activation; (2) The activated Penicillium reiterii and Talaromyces angelicae were inoculated into LB liquid culture medium respectively, and cultured at 32°C and 180 r / min for 24 h to obtain seed solution; (3) The seed liquid was transferred to new LB liquid culture medium respectively, and cultured at 37°C and 180 r / min for 48 h to obtain Penicillium reiterii fermentation liquid and Talaromyces angelicae fermentation liquid; (4) The fermentation liquid of Penicillium reiterii and the fermentation liquid of Talaromyces angelicae are mixed in a mass ratio of 1-3:2-4 to obtain a biological control agent for controlling tobacco bacterial wilt.

[0014] In the above preparation method, the number of viable bacteria in the fermentation broth of Penicillium rettii of the present invention is greater than 5×10 8 cfu / mL; the number of viable bacteria in the angelica tularensis fermentation liquid is greater than 1×10 8 cfu / mL; In this embodiment, when biological control is carried out, the biological control agent is applied 10 days after transplanting, and then applied again after an interval of 15 days, with each application amount being 80 ml / mu.

[0015] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0016] Example 1 Control effect of control agents on tobacco bacterial wilt 1. Test materials Drugs: Penicillium reiterii, Talaromyces angelicae, ε-polylysine, Brevibacillus laterosporus, Rheinheimerella aquatica, and sterile water as blank control.

[0017] The preparation of LB liquid medium includes the following steps: 950 ml of deionized water, 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl. Use 1 M NaOH to adjust the pH value to 7.0 and adjust the volume to 1 L. After the above steps, add 15 g of agar powder to the LB solid medium, sterilize it by high pressure at 121°C for 20 min, and pour the solid medium into a plate when it cools to 55°C; the liquid medium is divided for use.

[0018] The Penicillium reiterii and Talaromyces angelicae were inoculated into LB solid culture medium for activation; the activated Penicillium reiterii and Talaromyces angelicae were inoculated into LB liquid culture medium, and cultured at 30°C and 160r / min for 36h to obtain seed solution; the seed solution was transferred to new LB liquid culture medium, and cultured at 37°C and 200r / min for 48h to obtain fermentation liquid of Penicillium reiterii and Talaromyces angelicae; the number of viable bacteria in the fermentation liquid of Penicillium reiterii was greater than 5×10 8 cfu / mL; the number of viable bacteria in the angelica tularensis fermentation liquid is greater than 1×10 8 cfu / mL can be evenly mixed to obtain the test agent.

[0019] 2. Application method The test agent, ε-polylysine, Brevibacillus laterosporus, Rheinheimerella water, and sterile water were sprayed on the leaves 10 days after tobacco transplanting, and sprayed again 15 days later. After the disease began, the disease situation was investigated, and a five-point sampling survey was adopted, with 8 plants surveyed at each point, and a total of 40 plants surveyed in each plot. The number of diseased plants, the number of diseased leaves per plant, the total number of leaves surveyed, and the disease index of each leaf were recorded. The grading standard of tobacco bacterial wilt disease index is (grading survey based on leaves, 5 leaves per plant): Level 0: The whole plant is disease-free Level 1: Occasionally there are chlorotic spots on the stem, or less than half of the leaves on the diseased side wilt Level 3: There are black streaks on the stem, but not exceeding half of the stem height, or one-half to two-thirds of the leaves on the diseased side are withered Level 5: The black streaks on the stem exceed half of the stem height but do not reach the top of the stem, or more than two-thirds of the leaves on the diseased side wilt Level 7: Black streaks on the stem reach the top of the stem, or all leaves of the diseased plant wilt Level 9: Diseased plants are basically dead Table 1 Investigation on the efficacy of tobacco bacterial wilt control As can be seen from Table 1, the present invention applies fertilizer twice after transplanting, improves the stress resistance of tobacco, and has a good control effect on tobacco bacterial wilt, with a control efficiency of up to 80.96%.

[0020] It should be noted that, unless otherwise specified, tobacco planting, fertilization and field management are all carried out in accordance with conventional cultivation methods.

[0021] Comparison of the control effect of control agent 1 and other agents on tobacco bacterial wilt in the field 1. Test materials Drugs: The control agent of Example 1, 80% Bordeaux mixture wettable powder (commercially available), 77% copper calcium sulfate wettable powder (commercially available), and sterile water were used as blank controls.

[0022] 2. Application method 10 days after tobacco transplanting, the test agent was sprayed on tobacco leaves, and the control group was sprayed with the same amount of sterile water. After 15 days, it was sprayed again, and each treatment was repeated 3 times. After the onset of disease, the disease situation was investigated according to the classification standard and relative control effect in Example 1.

[0023] Table 2 Investigation of the efficacy of different pesticides It can be seen from Table 2 that the agent provided by the present invention has achieved good prevention effect, which is greatly improved compared with conventional prevention and control. At the same time, compared with chemical pesticides, the control agent provided by the present invention has improved the stress resistance of tobacco, and is safe, non-toxic, pollution-free, and environmentally friendly. It can be used as a biological pesticide for preventing and controlling tobacco wilt disease and put into research and production.

[0024] 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.

[0025] 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 biological control agent for tobacco bacterial wilt, characterized in that: The mass ratio of the fermentation liquid of Penicillium reiterii to the fermentation liquid of Talaromyces angelicae is 1~3:2~4.

2. The biological control agent for tobacco bacterial wilt according to claim 1, characterized in that: Penicillium reiterii Penicillium raperi Deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit number is: CGMCC No.41503; Angelicae Sinensis Talaromyces angelicae The deposit is in the General Microbiological Center of China Microorganism Culture Collection Administration Committee, with the deposit number of CGMCC No.41506. Penicillium raperi Talaromyces angelicae Talaromyces angelicae The mass ratio of the fermentation broth is 2:

3.

3. A method for preparing the biological control agent for tobacco bacterial wilt according to claim 1, characterized in that: The preparation of biological control agents includes the following steps: (1) Penicillium reiterii and Talaromyces angelicae were inoculated into LB solid culture medium for activation; (2) The activated Penicillium reiterii and Talaromyces angelicae were inoculated into LB liquid culture medium respectively, and cultured at 32°C and 180 r / min for 24 h to obtain seed solution; (3) The seed liquid was transferred to new LB liquid culture medium respectively, and cultured at 37°C and 180 r / min for 48 h to obtain Penicillium reiterii fermentation liquid and Talaromyces angelicae fermentation liquid; (4) The fermentation liquid of Penicillium reiterii and the fermentation liquid of Talaromyces angelicae are mixed in a mass ratio of 1-3:2-4 to obtain a biological control agent for controlling tobacco bacterial wilt.

4. The method for preparing the biological control agent for tobacco bacterial wilt according to claim 3, characterized in that: The number of viable bacteria in the fermentation broth of Penicillium reiterii is 5×10 8 cfu / mL; the number of viable bacteria in the angelica tularensis fermentation broth is 1×10 8 cfu / mL.

5. A biological control method for tobacco bacterial wilt using a biological control agent as claimed in claim 1, characterized in that: Apply the bio-fungus agent 10 days after transplanting and apply it again after an interval of 15 days.

6. The biological control method of the biological control agent for tobacco bacterial wilt according to claim 5, characterized in that: The application amount is 80ml / mu each time.

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