A microbial agent for preventing and treating tobacco bacterial wilt and preparation method thereof

Abstract

The present invention discloses a microbial agent for preventing and treating tobacco bacterial wilt and a preparation method thereof, and belongs to the field of microbial technology. The microbial agent of the present invention comprises a composite microbial fermentation product and a plant resistance promoter, and the mass ratio of the two is (30-50):0.1. The present invention screens a strain of calcoacetic acinetobacter CDWB36, and after mixing with Pseudomonas fluorescens in equal proportions and fermenting, the two show a significant synergistic effect, and the inhibitory effect on Ralstonia solanacearum is significantly enhanced; at the same time, the activity of the main defense enzyme of tobacco leaves is increased, thereby inducing tobacco to produce systemic resistance, and fundamentally enhancing the disease resistance of tobacco plants; the present invention adds naringenin and oligosaccharide to form a plant resistance promoter, and the two work together to effectively improve tobacco resistance. The microbial agent of the present invention has good effect in preventing and treating tobacco bacterial wilt, strong pertinence, no pollution, can be stored for a long time after being made into a microbial agent, is convenient for field application, and has potential practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a microbial agent for preventing and treating tobacco bacterial wilt and a preparation method thereof. Background Art

[0002] Tobacco is one of the important economic crops, with the largest planting area and yield in the world. However, tobacco diseases have a serious impact on the yield and quality of tobacco leaves. Among them, tobacco bacterial wilt is a common and important disease. The disease is not easy to be detected in the early stage of infecting the tobacco root system. It is easy to break out in a large area under high temperature and high humidity environment, causing significant economic losses. In recent years, due to large-scale continuous cropping, large-scale use of chemical pesticides and abnormal climate, the occurrence of tobacco bacterial wilt has increased year by year and has spread northward, becoming an important limiting factor for the high-quality production of tobacco leaves, seriously affecting the stable development and economic benefits of the tobacco industry.

[0003] Tobacco bacterial wilt is caused by infection with Ralstonia solanacearum, and the main primary source of infection is soil. The pathogen overwinters in soil or compost, and is spread by agricultural operations and insects, causing the roots of tobacco to turn black and rot, the stems to become hollow, and the pith to rot in a honeycomb shape, leading to the death of the entire plant. At present, the prevention and control measures for tobacco bacterial wilt are mainly chemical agents, and organochlorine, inorganic copper, and organic copper fungicides are mostly used, such as chlorobromoisocyanuric acid, basic copper sulfate, and thiophanate-methyl. However, the pathogen has a wide host range and strong survival ability, making it difficult to prevent and control. In addition, the long-term use of chemical pesticides can easily cause problems such as pesticide residues, imbalance of soil microorganisms, and resistance of pathogens. Biological control has become a research hotspot because of its green, high efficiency, and low resistance to drugs.

[0004] For example, Chinese patent application CN201610551285.7 discloses a method for preparing a composite bacterial preparation for preventing tobacco bacterial wilt: (1) Activate Acetobacter xylinum, Lactobacillus acidophilus, Bacillus megaterium, and Bacillus laterosporus respectively, and culture them on a slant; (2) Mix the activated microorganisms and put them into a triangular flask, place them in a shaker, and culture them at 28°C for 2 days, control the pH at 6.0-6.5, and make the bacterial count reach 500-600 million / ml, so as to obtain a microbial agent. The step (2) is preferably: the ratio of the number of microbial species is Acetobacter xylinum: Lactobacillus acidophilus: Bacillus megaterium: Bacillus laterosporus, which is 1:1.2-1.5:1.5-2.2:0.8.

[0005] The current technical solutions for microbial control of tobacco bacterial wilt are mostly to use a combination of multiple different microbial strains, which have a broad-spectrum antibacterial effect, but poor targeting and are difficult to effectively inhibit the occurrence of tobacco bacterial wilt. Therefore, how to develop an efficient microbial agent for tobacco bacterial wilt is a technical problem that needs to be solved urgently. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a composite high-efficiency tobacco bacterial wilt antagonist, which can effectively inhibit Ralstonia solanacearum on the one hand, and improve crop resistance on the other hand. Under the dual effects, the occurrence of tobacco bacterial wilt is effectively inhibited, and tobacco yield and quality are improved.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] A microbial agent for preventing and controlling tobacco bacterial wilt comprises a composite microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is (30-50):0.1.

[0009] Furthermore, the composite microbial fermentation product is Acinetobacter calcoaceticus CDWB36 ( Acinetobacter caicoaceticus ) and Pseudomonas fluorescens ( Pseudomonas fluorescens ) is obtained by spray drying the mixed fermentation broth; the preservation number of the calcoacetic Acinetobacter is CCTCC No.M2023964, which is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is June 8, 2023; the strain number of the fluorescing Pseudomonas is CGMCC No.1.15059, which was purchased from the General Microbiology Center of China National Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The Acinetobacter calcoaceticus CDWB36 of the present invention is isolated from the river sludge of Yihe Road, Linyi City, Shandong Province, and the isolation method is:

[0011] Use gradient dilution method and spread on NA medium to isolate bacteria in soil. Weigh 1g of dry sludge sample in 10mL of sterile water to prepare soil suspension, shake and culture for 30min, and then let stand for 30min. Dilute it to 100 times with sterile water to obtain soil dilution. Pipette 100μL and evenly spread it on the bacterial culture medium, invert it in a 28-30℃ incubator and culture it for 24h, then observe the growth of bacteria. Pick single colonies of different shapes, sizes, and colors and shake and culture them in NB liquid culture medium at 28-30℃ for 24h. Streak the cultured bacterial liquid on the bacterial culture medium plate and place it in a 28℃ incubator. After a single colony grows, streak culture is performed multiple times to obtain a pure culture of bacteria and preserve the strain.

[0012] Inoculate the spores of tobacco bacterial wilt pathogen into sterile water and shake to prepare a spore suspension. Evenly spread the spore suspension on the solid culture medium, and then use the point-grafting method to inoculate the screened test strain. Culture it at 30°C for 5 days, observe whether there is an inhibition zone, and use the test strain with an inhibition zone as the primary screening strain. During the rescreening, take a 6mm diameter bacterial block from the edge of the colony of the grass bacterial wilt pathogen that has been cultured for 5 days, move it to the middle of a blank PDA plate, and then inoculate the primary screening strain 6cm from the center. Each culture dish can be inoculated with two primary screening strains and cultured at 30°C for 5 days. Continuously observe and record the colony size and inhibition zone width of each primary screening strain. Repeat 3 times, and finally calculate the inhibition rate. The strain with the strongest antibacterial activity was finally screened out as calcoacetic acid Acinetobacter CDWB36. Figure 1 The schematic diagram of the colony morphology of Acinetobacter calcoaceticus CDWB36 is shown. The colony surface is moist and opaque, the colony is round, and the edges are irregular.

[0013] The fluorescent Pseudomonas of the present invention can be purchased through the strain catalog of the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, without the need for repeated biological preservation.

[0014] Furthermore, the preparation method of the composite microbial fermentation product is:

[0015] (1) Strain activation: The frozen Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens strains were inoculated into flasks containing 25 mL of LB liquid culture medium and incubated at 30°C and 200 r / min. -1 The activation was completed by culturing for 12 h under the conditions;

[0016] (2) Seed liquid preparation: The activated Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 After 30 h of cultivation, seed solutions of the two bacteria were obtained;

[0017] (3) Preparation of fermentation broth: The seed liquid of Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 Cultivate to OD 600 The standard seed solution of the two bacteria was obtained by setting the OD 600 The standard seed liquid of the two bacteria with a volume ratio of 1:1 was mixed and then inoculated into LB liquid medium at a volume fraction of 1%, and the mixture was inoculated at 30°C and 160 r·min. -1 After culturing for 36 hours, the composite microbial fermentation broth is obtained, and the composite microbial fermentation broth is spray-dried to obtain the composite microbial fermentation product.

[0018] Furthermore, the plant resistance promoter is obtained by mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1.

[0019] A method for preparing a microbial agent for preventing and treating tobacco bacterial wilt comprises the following preparation steps:

[0020] (1) preparing composite microbial fermentation products;

[0021] (2) mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1 to obtain a plant resistance promoter;

[0022] (3) The composite microbial fermentation product obtained in step (1) and the plant resistance promoter obtained in step (2) are mixed in a mass ratio of (30-50):0.1, and auxiliary materials are added to prepare granules.

[0023] Furthermore, in step (3), the auxiliary materials also include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, the plant resistance promoter, sodium dodecyl sulfate, starch, and kaolin is (30-50):0.1:0.1:5:15.

[0024] The biological bacterial agent of the present invention has an effective live bacterial count of ≥500 million / g and an application amount of 3-5g / strain.

[0025] Beneficial effects:

[0026] (1) The present invention screens a strain of Acinetobacter calcoaceticus CDWB36, which has a strong antagonistic effect on various plant pathogens such as tobacco bacterial wilt, and has a good inhibitory effect on the pathogen of tobacco bacterial wilt - Ralstonia solanacearum; at the same time, the selected Pseudomonas fluorescens, after being mixed with Acinetobacter calcoaceticus CDWB36 in equal proportions and fermented, the two exhibit a significant synergistic effect, and the inhibitory effect on Ralstonia solanacearum is significantly enhanced; at the same time, the Acinetobacter calcoaceticus screened by the present invention has a high efficiency in inhibiting bacteria, and after being used as a biocontrol agent to treat tobacco, the activity of the main defense enzymes in tobacco leaves is increased, thereby inducing tobacco to produce systemic resistance, thereby fundamentally enhancing the disease resistance of tobacco plants;

[0027] (2) Secondly, the present invention adds naringenin and galacto-oligosaccharide to form a plant resistance promoter. Naringenin can induce the deposition of hydrogen peroxide in leaves, causing active oxygen to burst and transmit the induction signal to the leaves, thereby improving crop resistance. At the same time, galacto-oligosaccharide is added. Galacto-oligosaccharide can stimulate the proliferation of active microorganisms, promote the growth of beneficial bacteria and inhibit harmful bacteria. The two work together to effectively improve tobacco resistance.

[0028] (3) The bacterial agent of the present invention has good effect in preventing and controlling tobacco bacterial wilt, is highly targeted, and is pollution-free. After being prepared into a bacterial agent, it can be stored for a long time, is convenient for field application, and has potential practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the colony morphology of Acinetobacter calcoaceticus CDWB36 of the present invention;

[0030] Figure 2 Figure 1 is a diagram showing the antibacterial effects of different fermentation broths of the present invention, wherein A is the microbial fermentation broth obtained in Example 1, B is the microbial fermentation broth obtained in Comparative Example 1, and C is the microbial fermentation broth obtained in Comparative Example 2;

[0031] Figure 3 This is a graph showing the disease index and control efficacy results of field experiments;

[0032] Figure 4 This is a graph showing the yield results of field experiments. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0034] Example 1

[0035] A microbial agent for preventing and treating tobacco bacterial wilt comprises a composite microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is 30:0.1.

[0036] The composite microbial fermentation product is Acinetobacter calcoaceticus CDWB36 ( Acinetobacter caicoaceticus ) and Pseudomonas fluorescens ( Pseudomonas fluorescens ) and the mixed fermentation liquid was spray-dried to obtain the mixture; the preservation number of the calcoacetic Acinetobacter is CCTCC No.M2023964, which is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; the strain number of the fluorescing Pseudomonas is CGMCC No.1.15059, which was purchased from the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Acinetobacter calcoaceticus CDWB36 was isolated from the sludge in the Yihe Road river in Linyi City, Shandong Province. The isolation method was as follows:

[0038] Use gradient dilution method and spread on NA medium to isolate bacteria in soil. Weigh 1g of dry sludge sample in 10mL of sterile water to prepare soil suspension, shake and culture for 30min, and then let stand for 30min. Dilute it to 100 times with sterile water to obtain soil dilution. Pipette 100μL and evenly spread it on the bacterial culture medium, invert it in a 28-30℃ incubator and culture it for 24h, then observe the growth of bacteria. Pick single colonies of different shapes, sizes, and colors and shake and culture them in NB liquid culture medium at 28-30℃ for 24h. Streak the cultured bacterial liquid on the bacterial culture medium plate and place it in a 28℃ incubator. After a single colony grows, streak culture is performed multiple times to obtain a pure culture of bacteria and preserve the strain.

[0039] The spores of tobacco bacterial wilt pathogen were inoculated into sterile water and shaken to prepare a spore suspension. The spore suspension was evenly spread on the solid culture medium, and then the selected strains were inoculated by the point method. The strains were cultured at 30°C for 5 days, and the inhibition zone was observed. The strains with inhibition zones were used as the primary screening strains. During the rescreening, a 6mm diameter bacterial block was taken from the edge of the colony of the grass bacterial wilt pathogen that had been cultured for 5 days, and it was moved to the middle of a blank PDA plate. Then, the primary screening strains were inoculated 6cm from the center. Two primary screening strains could be inoculated in each culture dish, and cultured at 30°C for 5 days. The colony size and the width of the inhibition zone of each primary screening strain were continuously observed and recorded. Repeated 3 times, and the inhibition rate was finally calculated. The strain with the strongest antibacterial activity was finally screened as Calcium Acinetobacter CDWB36. Pseudomonas fluorescens can be purchased through the strain catalog of the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, without the need for repeated biological preservation.

[0040] The preparation method of the composite microbial fermentation product is as follows:

[0041] (1) Strain activation: The frozen Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens strains were inoculated into flasks containing 25 mL of LB liquid culture medium and incubated at 30°C and 200 r / min. -1 The activation was completed by culturing for 12 h under the conditions;

[0042] (2) Seed liquid preparation: The activated Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 After 30 h of cultivation, seed solutions of the two bacteria were obtained;

[0043] (3) Preparation of fermentation broth: The seed liquid of Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 Cultivate to OD 600The standard seed solution of the two bacteria was obtained by setting the OD 600 The standard seed liquid of the two bacteria with a volume ratio of 1:1 was mixed and then inoculated into LB liquid medium at a volume fraction of 1%, and the mixture was inoculated at 30°C and 160 r·min. -1 After culturing for 36 hours, the composite microbial fermentation broth is obtained, and the composite microbial fermentation broth is spray-dried to obtain the composite microbial fermentation product.

[0044] The plant resistance promoter is obtained by mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1.

[0045] A method for preparing a microbial agent for preventing and treating tobacco bacterial wilt comprises the following preparation steps:

[0046] (1) preparing composite microbial fermentation products;

[0047] (2) mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1 to obtain a plant resistance promoter;

[0048] (3) The composite microbial fermentation product obtained in step (1) and the plant resistance promoter are mixed in a mass ratio of 30:0.1, and auxiliary materials are added to prepare granules.

[0049] The auxiliary materials in step (3) also include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, the plant resistance promoter, sodium dodecyl sulfate, starch, and kaolin is 30:0.1:0.1:5:15.

[0050] Example 2

[0051] A microbial agent for preventing and treating tobacco bacterial wilt comprises a composite microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is 40:0.1.

[0052] The composite microbial fermentation product is Acinetobacter calcoaceticus CDWB36 ( Acinetobacter caicoaceticus ) and Pseudomonas fluorescens ( Pseudomonas fluorescens ) and the mixed fermentation liquid was spray-dried; the preservation number of the calcoacetic Acinetobacter is CCTCC No.M2023964, which is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; the strain number of the fluorescing Pseudomonas is CGMCC No.1.15059, which was purchased from the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0053] In this example, Acinetobacter calcoaceticus CDWB36 was isolated from the sludge in the Yihe Road river in Linyi City, Shandong Province, and the isolation method was the same as that in Example 1.

[0054] The preparation method of the composite microbial fermentation product is the same as that in Example 1.

[0055] The plant resistance promoter is obtained by mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1.

[0056] A method for preparing a microbial agent for preventing and treating tobacco bacterial wilt comprises the following preparation steps:

[0057] (1) preparing composite microbial fermentation products;

[0058] (2) mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1 to obtain a plant resistance promoter;

[0059] (3) The composite microbial fermentation product obtained in step (1) and the plant resistance promoter are mixed in a mass ratio of 40:0.1, and auxiliary materials are added to prepare granules.

[0060] The auxiliary materials in step (3) also include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, the plant resistance promoter, sodium dodecyl sulfate, starch, and kaolin is 40:0.1:0.1:5:15.

[0061] Example 3

[0062] A microbial agent for preventing and treating tobacco bacterial wilt comprises a composite microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is 50:0.1.

[0063] The composite microbial fermentation product is obtained by spray drying a mixed fermentation liquid of Acinetobacter caicoaceticus CDWB36 and Pseudomonas fluorescens; the preservation number of the Acinetobacter caicoaceticus is CCTCC No.M2023964, which is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; the strain number of the Pseudomonas fluorescens is CGMCC No.1.15059, which is purchased from the General Microbiology Center of the China National Committee for the Preservation of Microbiological Cultures, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0064] In this example, Acinetobacter calcoaceticus CDWB36 was isolated from the sludge in the Yihe Road river in Linyi City, Shandong Province, and the isolation method was the same as that in Example 1.

[0065] The preparation method of the composite microbial fermentation product is the same as that in Example 1.

[0066] The plant resistance promoter is obtained by mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1.

[0067] A method for preparing a microbial agent for preventing and treating tobacco bacterial wilt comprises the following preparation steps:

[0068] (1) preparing composite microbial fermentation products;

[0069] (2) mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1 to obtain a plant resistance promoter;

[0070] (3) The composite microbial fermentation product obtained in step (1) and the plant resistance promoter are mixed in a mass ratio of 50:0.1, and auxiliary materials are added to prepare granules.

[0071] The auxiliary materials in step (3) also include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, the plant resistance promoter, sodium dodecyl sulfate, starch, and kaolin is 50:0.1:0.1:5:15.

[0072] Comparative Example 1

[0073] A microbial agent for preventing and treating tobacco bacterial wilt comprises a microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is 30:0.1.

[0074] The microbial fermentation product is Acinetobacter calcoaceticus CDWB36 ( Acinetobacter caicoaceticus ) is obtained by spray drying the fermentation broth; the preservation number of the calcoacetic acid Acinetobacter is CCTCC No.M2023964, and it is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China.

[0075] Acinetobacter calcoaceticus CDWB36 was isolated from the sludge in the Yihe Road river in Linyi City, Shandong Province, and the isolation method was the same as in Example 1.

[0076] The preparation method of the microbial fermentation product is as follows:

[0077] (1) Strain activation: The frozen Acinetobacter calcoaceticus CDWB36 was inoculated into a flask containing 25 mL of LB liquid medium and incubated at 30°C and 200 r / min. -1 The activation was completed by culturing for 12 h under the conditions;

[0078] (2) Seed liquid preparation: The activated Acinetobacter calcoaceticus CDWB36 was inoculated into LB liquid medium at a volume fraction of 1% and incubated at 30°C and 200 r / min. -1 After culturing for 30 h, seed solution was obtained;

[0079] (3) Preparation of fermentation broth: The seed liquid of Acinetobacter calcoaceticus CDWB36 was inoculated into LB liquid medium at a volume fraction of 1% and incubated at 30°C and 200 r / min. -1Cultivate to OD 600 The standard seed solution was obtained by setting the OD 600 The standard seed solution with a volume fraction of 1 was inoculated into LB liquid medium at 30°C and 160 r·min. -1 After culturing for 36 hours, the microbial fermentation liquid is obtained, and the microbial fermentation liquid is spray-dried to obtain the microbial fermentation product.

[0080] The plant resistance promoter is obtained by mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1.

[0081] A method for preparing a microbial agent for preventing and treating tobacco bacterial wilt comprises the following preparation steps:

[0082] (1) preparing composite microbial fermentation products;

[0083] (2) mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1 to obtain a plant resistance promoter;

[0084] (3) The composite microbial fermentation product obtained in step (1) and the plant resistance promoter are mixed in a mass ratio of 30:0.1, and auxiliary materials are added to prepare granules.

[0085] The auxiliary materials in step (3) also include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, the plant resistance promoter, sodium dodecyl sulfate, starch, and kaolin is 30:0.1:0.1:5:15.

[0086] In this comparative example, except that only calcoaceticus was used to prepare the microbial fermentation product, the other raw materials and preparation method were the same as those in Example 1.

[0087] Comparative Example 2

[0088] A microbial agent for preventing and treating tobacco bacterial wilt comprises a microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is 30:0.1.

[0089] The microbial fermentation product is obtained by spray drying the fermentation liquid of Pseudomonas fluorescens; the strain number of the Pseudomonas fluorescens is CGMCC No.1.15059, which is purchased from the General Microbiology Center of China National Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0090] The preparation method of the microbial fermentation product is as follows:

[0091] (1) Strain activation: The frozen Pseudomonas fluorescens was inoculated into a flask containing 25 mL of LB liquid culture medium and incubated at 30°C and 200 r / min.-1 The activation was completed by culturing for 12 h under the conditions;

[0092] (2) Seed liquid preparation: The activated Pseudomonas fluorescens was inoculated into LB liquid culture medium at a volume fraction of 1% and incubated at 30°C and 200 r / min. -1 After culturing for 30 h, seed solution was obtained;

[0093] (3) Fermentation broth preparation: The seed solution of Pseudomonas fluorescens was inoculated into LB liquid medium at a volume fraction of 1%, and the culture medium was incubated at 30°C and 200 r / min. -1 Cultivate to OD 600 The standard seed solution was obtained by setting the OD 600 The standard seed solution with a volume fraction of 1 was inoculated into LB liquid medium at 30°C and 160 r·min. -1 After culturing for 36 hours, the microbial fermentation liquid is obtained, and the microbial fermentation liquid is spray-dried to obtain the microbial fermentation product.

[0094] The plant resistance promoter is obtained by mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1.

[0095] A method for preparing a microbial agent for preventing and treating tobacco bacterial wilt comprises the following preparation steps:

[0096] (1) preparing composite microbial fermentation products;

[0097] (2) mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1 to obtain a plant resistance promoter;

[0098] (3) The composite microbial fermentation product obtained in step (1) and the plant resistance promoter are mixed in a mass ratio of 30:0.1, and auxiliary materials are added to prepare granules.

[0099] The auxiliary materials in step (3) also include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, the plant resistance promoter, sodium dodecyl sulfate, starch, and kaolin is 30:0.1:0.1:5:15.

[0100] In this comparative example, except that only Pseudomonas fluorescens was used to prepare the microbial fermentation product, the other raw materials and preparation methods were the same as those in Example 1.

[0101] Comparative Example 3

[0102] In this comparative example, except for changing the volume ratio of the standard seed solution of the two bacteria in the preparation method of the composite microbial fermentation product, that is, changing the dosage ratio of the calcoacetic acid Acinetobacter CDWB36 and the fluorescing Pseudomonas strain, the other raw materials and preparation method are the same as Example 1.

[0103] A microbial agent for preventing and treating tobacco bacterial wilt comprises a composite microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is 30:0.1.

[0104] The preparation method of the composite microbial fermentation product is as follows:

[0105] (1) Strain activation: The frozen Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens strains were inoculated into flasks containing 25 mL of LB liquid culture medium and incubated at 30°C and 200 r / min. -1 The activation was completed by culturing for 12 h under the conditions;

[0106] (2) Seed liquid preparation: The activated Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 After 30 h of cultivation, seed solutions of the two bacteria were obtained;

[0107] (3) Preparation of fermentation broth: The seed liquid of Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 Cultivate to OD 600 The standard seed solution of the two bacteria was obtained by setting the OD 600 The standard seed liquid of the two bacteria with a volume ratio of 2:1 was mixed and then inoculated into LB liquid medium at a volume fraction of 1%, and the mixture was incubated at 30°C and 160 r·min. -1 After culturing for 36 hours, the composite microbial fermentation broth is obtained, and the composite microbial fermentation broth is spray-dried to obtain the composite microbial fermentation product.

[0108] Comparative Example 4

[0109] In this comparative example, except for changing the volume ratio of the standard seed solution of the two bacteria in the preparation method of the composite microbial fermentation product, that is, changing the dosage ratio of the calcoacetic acid Acinetobacter CDWB36 and the fluorescing Pseudomonas strain, the other raw materials and preparation method are the same as Example 1.

[0110] A microbial agent for preventing and treating tobacco bacterial wilt comprises a composite microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the two is 30:0.1.

[0111] The preparation method of the composite microbial fermentation product is as follows:

[0112] (1) Strain activation: The frozen Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens strains were inoculated into flasks containing 25 mL of LB liquid culture medium and incubated at 30°C and 200 r / min.-1 The activation was completed by culturing for 12 h under the conditions;

[0113] (2) Seed liquid preparation: The activated Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 After 30 h of cultivation, seed solutions of the two bacteria were obtained;

[0114] (3) Preparation of fermentation broth: The seed liquid of Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 Cultivate to OD 600 The standard seed solution of the two bacteria was obtained by setting the OD 600 The standard seed liquid of the two bacteria with a volume ratio of 1:2 was mixed and then inoculated into LB liquid medium at a volume fraction of 1%, and the mixture was incubated at 30°C and 160 r·min. -1 After culturing for 36 hours, the composite microbial fermentation broth is obtained, and the composite microbial fermentation broth is spray-dried to obtain the composite microbial fermentation product.

[0115] Comparative Example 5

[0116] In this comparative example, except that naringenin is not added to the plant resistance promoter, the other raw materials and preparation methods are the same as those in Example 1. That is:

[0117] The plant resistance promoter is galacto-oligosaccharide.

[0118] Comparative Example 6

[0119] In this comparative example, except that galacto-oligosaccharide is not added to the plant resistance promoter, the other raw materials and preparation methods are the same as those in Example 1. That is:

[0120] The plant resistance promoter is naringenin.

[0121] Comparative Example 7

[0122] In this comparative example, except that no plant resistance promoter is added, the rest of the raw materials and preparation method are the same as those in Example 1. That is:

[0123] A microbial agent for preventing and treating tobacco bacterial wilt, comprising a composite microbial fermentation product.

[0124] A method for preparing a microbial agent for preventing and treating tobacco bacterial wilt comprises the following preparation steps:

[0125] (1) preparing composite microbial fermentation products;

[0126] (2) Add the composite microbial fermentation product obtained in step (1) to auxiliary materials to prepare granules.

[0127] The auxiliary materials in step (3) also include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, sodium dodecyl sulfate, starch, and kaolin is 30:0.1:5:15.

[0128] Performance Testing

[0129] Identification of antibacterial activity of bacterial fermentation broth

[0130] The microbial fermentation liquids obtained in Example 1 (Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens), Comparative Example 1 (Acinetobacter calcoaceticus CDWB36), and Comparative Example 2 (Pseudomonas fluorescens) were used as test liquids.

[0131] Take 200 μL of tobacco bacterial wilt pathogen suspension and spread it evenly on the NA plate, add 10 μL of the test bacteria solution in the middle of the plate, and after the culture medium is fully absorbed, invert it in a 28-30℃ constant temperature incubator for 24 hours, observe the antibacterial effect and measure the diameter of the antibacterial circle. Set it to repeat 3 times and take the average value.

[0132] Table 1 Antibacterial test results

[0133]

[0134] From the data in Table 1 and Figure 2 We can see that the calcoacetic acid Acinetobacter CDWB36 and Pseudomonas fluorescens of the present invention both have good inhibitory effects on tobacco bacterial wilt pathogens, and the antibacterial effect of the mixed fermentation broth is significantly higher than that of a single strain. It can be seen that the two bacteria have a mutual synergistic and promoting effect after mixing.

[0135] Pot experiment:

[0136] Tobacco variety: Yunyan 87;

[0137] Plant material cultivation: Sow tobacco seeds in a 72-hole tray filled with peat soil (2 seeds per hole), cover it with 0.5 cm of peat soil, place the tray in a tray, and add 1L of water. After the seeds germinate, water them twice a week, 1L of water each time. When the tobacco seedlings grow to the four-leaf one-heart stage, transplant them into pots (20 cm high, 20 cm upper diameter), and conduct potted experiments 30 days later.

[0138] Experimental methods:

[0139] Take the tobacco planted 30 days after transplantation, and dig a 10 cm deep and 2 cm wide longitudinal incision in the soil 2 cm away from the stem base with a blade, and add 3 g of the bacterial agent of the present invention or the comparative example into the incision. 2 days later, inoculate the incision with 20 mL of spore suspension of bacterial wilt pathogen, and the concentration of bacterial wilt pathogen is 1×10 9 CFU·mL -1 A blank control was set up, and no biological control measures were applied to the blank control. The defensive enzyme activity of tobacco leaves was measured 10 days after treatment, and the disease index and control effect were tested 2 weeks later.

[0140] The treatment groups were:

[0141] A1: Example 1 + bacterial wilt pathogen;

[0142] A2: Example 2 + bacterial wilt pathogen;

[0143] A3: Example 3 + bacterial wilt pathogen;

[0144] A4: Comparative Example 1 + bacterial wilt pathogen;

[0145] A5: Comparative Example 2 + bacterial wilt pathogen;

[0146] A6: Comparative Example 3 + bacterial wilt pathogen;

[0147] A7: Comparative Example 4 + bacterial wilt pathogen;

[0148] A8: Comparative Example 5 + bacterial wilt pathogen;

[0149] A9: Comparative Example 6 + bacterial wilt pathogen;

[0150] A10: Comparative Example 7 + bacterial wilt pathogen;

[0151] CK: bacterial wilt pathogen;

[0152] There were 10 pots in each treatment, and the results were averaged;

[0153] According to the "Grading and Investigation Methods of Tobacco Pests and Diseases" (GB / T23222-2008), the disease situation of potted seedlings was recorded. The incidence rate, disease index and biological control effect were calculated as follows:

[0154] Disease index = ∑(number of plants at each disease level × each disease index) / (total number of plants surveyed × highest disease index of bacterial wilt) × 100%

[0155] Biocontrol effect = (control disease index - treatment disease index) / control disease index × 100%

[0156] Table 2 Potted plant experiment results

[0157]

[0158] Protective enzyme activity assay:

[0159] The activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), chitinase and β-1,3-glucanase were determined according to the methods in the literature.

[0160] Phenylalanine ammonia lyase: Tian Libo, Yang Yan, Shang Sang, et al., Powdery mildew resistance of different bitter melon strains and its correlation with defense enzyme activities, Journal of Shenyang Agricultural University, 2015, 46(3):284-291.

[0161] Polyphenol oxidase: Hu Huihui, Zhang Hui, Zhang Wenna, et al., Ice temperature storage improves the quality of fresh Ruoqiang winter dates [J], Modern Food Science and Technology, 2020, 36(3): 106-112.

[0162] Chitinase: Lv Huifang, Bie Zhilong, Effects of intercropping wheat on physiological parameters of watermelon after infection with Fusarium wilt[J], Chinese Vegetables, 2019, 32(7), 14-18.

[0163] β-1,3-Glucanase: Zeng Yuruikang, Bai Yujia, Wang Qianqian, et al., Changes in the activity of key disease-resistant enzymes in post-harvest Kashi melon infected with Alternaria alternata[J]. Food Research and Development, 2022, 43(16): 8-17.

[0164] Table 3 Effects of defense enzyme activity

[0165]

[0166] From the data in Table 2-3, we can see that the tobacco leaves obtained from the treatment group of the embodiment of the present invention have a low incidence of bacterial wilt, a prevention effect of about 80%, and the activity of tobacco leaf defense enzymes has also been significantly improved. However, in Comparative Examples 1-4, which have changed the composition of microbial strains, and Comparative Examples 5-7, which have changed the composition of plant resistance promoters, the synergistic effect between strains is broken and the resistance promotion effect is weakened, resulting in a decrease in tobacco leaf disease resistance and a decrease in bacterial wilt prevention effect.

[0167] For further verification, field experiments were conducted in Xinyun Demonstration Area, Huidong County, Liangshan Prefecture, Sichuan Province and Dazhai Tobacco Station, Gulin County, Luzhou City, Sichuan Province. The test site is a hilly mountainous area with sufficient sunlight, uniform and loose soil, and tobacco has been planted for many years. It is a plot where tobacco bacterial wilt occurs all year round. The tested tobacco variety: Yunyan 87.

[0168] A randomized block design was used with a total of 10 treatments. The control group (CK) was treated with local conventional fertilization (nitrogen application rate of 105 kg / hm2).2 , the fertilizer ratio is m(N):m(P2O5):m(K2O)=1:1.2:3);

[0169] The treatment groups were:

[0170] A1: Example 1+conventional fertilization;

[0171] A2: Example 2+conventional fertilization;

[0172] A3: Example 3+conventional fertilization;

[0173] A4: Comparative Example 1 + conventional fertilization;

[0174] A5: Comparative Example 2 + conventional fertilization;

[0175] A6: Comparative Example 3 + conventional fertilization;

[0176] A7: Comparative Example 4 + conventional fertilization;

[0177] A8: Comparative Example 5 + conventional fertilization;

[0178] A9: Comparative Example 6 + conventional fertilization;

[0179] A10: Comparative Example 7 + conventional fertilization;

[0180] Each treatment group was repeated three times, with 5 rows in each plot, 12 tobacco plants in each row, 1.20m row spacing, and 0.55m plant spacing. Transplanting was carried out on May 1, 2022. The treatment group applied fungicide (3g / plant) 25 days after transplanting the tobacco seedlings. Other cultivation and management measures were carried out according to local conventional methods.

[0181] Field survey:

[0182] Refer to GB / T 23222-2008 "Grading and Investigation Methods of Tobacco Diseases and Insects" to investigate the occurrence of tobacco bacterial wilt at maturity, calculate the incidence rate, disease index and control effect; refer to GB 2635-1992 "Flue-cured Tobacco" to record and calculate the yield. Figures 3 - 4 shown.

[0183] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A microbial agent for preventing and treating tobacco bacterial wilt, characterized in that: The invention comprises a composite microbial fermentation product and a plant resistance promoter, wherein the mass ratio of the composite microbial fermentation product and the plant resistance promoter is (30-50):0.1; the composite microbial fermentation product is Acinetobacter calcoaceticus CDWB36 ( Acinetobacter caicoaceticus ) and Pseudomonas fluorescens ( Pseudomonas fluorescens ) and the mixed fermentation liquid is spray-dried to obtain the mixture; the preservation number of the calcoacetic acinetobacter is CCTCC No.M2023964, which is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; the strain number of the fluorescing Pseudomonas is CGMCC No.1.15059, which is purchased from the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the preparation method of the composite microbial fermentation product is: (1) Strain activation: The frozen Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens strains were inoculated into flasks containing 25 mL of LB liquid culture medium and incubated at 30°C and 200 r / min. -1 The activation was completed by culturing for 12 h under the conditions; (2) Seed liquid preparation: The activated Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 After 30 h of cultivation, seed solutions of the two bacteria were obtained; (3) Preparation of fermentation broth: The seed liquid of Acinetobacter calcoaceticus CDWB36 and Pseudomonas fluorescens were inoculated into LB liquid culture medium at a volume fraction of 1%, respectively, at 30°C and 200 r·min -1 Cultivate to OD 600 The standard seed solution of the two bacteria was obtained by setting the OD 600 The standard seed liquid of the two bacteria with a volume ratio of 1:1 was mixed and then inoculated into LB liquid medium at a volume fraction of 1%, and the mixture was inoculated at 30°C and 160 r·min. -1 The culture was performed for 36 hours to obtain a composite microbial fermentation broth, which was spray-dried to obtain a composite microbial fermentation product; the plant resistance promoter was obtained by mixing naringenin and oligogalactose in a mass ratio of 1:

1.

2. A method for preparing the microbial agent for preventing and treating tobacco bacterial wilt according to claim 1, characterized in that: The method comprises the following preparation steps: (1) preparing composite microbial fermentation products; (2) mixing naringenin and galacto-oligosaccharide in a mass ratio of 1:1 to obtain a plant resistance promoter; (3) The composite microbial fermentation product obtained in step (1) and the plant resistance promoter obtained in step (2) are mixed in a mass ratio of (30-50):0.1, and auxiliary materials are added to prepare granules.

3. The method for preparing the microbial agent for preventing and treating tobacco bacterial wilt according to claim 2, characterized in that: The auxiliary materials in step (3) include sodium dodecyl sulfate, starch, and kaolin, wherein the mass ratio of the composite microbial fermentation product, the plant resistance promoter, sodium dodecyl sulfate, starch, and kaolin is (30-50):0.1:0.1:5:15.

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