Complex microbial inoculant for preventing and treating tobacco bacterial wilt and application of complex microbial inoculant

Abstract

The invention relates to a complex microbial inoculant for preventing and treating tobacco bacterial wilt and application of the complex microbial inoculant. Strain in the complex microbial inoculant comprises a mulberry enterobacter 65D6 strain, a Ralstonia 56D2 strain, an agrobacterium 18A6 strain, an arthrobacter uretogenes 05-507 strain and a bacillus safensis 05-2101 strain. The five specific strains are compounded and combined for use, an excellent tobacco bacterial wilt prevention and treatment effect is achieved, and experimental research shows that the disease occurrence of tobacco plants treated by the complex microbial inoculant is obviously slowed down, and the disease index is obviously lower than that of a clear water control group and that of a thiediazole copper and chloriure.copper sulfate chemical agent control group. Meanwhile, the five strains can be mutually matched and promoted, a synergistic effect is achieved in the aspect of preventing and treating tobacco bacterial wilt, the WY11 strain is further added for combined use, the effect of preventing and treating the tobacco bacterial wilt is improved, and the better effect of promoting the growth of tobacco plants can be achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biocontrol agents, and relates to a composite microbial agent for preventing and treating tobacco bacterial wilt and application thereof. BACKGROUND

[0002] Bacterial wilt is mainly caused by plant pathogen Ralstonia spp. and is an important soil-borne disease of crops. Ralstonia spp. can infect more than 400 crops, and causes serious damage to banana, tomato, potato and tobacco. The pathogenic bacteria of tobacco bacterial wilt are R. nicotianae, R. solanacearum, R. pseudosolanacearum and R. syzygii. In China, R. nicotianae is the main pathogen of tobacco bacterial wilt, and the disease occurs commonly in the Yangtze River Basin and other tobacco-growing areas to the south. The disease occurs in 43 districts (counties) of 12 cities, including Wenshan, Baoshan, Lincang, Honghe, Kunming, Yuxi, Qujing, Zhaotong, Dali, Lijiang, Chuxiong and Dehong in Yunnan Province. Although tobacco bacterial wilt causes serious damage, there is a lack of disease-resistant resources, and there is no specific chemical control, so the disease has been an important factor restricting the improvement of tobacco yield and quality.

[0003] Biological control has attracted widespread attention due to its environmental friendliness. A large number of biocontrol resources have been intensively studied as a means of biological control of soil-borne diseases. For example, the reported biocontrol bacteria for bacterial wilt mainly include Pseudomonas spp., Bacillus spp., Streptomyces spp., Acinetobacter spp., Burkholderia spp. and Paenibacillus spp. In China, 12 biocontrol bacteria have been developed and registered for tobacco bacterial wilt, mainly including Pseudomonas fluorescens, Bacillus amyloliquefaciens, Paenibacillus polymyxa and Bacillus subtilis, most of which have the ability to antagonize Ralstonia spp.

[0004] In China, the pathogen diversity is complex in different planting areas in terms of latitude, altitude and flue-cured tobacco varieties, and there is an urgent need to develop composite biocontrol bacteria with strong adaptability and good control effect to control the spread of diseases. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a composite microbial agent for preventing and treating tobacco bacterial wilt and application thereof.

[0006] To achieve the object of the present application, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a complex microbial agent for preventing and treating tobacco bacterial wilt, wherein the strains in the complex microbial agent include: Enterobacter mori 65D6 strain with a preservation number of GDMCC No: 64022, Ralstonia mannitolilytica 56D2 strain with a preservation number of CCTCC NO: M 2019921, Agrobacterium deltaense 18A6 strain with a preservation number of CCTCC NO: M 2020041, Paenarthrobacter ureafaciens 05-507 strain with a preservation number of CCTCC NO: M 20211381, and Bacillus safensis 05-2101 strain with a preservation number of CCTCC NO: M 2022547.

[0008] The present application creatively develops a brand-new microbial compounding mode and a strategy for preventing and treating tobacco bacterial wilt, i.e., the above five specific strains are compounded and used in combination, which has excellent effect of preventing and treating tobacco bacterial wilt. Experimental research shows that tobacco plants treated by the complex microbial agent are obviously slowed down in disease occurrence, and the disease index is obviously lower than that of the water control group and the thiodiazole copper and chlorure copper sulfate chemical agent control groups. Meanwhile, the five strains can cooperate and promote each other, and play a synergistic role in preventing and treating tobacco bacterial wilt. Under the condition of consistent use of the amount of bacteria, the effect is obviously better than that of other strain combination intervention modes.

[0009] Preferably, the viable bacterial count ratio of the 65D6 strain, the 56D2 strain, the 18A6 strain, the 05-507 strain and the 05-2101 strain is (1-5):(1-5):(1-5):(1-5):(1-5), wherein the specific point values in (1-5) can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and other specific point values in the range can be selected, which will not be described one by one here.

[0010] Preferably, the total number of bacteria in the complex microbial agent is not less than 1×10 7 CFU / mL or 1×10 7 CFU / g, for example, 1×10 7 CFU / g (CFU / mL), 2×10 7 CFU / g (CFU / mL), 5×10 7 CFU / g (CFU / mL), 8×10 7CFU / g (CFU / mL), 1x10 8 CFU / g (CFU / mL), 5x10 8 CFU / g (CFU / mL), 1x10 9 CFU / g (CFU / mL), 1x10 10 CFU / g (CFU / mL), 1x10 11 CFU / g (CFU / mL), 1x10

[0011] Preferably, the dosage form of the complex microbial agent includes a powder or a solution.

[0012] Further preferably, the strains in the complex microbial agent further include a Bacillus amyloliquefaciens WY11 strain with a preservation number of CGMCC No. 6663.

[0013] The present application also creatively finds that the further addition of the WY11 strain for combination use not only improves the effect of the complex microbial agent in preventing and treating tobacco bacterial wilt, but also obtains a better effect of promoting tobacco plant growth.

[0014] Preferably, the ratio of the viable bacterial counts of the 65D6 strain, the 56D2 strain, the 18A6 strain, the 05-507 strain, the 05-2101 strain, and the WY11 strain is (1-5):(1-5):(1-5):(1-5):(1-5):(1-5); wherein the specific point values in (1-5) can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and other specific point values in this numerical range can also be selected, and thus it is not necessary to repeat them one by one here.

[0015] Preferably, the complex microbial agent further contains an auxiliary agent, and the auxiliary agent includes any one or a combination of at least two of a stabilizer, a penetration promoter, a bacterial body protective agent, or a preservative.

[0016] In a second aspect, the present application provides an application of the complex microbial agent in the first aspect in the preparation of a product for preventing and treating tobacco bacterial wilt and / or promoting tobacco growth.

[0017] Preferably, the tobacco bacterial wilt is caused by Ralstonia solanacearum, Ralstonia psuedosolanacearum, Ralstonia atrosepticum, or Ralstonia insidiosa.

[0018] In a third aspect, the present application provides a method for preventing and treating tobacco bacterial wilt and / or promoting tobacco growth, and the method includes applying the complex microbial agent in the first aspect to tobacco seedlings.

[0019] Preferably, the application amount is not less than 1x10 7 CFU, for example, 1x10 7 CFU, 3x10 7 CFU, 5x10 7 CFU, 1x10 8 CFU, 1x10 9 CFU, 5x10 9 CFU, 8x10 9 CFU, 1x10 10 CFU, etc., and other specific point values in the numerical range can be selected, which will not be described here one by one.

[0020] Preferably, the application period is selected from any one or a combination of at least two of transplanting, clump stage or vigorous growth stage.

[0021] Preferably, the application method includes root irrigation treatment, root dipping treatment or root soaking treatment.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application creatively develops a brand new microbial compound method and a strategy for preventing and treating tobacco bacterial wilt, that is, five specific strains of Enterobacter mori 65D6 strain, Ralstonia mannitolilytica 56D2 strain, Agrobacterium deltaense 18A6 strain, Paenarthrobacter ureafaciens 05-507 strain and Bacillus safensis 05-2101 strain are compounded and used in combination, which has excellent effect of preventing and treating tobacco bacterial wilt. Experimental research shows that the disease of tobacco plants treated by the compound microbial agent is obviously slowed down, and the disease index is obviously lower than that of the water control group and the thiodiazole copper and chlorure copper sulfate chemical agent control group. At the same time, the five strains can cooperate and promote each other, and play a synergistic effect in preventing and treating tobacco bacterial wilt. Under the condition of consistent use of bacteria, the effect is obviously better than that of other strain combination intervention methods. Further adding WY11 strain for combination, the compound microbial agent not only improves the effect of preventing and treating tobacco bacterial wilt, but also obtains better effect of promoting tobacco plant growth. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a graph of the effect of the application of the compound microbial agent on the agronomic traits of tobacco plants in the Yuxi Eshan test point in Example 5;

[0025] Figure 2is a comparison chart of the effects of applying the compound microbial inoculant and chemical agents on the incidence, disease index and severity of tobacco bacterial wilt in the test point in Lincang Lianxiang District in Example 5;

[0026] Figure 3 is a comparison chart of the effects of applying the compound microbial inoculant and chemical agents on the agronomic traits of tobacco plants in the test point in Lincang Lianxiang District in Example 5;

[0027] Figure 4 is a comparison chart of the effects of each microbial agent on the growth of Ralstonia solanacearum plate in Example 6;

[0028] The classification and naming of the 65D6 strain involved in the application is Enterobacter mori, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 64022, the preservation date is November 14, 2023, and the preservation address is No. 59, Building 5, Guangzhou Xianlie Middle Road 100 Courtyard.

[0029] The classification and naming of the 56D2 strain involved in the application is Ralstonia mannitolilytica 56D2, the preservation unit is China Center for Type Culture Collection, the preservation time is November 12, 2019, the preservation number is CCTCC NO: M2019921, and the address is China. Wuhan. Wuhan University.

[0030] The classification and naming of the 18A6 strain involved in the application is Agrobacterium deltaense 18A6, the preservation unit is China Center for Type Culture Collection, the preservation time is January 13, 2020, the preservation number is CCTCC NO: M2020041, and the address is China. Wuhan. Wuhan University.

[0031] The classification and naming of the 05-507 strain involved in the application is Paenarthrobacter ureafaciens 05-507, the preservation unit is China Center for Type Culture Collection, the preservation time is November 08, 2021, the preservation number is CCTCC NO: M 20211381, and the address is China. Wuhan. Wuhan University.

[0032] The classification and naming of the 05-2101 strain involved in the application is Bacillus safensis 05-2101, the preservation unit is China Center for Type Culture Collection, the preservation time is May 3, 2022, the preservation number is CCTCC NO: M2022547, and the address is China. Wuhan. Wuhan University.

[0033] The WY11 strain of the present application is classified as Bacillus amyloliquefaciens, and is preserved in the China General Microbiological Culture Collection Center on October 11, 2012, with a preservation number of CGMCC No. 6663, and an address of No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0034] The technical solutions of the present application are further illustrated by specific embodiments. It should be understood by those skilled in the art that the embodiments are only used to help understand the present application, and should not be regarded as specific limitations on the present application.

[0035] The calculation formulas of "morbidity", "disease index", "control effect", "empty pond rate" and "severity" involved in the following examples are as follows:

[0036] Morbidity = (number of diseased plants / total number of investigated plants) × 100%;

[0037] Disease index = [Σ(disease level × number of strains at the level) / (highest level × total number of strains)] × 100;

[0038] Control effect = (disease index of control-disease index of treatment) / disease index of control × 100%.

[0039] The disease index is investigated according to the Tobacco Disease Grading and Investigation Method (GB / T 23222-2008) of the People's Republic of China Tobacco Industry Standard, and the disease grading is as follows: 0 level for no disease on the whole plant, 1 level for wilting of one-half or less of the leaves on the diseased side, 3 level for wilting of one-half to two-thirds of the leaves on the diseased side, 5 level for wilting of more than two-thirds of the leaves on the diseased side, 7 level for wilting of all the leaves on the diseased plant, and 9 level for almost dead diseased plant.

[0040] Empty pond rate = 100% × (number of dead seedling plants / total number of transplanted plants).

[0041] Severity = ∑(number of plants at each level × representative value at each level) / total number of investigated plants

[0042] Example 1

[0043] Evaluation of the indoor control effect of the complex microbial agent on tobacco bacterial wilt:

[0044] (1) Seed liquid culture: pick one ring of each of the biocontrol bacteria 65D6, 56D2, 18A6, 05-507 and 05-2101, and inoculate into a 500 mL centrifuge tube containing 500 mL of seed culture solution (10.0 g / L of proteose peptone, 3.0 g / L of beef extract, 5.0 g / L of sodium chloride, 15.0 g / L of agar, pH 7.0), and place in a 28°C constant temperature shaker for 16 h.

[0045] (2) Fermentation: The various sub-solutions were transferred to 150 L fermentation medium (sucrose 10 g / L, bone peptone 5 g / L, corn syrup dry powder 5 g / L, soybean meal powder 15 g / L, magnesium sulfate 0.5 g / L, pH 7.2) at a ratio of 1:100 for fermentation. The fermentation process parameters were as follows: (1) sterilization: the emptying condition was 0.15 MPa, 123°C, 1 h; the actual sterilization condition was 0.15 MPa, 123°C, 35 min; (2) inoculation: after sterilization, when the culture solution temperature dropped to 37°C, inoculation was started; (3) fermentation: the rotation speed was kept at 120 r / min until the end of fermentation; the aeration amount was kept at 15 m 3 / h until the end of fermentation; the tank pressure was kept at 0.05 Mpa throughout the fermentation process, and the temperature was kept at 35°C, with an upper and lower floating of 0.5°C. The fermentation was run for 28 h.

[0046] (3) Preparation of test bacterial agent: the fermentation broth of 65D6, 56D2, 18A6, 05-507, and 05-2101 was diluted and mixed at a ratio of 1:1:1:1:1 of viable bacteria number, to obtain a complex bacterial agent 1 with a concentration of 1×10 7 CFU / mL; the fermentation broth of 56D2, 18A6, 05-507, and 05-2101 was diluted and mixed at a ratio of 1:1:1:1 of viable bacteria number, to obtain a complex bacterial agent 2 with a concentration of 1×10 7 CFU / mL; the fermentation broth of 65D6, 18A6, 05-507, and 05-2101 was diluted and mixed at a ratio of 1:1:1:1 of viable bacteria number, to obtain a complex bacterial agent 3 with a concentration of 1×10 7 CFU / mL; the fermentation broth of 65D6, 56D2, 05-507, and 05-2101 was diluted and mixed at a ratio of 1:1:1:1 of viable bacteria number, to obtain a complex bacterial agent 4 with a concentration of 1×10 7 CFU / mL; the fermentation broth of 65D6, 56D2, 18A6, and 05-2101 was diluted and mixed at a ratio of 1:1:1:1 of viable bacteria number, to obtain a complex bacterial agent 5 with a concentration of 1×10 7 CFU / mL; the fermentation broth of 65D6, 56D2, 18A6, and 05-507 was diluted and mixed at a ratio of 1:1:1:1 of viable bacteria number, to obtain a complex bacterial agent 6 with a concentration of 1×10 7 CFU / mL.

[0047] (4) Test grouping:

[0048] The biocontrol bacteria treatment groups 1-6 (root irrigation with the compound microbial agents 1-6 in turn), the conventional control group (root irrigation with the water dilution of LB medium), and the chemical agent chlorure copper sulfate control group, each treatment group with 3 repetitions, and each repetition with 10 tobacco plants.

[0049] (5) Experimental process: The experiment was carried out in a laboratory temperature-controlled (28°C) greenhouse, and tobacco plants (variety: Honghuadajinyuan) were used as the culture medium. After 50 days of cultivation, the biocontrol bacteria treatment groups 1-6 were root irrigated with 200 mL of compound microbial agent 1-6, the conventional control group was root irrigated with 200 mL of water dilution of LB medium, and the chemical agent chlorure copper sulfate control group was root irrigated with 200 mL of chlorure copper sulfate (pesticide registration certificate number PD20160275, produced by Nanjing Nanfeng Pesticide Technology Development Co., Ltd.) diluted 1000 times with tap water according to the usage instructions.

[0050] The experiment selected a strong pathogenicity Ralstonia nicotianae strain QBRS-1 from Qianbain County, Wenshan City, Yunnan Province, which was preserved in the laboratory as the pathogenic bacteria. After 24 hours, the Ralstonia nicotianae strain QBRS-1 cultured in CG medium for 24 hours was diluted, and each tobacco plant was root irrigated with 100 mL of the dilution (inoculation concentration of 10 7 CFU / mL).

[0051] (6) Test results: After 22 days, the area under the disease index curve (average) of each group and the control effect (%) relative to the conventional control group were calculated, and the results are shown in Table 1.

[0052] Table 1

[0053]

[0054] As can be seen from the data results in Table 1, after the tobacco plants were pretreated with the compound microbial agent involved in the present application and then inoculated with Ralstonia nicotianae QBRS-1, tobacco bacterial wilt could be effectively controlled. The area under the disease index curve of the control group was 595.6±128.8 after 22 days of inoculation, the area under the disease index curve of the control agent group was 273.3±82.6, and the relative control effect was 54.11%; the area under the disease index curve of the compound microbial agent 1 treatment group was only 147.80±64.95, and the control effect was 75.18%. The above results show that the control effect of the compound microbial agent involved in the present application on tobacco bacterial wilt is better than that of the chemical agent chlorure copper sulfate. At the same time, compared with the compound microbial agents 2-6, the effect of the compound microbial agent 1 is obviously better, which shows that the five strains of 65D6, 56D2, 18A6, 05-507, and 05-2101 have synergistic effects in the prevention and control of tobacco bacterial wilt.

[0055] Example 2

[0056] Evaluation of the effect of the complex microbial agent on tobacco bacterial wilt in the greenhouse:

[0057] (1) Seed liquid culture is the same as in Example 1.

[0058] (2) Fermentation is the same as in Example 1.

[0059] (3) Preparation of the test microbial agent: dilute the fermentation broth of 65D6, 56D2, 18A6, 05-507, and 05-2101, and mix them in a ratio of 1:1:1:1:1 of viable bacterial numbers to obtain a complex microbial agent C3 with a concentration of 1 x 10 8 CFU / mL.

[0060] (4) Grouping of the test:

[0061] The biocontrol agent treatment group (root irrigation with the complex microbial agent C3), the water control group (root irrigation with an aqueous dilution of the LB medium), the chemical agent chlorothalonil and copper sulfate control group, and the chemical agent thiodiazole copper control group, each with 3 repetitions and 10 tobacco plants per repetition.

[0062] (5) Experimental process: the experiment was carried out in a greenhouse at 28-30°C. After 50 days of cultivation in tobacco floating seedling substrate (tobacco special, produced by Yunnan Mile Tobacco Material Co., Ltd.), the tobacco plants (variety: Honghua Dajinyuan) were transplanted into a substrate mixed at a ratio of 3:1 of red soil and organic matter;

[0063] After transplantation, the biocontrol agent treatment group was irrigated with 200 mL of the complex microbial agent C3; the water control group was irrigated with 200 mL of an aqueous dilution of the LB medium; the chemical agent chlorothalonil and copper sulfate control group was irrigated with 200 mL of a 1000-fold dilution of chlorothalonil and copper sulfate (pesticide registration certificate number PD20160275, produced by Nanjing Nanfeng Pesticide Technology Development Co., Ltd.) in tap water according to the usage instructions; and the chemical agent thiodiazole copper control group was irrigated with 200 mL of a 1000-fold dilution of thiodiazole copper (pesticide registration certificate number PD20086024, Zhejiang Longwan Chemical Co., Ltd.) in tap water according to the usage instructions.

[0064] The test selected a highly pathogenic Ralstonia nicotianae strain QBRS-1 from Qianbain County, Wenshan Prefecture, Yunnan Province, as the pathogenic bacteria. After 24 hours, the Ralstonia nicotianae strain QBRS-1 cultured in CG medium for 24 hours was diluted, and each tobacco plant was irrigated with 100 mL of the dilution (inoculum concentration of 10 7 CFU / mL).

[0065] (6) Test results: After inoculation of pathogenic bacteria, the disease index was investigated every 7 days, and a total of 5 times. After the end of the test, the disease index, the area under the disease index curve and the control effect were counted, and the results are shown in Table 2.

[0066] Table 2

[0067]

[0068] From the data results in Table 2, under the condition of soil-based cultivation medium in the greenhouse, the disease occurrence of tobacco plants treated with the compound microbial agent C3 was significantly slowed down, especially within the first 21 days after inoculation. The disease index of the three repeats in the compound microbial agent C3 treatment group was significantly lower than that in the water control group and the thiacopper and chlorure copper sulfate chemical agent control groups. During the observation period, the area under the disease index curve of the compound microbial agent C3 treatment group was 1069.00±95.52, the relative control effect was 38.88% compared with the water control group, and the relative control effects of the chemical agents chlorure copper sulfate and thiacopper were only 14.29% and 7.78%, respectively. The above results show that the effect of the compound microbial agent C3 in controlling tobacco bacterial wilt is better than that of the two control agents.

[0069] Example 3

[0070] Evaluation of the greenhouse effect of the compound microbial agent on tobacco bacterial wilt:

[0071] (1) Seed liquid culture as in Example 2, and adding WY11 strain.

[0072] (2) Fermentation as in Example 2, and adding WY11 strain.

[0073] (3) Preparation of microbial agent: dilute the fermentation broth of 65D6, 56D2, 18A6, 05-507 and 05-2101, and mix them in a ratio of 1:1:1:1:1 of viable bacteria number, to obtain a compound microbial agent C3 with a concentration of 1×10 7 CFU / mL. Dilute the fermentation broth of 05-507, 05-2101 and WY11, and mix them in a ratio of 1:1:1 of viable bacteria number, to obtain a compound microbial agent ST6 with a concentration of 1×10 7 CFU / mL.

[0074] (4) Test grouping:

[0075] The biocontrol agent treatment group (mixed with compound microbial agent C3+ST6 in a volume ratio of 1:1 for root irrigation), the water control group (irrigated with water diluted with the same volume of LB medium), the chemical agent chlorure copper sulfate control group, and the chemical agent thiacopper control group, each with 3 repeats, 10 tobacco plants per repeat.

[0076] (5) Experimental process: The experiment was carried out in a greenhouse at 28-30℃. The tobacco plants (variety: Honghuadajinyuan) were cultivated for 50 days in tobacco floating seedling substrate (tobacco special, produced by Yili City Tobacco Materials Co., Ltd. in Yunnan Province) as culture medium, and then transplanted into the substrate mixed by red soil and organic matter at a ratio of 3:1;

[0077] After transplanting, the biocontrol bacteria treatment group was irrigated with 200 mL of compound microbial agent C3+ST6; the water control group was irrigated with 200 mL of LB medium diluted with water; the chlorothalonil+copper sulfate control group was irrigated with 200 mL of chlorothalonil+copper sulfate (pesticide registration certificate number PD20160275, produced by Nanjing Nanfeng Pesticide Technology Development Co., Ltd.) diluted 1000 times with tap water according to the usage instructions; the thiodiazole copper control group was irrigated with 200 mL of thiodiazole copper (pesticide registration certificate number PD20086024, Zhejiang Longwan Chemical Co., Ltd.) diluted 1000 times with tap water according to the usage instructions;

[0078] The experiment selected a strong pathogenicity Ralstonia nicotianae strain QBRS-1 from Qianbain County, Wenshan City, Yunnan Province, which was preserved in the laboratory as the pathogenic bacteria. After 24 h, the Ralstonia nicotianae strain QBRS-1 cultured in CG medium for 24 h was diluted, and each tobacco plant was irrigated with 100 mL of the diluted solution (inoculum concentration of 10 7 CFU / mL).

[0079] (6) Test results: The disease index was investigated every 7 days after inoculation of the pathogenic bacteria, and a total of 6 times. After the end of the experiment, the disease index, area under the disease index curve, and control effect were counted, and the results are shown in Table 3.

[0080] Table 3

[0081]

[0082] As shown by the data results in Table 3, within 42 days after inoculation of the pathogenic bacteria, the disease index of the three replicates of the water control group was all above 70%, and the corresponding area under the disease index curve was 1345.00±152.10. Neither chlorothalonil+copper sulfate nor thiodiazole copper could effectively control the bacterial wilt in this experiment, and the area under the disease index curve was all higher than that of the water control, and the corresponding control effect was negative, which also reflected the instability of the control effect of chemical agents. The disease progress and disease index of the tobacco plants treated with the compound microbial agent C3+ST6 were lower than those of the water control and the two control agents at the same investigation time point, and the area under the disease index curve during the investigation period was 669.20±122.00, which was significantly lower than that of the controls, and the relative control effect reached 50.25%. The above results showed that the microbial agent composed of compound microbial agents C3 and ST6 had a good control effect on bacterial wilt.

[0083] Example 4

[0084] Evaluation of the indoor control effect of the complex microbial agent (in the form of microbial powder) on tobacco bacterial wilt:

[0085] (1) Seed liquid culture: same as Example 3.

[0086] (2) Fermentation: the fermentation parameters of the test bacteria are shown in Table 4.

[0087] Table 4

[0088]

[0089] (3) Preparation of microbial powder: based on whether the spores are produced by the strains, the production process of the microbial powder of different strains is optimized. 56D2, 65D6, 18A6 and 05-507 do not produce spores, and the microbial powder is prepared by physical adsorption. WY11 and 05-2101 are prepared by spray drying. The physical adsorption process is as follows: centrifuge the fermentation broth of the non-spore-producing strain, collect the centrifuged bacteria as raw material, mix with diatomite at a mass ratio of 1:1 and add 5‰ of sucrose as nutrient substance, and place the mixture in a cool, dry and ventilated place to dry. When the water content is about 10%, crush and package. The spray drying process is as follows: centrifuge the fermentation broth of the target strain, mix the centrifuged bacteria with the adsorbent in proportion to prepare the spray drying material, and then spray dry. The effective viable cell counts of the above strain fermentation broths are 56D2 2.0 billion / mL, 65D6 185.8 billion / mL, 18A6 241.8 billion / mL, WY11 43.6 billion / mL, 05-2101 178.4 billion / mL and 05-507 750.8 billion / mL. The effective viable cell counts of the microbial powder prepared by physical adsorption are as follows: 56D2 182.3 billion / g, 65D6 130.9 billion / g, 18A6 133.5 billion / g and 05-507 356.5 billion / g. The effective viable cell counts of the microbial powder prepared by spray drying are WY11 341.3 billion / g and 05-2101 32.4 billion / g.

[0090] (4) Preparation of complex powder: mix 56D2, 65D6, 18A6, 05-507 and 05-2101 powders in equal proportions by mass to obtain complex powder C3; mix WY11, 05-2101 and 05-507 powders in equal proportions by mass to obtain complex powder ST6; mix complex powder C3 and complex powder ST6 in equal proportions to obtain complex microbial agent C3+ST6.

[0091] (5) Test grouping:

[0092] Compound powder C3+ST6, compound powder C3, compound powder ST6, water control group (water dilution of LB medium), chemical agent chlorothene + copper sulfate control group, each treatment group repeated 10 tobacco plants.

[0093] (6) Experimental process: The experiment was carried out in a laboratory temperature-controlled (28°C) greenhouse, and tobacco plants (variety: Honghuadajinyuan) were used as culture medium. After 50 days of cultivation, compound powder C3+ST6, compound powder C3, and compound powder ST6 were each applied to each tobacco seedling at about 1x10 11 CFU, diluted with 200 mL of water for root irrigation; the water control group was irrigated with 200 mL of water dilution of LB medium; the chemical agent chlorothene + copper sulfate control group was diluted 1000 times with tap water according to the usage instructions, and each tobacco seedling was irrigated with 200 mL.

[0094] The experiment selected a strong pathogenic Ralstonia nicotianae strain QBRS-1 from Qianxinan, Wenshan, Yunnan Province, as the pathogenic bacteria, and after 24 hours, the Ralstonia nicotianae strain QBRS-1 cultured in CG medium for 24 hours was diluted, and each tobacco plant was irrigated with 100 mL of the dilution (inoculation concentration of 10 7 CFU / mL).

[0095] (6) Test results: After 35 days of inoculation of the pathogenic bacteria, the area under the disease index curve (average) and the relative control effect (%) of each group were calculated, and the results are shown in Table 5.

[0096] Table 5

[0097]

[0098] As shown by the data in Table 5, the tobacco plants treated with each bacterial agent or chlorothene + copper sulfate had good control effect on bacterial wilt, among which the area under the disease index curve of the compound bacterial agent ST6+C3 was the smallest, only 183.40±68.24, while the area under the disease index curve of the water control group was 793.30±44.16, and the relative control effect of the compound bacterial agent ST6+C3 was 76.88%; the control effect of the compound bacterial agent ST6 was 68.13%, which was better than that of chlorothene + copper sulfate and the compound bacterial agent C3. The above results show that the effect of the compound bacterial agent C3 and ST6 after compounding is better than that of any single compound bacterial agent.

[0099] Example 5

[0100] Field evaluation of the complex microbial inoculant on tobacco bacterial wilt:

[0101] (1) Seed liquid culture: same as Example 4.

[0102] (2) The fermentation medium of the tested strains is shown in Table 6.

[0103] Table 6

[0104]

[0105] Liquid fermentation of the strains was carried out in a 10-ton fermentation tank, and the process parameters were as follows: ① sterilization: the emptying condition was 0.15 MPa, 123℃, 1 h; the actual sterilization condition was 0.15 MPa, 123℃, 35 min. ② inoculation: when the temperature of the culture solution decreased to 37℃ after sterilization, inoculation was started, and the inoculation ratio was about 1:120 of the strain / culture solution. ③ fermentation control: the rotation speed was maintained at 120 r / min until the end of fermentation; the aeration amount was maintained at 15 m 3 / h until the end of fermentation; the tank pressure was maintained at 0.05 MPa throughout the process; due to equipment reasons, the aeration amount was allowed to fluctuate for a short time. The temperature was maintained at 35℃ throughout the fermentation process, with a fluctuation of 0.5℃ up and down. The fermentation time was 25-42 h. ④ detection: before inoculation, the strain was detected for purity by streak culture and microscopic examination; when the temperature of the culture solution decreased to 37℃ after sterilization, the first sampling was carried out before inoculation to detect the completeness of sterilization by streak culture and microscopic examination; the second sampling was carried out 8 h after inoculation to detect whether the fermentation was contaminated by streak culture and microscopic examination; the third sampling was carried out 24 h after fermentation to detect whether the fermentation was contaminated and the spore rate, and thereafter, sampling was carried out every 6 h until the spore rate reached more than 95% to complete the fermentation by streak culture and microscopic examination; after the completion of fermentation, the fermentation broth was sampled to detect the bacterial amount by dilution plate method, which was diluted to 50 billion / mL.

[0106] (3) Preparation of the complex microbial inoculant: the fermentation broths of 56D2, 65D6, 18A6, 05-507 and 05-2101 were mixed in equal proportions by mass, and the adjuvants xanthan gum 0.2%, aminoglycol 0.5%, glycerol 3.0%, methyl p-hydroxybenzoate 0.1% and DP-10 (octylphenol polyoxyethylene ether) 2.0% were added to obtain the complex microbial inoculant C3; the fermentation broths of WY11, 05-2101 and 05-507 were mixed in equal proportions by mass, and the adjuvants xanthan gum 0.2%, aminoglycol 0.5%, glycerol 3.0%, methyl p-hydroxybenzoate 0.1% and DP-10 (octylphenol polyoxyethylene ether) 2.0% were added to obtain the complex microbial inoculant ST6; the complex microbial inoculants C3 and ST6 were mixed in equal proportions to obtain the complex microbial inoculant C3+ST6, which was used as the sample to be tested.

[0107] (4) The test is carried out in Eshan County of Yuxi City, Linxiang District of Lincang City and Qiubei County of Wenshan Zhuang Autonomous Prefecture.

[0108] (4.1) Eshan test point in Yuxi:

[0109] The heavy disease field of bacterial wilt, the incidence of bacterial wilt in previous years is more than 40%. The test sets up two treatments of compound microbial agent C3+ST6 treatment group and water control group, each about half area, the treatment group is applied with compound microbial agent C3+ST6 20 times dilution liquid, 500 mL per plant at transplanting, clump and vigorous growth, and the film is uncovered and the soil is cultivated at clump period. The occurrence degree of bacterial wilt is investigated at clump, vigorous growth and maturity period, and the agronomic characters of tobacco plant are investigated after the top is punched. The results are shown in Table 7 and Figure 1 .

[0110] Table 7

[0111]

[0112] From the results of Table 7, the bacterial wilt of Eshan test site is significantly reduced compared with previous years. From the disease investigation results, the application of the biocontrol agent involved in the application achieves 100, 84.87, 88.65% of bacterial wilt control effect at clump, vigorous growth and maturity period, respectively.

[0113] From the Figure 1 results, it can be seen that the growth of tobacco plants treated with the biocontrol agent involved in the application is stronger than that of the control group by observing the growth of tobacco plants at clump period, uncovering and cultivating the film, vigorous growth and after the top is punched. After the top is punched, one tobacco plant is investigated every 7 plants, and the results show that the average plant height of the treatment group is 94.87±8.11 cm, and the average plant height of the control group is 87.53±5.87 cm, and the plant height of the treatment group is increased by 8.38% compared with the control group; the average leaf area of the largest leaf of the tobacco plant in the control group is 1286.17±190.31 cm 2 (n=15), and the average leaf area of the largest leaf of the tobacco plant in the treatment group is 1516.18±242.36 cm 2 (n=15), which is increased by 17.88% compared with the control group. There is a significant difference in plant height and maximum leaf area between the treatment group and the control group. The above results show that the compound microbial agent involved in the application has good control effect on bacterial wilt under field conditions, and has obvious growth promoting effect.

[0114] (4.2) Test point in Linxiang District of Lincang City:

[0115] The common occurrence of bacterial wilt, bacterial wilt is moderate incidence of field, test area 3 mu. Test set: composite microbial agent C3 + ST6 treatment group (3 repeats), chlorure copper sulfate group (2 repeats), water control group (2 repeats). Respectively, transplant, team and vigorous growth, composite microbial agent C3 + ST6 dilution 20 times, 500 mL per plant; Chlorure copper sulfate according to the 3 times the amount of use, 500 mL per plant, the test did not produce drug damage. After the top of the investigation of bacterial wilt and tobacco agronomic traits. Results as shown in Figure 2 and Figure 3

[0116] From Figure 2 It can be seen that the disease survey results show that the incidence of bacterial wilt, severity and disease index of composite microbial agent treatment group are significantly lower than those of water control group, the control effect of composite microbial agent treatment group relative to control group is 89.31%, and there is no significant difference in the incidence of bacterial wilt, severity and disease index between chlorure copper sulfate group and water control group.

[0117] From Figure 3 It can be seen that after the top of the field observation of tobacco growth, it is found that the growth of tobacco in the three repeats of composite microbial agent treatment group is better than that in water control group and chlorure copper sulfate group. The investigation and analysis of agronomic traits show that there is extremely significant difference in plant height, maximum leaf area, effective leaf number, stem circumference and pitch between composite microbial agent treatment group and chlorure copper sulfate and water control group. The plant height, leaf area, effective leaf number, pitch and stem circumference of composite microbial agent treatment group are increased by 11.72%, 11.41%, 6.43%, 6.42% and 5.13% respectively compared with those of control group. There is no significant difference between chlorure copper sulfate and control group.

[0118] (4.3) Wenshan Qiongbei test point:

[0119] The tobacco field with severe occurrence of bacterial wilt, test site 3 mu. Set the treatment: composite microbial agent C3 + ST6 20 times dilution liquid was applied twice at transplanting and team period, 500 mL per plant (two repeats, 2 mu); 3 times the conventional amount of chlorure copper sulfate (0.5 mu) was applied according to the product use instruction; the untreated tobacco was set as control group (0.5 mu). At the mature period, the disease grade of each tobacco was investigated, and the disease index and control effect were calculated. The results are shown in Table 8.

[0120] Table 8

[0121]

[0122] ​From the results of Table 8, the disease investigation results show that the early stage of the bacterial wilt occurs relatively lightly. The control incidence rate is 26.58%, the incidence rates of the two groups of compound microbial agents are 11.23% and 9.01% respectively, and the incidence rate of the chlorure copper sulfate agent treatment group is 15.63%. The bacterial wilt disease index of the two groups of compound microbial agent treatment groups is 2.53 and 1.61 respectively, compared with the blank control, the prevention effects are 69.98% and 80.83% respectively, and the prevention effect of chlorure copper sulfate is also 50.55%.

[0123] Example 6

[0124] Evaluation of the ability of the compound microbial agent to antagonize Ralstonia bacteria:

[0125] (1) Preparation of bacterial plate: Ralstonia bacteria QBRS-1 was inoculated into CG medium and cultured at 28°C and 180 r / min for 24 h (bacterial concentration 10 9 CFU / mL), gradient diluted to 10 -4 ; 100 μL of the diluted solution was placed on the surface of CGA (0.1% acid hydrolyzed casein, 0.5% glucose and 2% peptone, 1.5% agar) medium, and evenly coated with a coating bead, and dried on a clean bench;

[0126] (2) Antagonism of biocontrol bacteria: LB liquid medium was inoculated with 65D6, 56D2, 05-507, 05-2101 and 18A6 strains respectively, and cultured at 28°C and 180 r / min for 24 h; 1 mL of each strain culture solution was taken into 3 independent centrifuge tubes and centrifuged at 12000 r / min for 1 min; the supernatant was discarded and the bacterial agent was resuspended with 1 mL of sterile water; 100 μL of the culture solution of each of the 5 strains was added to another new centrifuge tube, mixed evenly to prepare a compound microbial agent C3 bacterial suspension, centrifuged at 12000 r / min for 1 min, and the supernatant was discarded, and the bacterial agent was resuspended with 1 mL of sterile water;

[0127] 10 μL of single bacterial solution or mixed bacterial solution was dropped on the plate containing the above Ralstonia bacteria QBRS-1, 3 points were symmetrically connected per dish; after drying, it was placed in a 28°C incubator for culture for 48 h, and the inhibition zone of the strain was measured (the distance from the edge of the pathogenic bacteria to the edge of the biocontrol bacteria colony), and the results are shown in Figure 4 and Table 9.

[0128] Table 9

[0129]

[0130] From the results of Table 8, the disease investigation results show that the early stage of the bacterial wilt occurs relatively lightly. The control incidence rate is 26.58%, the incidence rates of the two groups of compound microbial agents are 11.23% and 9.01% respectively, and the incidence rate of the chlorure copper sulfate agent treatment group is 15.63%. The bacterial wilt disease index of the two groups of compound microbial agent treatment groups is 2.53 and 1.61 respectively, compared with the blank control, the prevention effects are 69.98% and 80.83% respectively, and the prevention effect of chlorure copper sulfate is also 50.55%. Figure 4As can be seen from the results of Table 9, when single bacteria were inoculated, 56D2 and 05-2101 had weak inhibition effect on Ralstonia solanacearum, but 65D6, 05-507 and 18A6 had no effect on the growth of the pathogenic bacteria; when the compound microbial inoculant C3 was inoculated, the mixed solution C3 had no antagonistic effect on Ralstonia solanacearum. It is indicated that the compound microbial inoculant C3 does not control tobacco bacterial wilt by antagonism as the main mechanism, and it is possible that the five strains of bacteria control the disease by synergistic effect, competition for ecological niche, regulation of tobacco rhizosphere microbial community, induction of plant resistance or other mechanisms.

[0131] The applicant declares that the technical solutions of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0132] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0133] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable means without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A compound microbial agent for controlling tobacco bacterial wilt, characterized in that, The strains in the compound microbial agent include: Enterobacter mori strain 65D6 with accession number GDMCC No: 64022, Ralstonia mannitolilytica strain 56D2 with accession number CCTCC NO: M 2019921, Agrobacterium deltaense strain 18A6 with accession number CCTCC NO: M 2020041, Paenarthrobacter ureafaciens strain 05-507 with accession number CCTCC NO: M20211381, and Bacillus safensis strain 05-2101 with accession number CCTCC NO: M 2022547.

2. The compound microbial agent according to claim 1, characterized in that, The ratio of viable counts of the 65D6 strain, 56D2 strain, 18A6 strain, 05-507 strain, and 05-2101 strain is (1-5):(1-5):(1-5):(1-5):(1-5).

3. The compound microbial agent according to claim 1, characterized in that, The total bacterial count in the compound microbial agent is not less than 1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

4. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent can be in the form of powder or solution.

5. The compound microbial agent according to any one of claims 1-4, characterized in that, The bacterial strains in the compound bacterial agent also include: Bacillus amyloliquefaciens strain WY11 with preservation number CGMCC No. 6663.

6. The compound microbial agent according to claim 5, characterized in that, The ratio of viable counts of the 65D6 strain, 56D2 strain, 18A6 strain, 05-507 strain, 05-2101 strain, and WY11 strain is (1-5):(1-5):(1-5):(1-5):(1-5):(1-5).

7. The compound microbial agent according to any one of claims 1-6, characterized in that, The compound microbial agent also contains adjuvants, which include any one or a combination of at least two of the following: stabilizers, penetration enhancers, cell protectants, or preservatives.

8. The use of the compound microbial agent according to any one of claims 1-7 in the preparation of products for the prevention and control of tobacco bacterial wilt and / or the promotion of tobacco growth.

9. The application according to claim 8, characterized in that, The tobacco bacterial wilt is caused by *Ralstonia tobaccoensis*, *Ralstonia syringae*, *Ralstonia solanaceae*, or *Ralstonia pseudosolanum*.

10. A method for preventing and controlling tobacco bacterial wilt and / or promoting tobacco growth, characterized in that, The method includes: applying the compound microbial agent according to any one of claims 1-7 to tobacco seedlings; Preferably, the application rate is no less than 1×10⁻⁶ per tobacco seedling. 7 CFU; Preferably, the application period is selected from any one or a combination of at least two of the following: transplanting, seedling stage, or vigorous growth stage; Preferably, the application methods include root drenching, root dipping, or root soaking.

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