Compound Bacterial Agent for Preventing and Controlling Tobacco Target Spot Disease, Preparation Thereof and Application Thereof

Abstract

The present invention discloses a compound bactericide for preventing and controlling tobacco target spot disease, its preparation and application. The compound bactericide includes Achromobacter, Stenotrophomonas and Bacillus subtilis. The number of Achromobacter accounts for 10-25% of the total number of bacteria in the compound bactericide, the number of Stenotrophomonas accounts for 45-70% of the total number of bacteria in the compound bactericide, and the number of Bacillus subtilis accounts for 20-30% of the total number of bacteria in the compound bactericide. The above compound bactericide for preventing and controlling tobacco target spot disease has a reasonable bactericide ratio, can reduce the number of pathogenic species of tobacco target spot disease, reduce the pathogenicity of pathogens and inhibit the occurrence of diseases, and has excellent and stable control effects on target spot disease.

Description

Technical Field

[0001] The present invention relates to the technical field of tobacco target spot disease control, and particularly to a composite bactericide for controlling tobacco target spot disease, its preparation and application. Background Art

[0002] Target spot disease is a relatively common disease of tobacco, and chemical control is usually carried out by spraying chemical agents. However, chemical control is likely to cause problems of pesticide residues and serious ecological environmental pollution.

[0003] Biological control measures can reduce the inoculum quantity of these pathogens by regulating the microbial environment around plants, reduce the pathogenicity of these pathogens and inhibit the occurrence of diseases, so as to achieve the purpose of "controlling bacteria with bacteria". Therefore, there is an urgent need to develop a biological control bactericide for tobacco target spot disease. Summary of the Invention

[0004] Based on this, in view of the problem that chemical control is likely to cause pesticide residues, it is necessary to provide a composite bactericide for controlling tobacco target spot disease, its preparation and application.

[0005] A composite bactericide for controlling tobacco target spot disease, comprising Achromobacter, Stenotrophomonas and Bacillus subtilis. The number of Achromobacter accounts for 10 - 25% of the total number of bacteria in the composite bactericide, the number of Stenotrophomonas accounts for 50 - 70% of the total number of bacteria in the composite bactericide, and the number of Bacillus subtilis accounts for 20 - 30% of the total number of bacteria in the composite bactericide.

[0006] Optionally, the number of Achromobacter accounts for 22.92% of the total number of bacteria in the composite bactericide, the number of Stenotrophomonas accounts for 49.45% of the total number of bacteria in the composite bactericide, and the number of Bacillus subtilis accounts for 27.63% of the total number of bacteria in the composite bactericide.

[0007] Optionally, the composite bactericide further comprises auxiliaries, and the auxiliaries include one or more of silica white carrier accounting for 2% of the mass of the bacteria in the composite bactericide, sodium lignosulfonate accounting for 1% of the mass of the bacteria in the composite bactericide, Tween 80 accounting for 1% of the mass of the bacteria in the composite bactericide, K2HPO4 accounting for 0.2% of the mass of the bacteria in the composite bactericide, and carboxymethyl cellulose accounting for 0.01% of the mass of the bacteria in the composite bactericide.

[0008] A preparation method of a composite bactericide for controlling tobacco target spot disease, comprising the following steps:

[0009] Ferment the mixed bacterial strains corresponding to the above-mentioned composite bactericide to obtain a fermentation broth.

[0010] Freeze - centrifuge the fermentation broth to separate the mixed bacteria, and freeze - dry the mixed bacteria to obtain the composite bactericide.

[0011] Optionally, the fermentation conditions in the step of obtaining the fermentation broth by fermentation are to ferment the mixed strains at pH 7-8, 160-180 r / min, and 35-37 °C for 36-48 h.

[0012] Optionally, the step of separating the mixed bacteria from the fermentation broth by freeze centrifugation includes:

[0013] Freeze centrifuge the fermentation broth for 10-20 min.

[0014] Optionally, the step of freeze-drying the mixed bacteria includes:

[0015] Pre-freeze the mixed bacteria at -80 °C for 4-5 h.

[0016] Optionally, it further includes mixing the freeze-dried mixed bacteria with an auxiliary agent.

[0017] Application of the above-mentioned composite microbial inoculum in preventing and controlling tobacco target spot disease.

[0018] The above-mentioned composite microbial inoculum for preventing and controlling tobacco target spot disease has a reasonable ratio of microbial agents, can reduce the number of pathogen species of tobacco target spot disease, reduce the pathogenicity of pathogens and inhibit the occurrence of diseases, and has excellent and stable prevention and control effects on target spot disease. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the distribution of each group in the test tobacco field provided in Example 5 of the present invention. Detailed Embodiments

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0021] A composite microbial inoculum for preventing and controlling tobacco target spot disease, comprising Achromobacter, Stenotrophomonas and Bacillus subtilis. The number of Achromobacter accounts for 10-25% of the total number of bacteria in the composite microbial inoculum, the number of Stenotrophomonas accounts for 45-70% of the total number of bacteria in the composite microbial inoculum, and the number of Bacillus subtilis accounts for 20-30% of the total number of bacteria in the composite microbial inoculum.

[0022] Among them, the dominant populations of the composite microbial inoculum are Achromobacter and Stenotrophomonas, accounting for 22.92% and 49.45% respectively, as well as a small part of Bacillus subtilis and the like.

[0023] Among them, the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0024] The Achromobacter sp. used in the embodiments of the present application is Achromobacter sp. strain CCTCC AB 2013198, which is purchased from the China Center for Type Culture Collection.

[0025] The Stenotrophomonas sp. strain used in the embodiments of the present application is Stenotrophomonas sp. strain CCTCC AB 205694, which is purchased from the China Center for Type Culture Collection.

[0026] The Bacillus subtilis strain used in the embodiments of the present application is Bacillus subtilis strain CCTCC AB 130001, which is purchased from the China Center for Type Culture Collection.

[0027] Optionally, the number of Achromobacter accounts for 22.92% of the total number of bacteria in the compound microbial agent, the number of Stenotrophomonas accounts for 49.45% of the total number of bacteria in the compound microbial agent, and the number of Bacillus subtilis accounts for 27.63% of the total number of bacteria in the compound microbial agent.

[0028] Optionally, the compound microbial agent further includes auxiliaries, and the auxiliaries include one or more of silica white carrier accounting for 2% of the mass of the bacteria in the compound microbial agent, sodium lignosulfonate accounting for 1% of the mass of the bacteria in the compound microbial agent, Tween 80 accounting for 1% of the mass of the bacteria in the compound microbial agent, K2HPO4 accounting for 0.2% of the mass of the bacteria in the compound microbial agent, and carboxymethyl cellulose accounting for 0.01% of the mass of the bacteria in the compound microbial agent.

[0029] For the convenience of spraying, the compound microbial agent can also be formulated into the form of a wettable biocontrol powder. Specifically, the mixed bacteria can be uniformly mixed with auxiliaries such as 2% silica white carrier, 1% sodium lignosulfonate, 1% Tween 80, 0.2% K2HPO4, and 0.01% carboxymethyl cellulose.

[0030] Among them, silica white can improve the fluidity of the powder and can also be used as an anti-caking agent for easily caking powder substances. Lignin has a large specific surface area and contains various active groups, and can be used as a slow-release agent for microbial agents. Tween-80 is a solubilizer or emulsifier. Carboxymethyl cellulose can be used as an emulsifier, thickener, stabilizer, sizing agent, film-forming agent, and binder. K2HPO4 is an inducer for inducing disease resistance in host plants.

[0031] A preparation method of a composite bactericide for preventing and controlling tobacco target spot disease, comprising the following steps:

[0032] S100: Ferment the mixed bacterial strains corresponding to the composite bactericide to obtain a fermentation broth.

[0033] S200: Freeze-centrifuge the fermentation broth to separate the mixed bacterial cells, and freeze-dry the mixed bacterial cells to obtain the composite bactericide.

[0034] Optionally, the fermentation conditions in the step of fermenting to obtain the fermentation broth are to ferment the mixed bacterial strains at pH 7-8, 160-180 r / min, and 35-37 °C for 36-48 h.

[0035] Optionally, the step of freeze-centrifuging the fermentation broth to separate the mixed bacterial cells includes:

[0036] Freeze-centrifuge the fermentation broth for 10-20 min.

[0037] Optionally, the step of freeze-drying the mixed bacterial cells includes:

[0038] Pre-freeze the mixed bacterial cells at -80 °C for 4-5 h.

[0039] Optionally, it further includes mixing the freeze-dried mixed bacterial cells with an auxiliary agent.

[0040] An application of the above composite bactericide in preventing and controlling tobacco target spot disease.

[0041] Example 1

[0042] Before fermentation in a 50 L fermenter, inoculate the mixed bacterial strains, which include Achromobacter, Stenotrophomonas, and Bacillus subtilis. Among them, the number of Achromobacter accounts for 10% of the total number of bacterial cells in the mixed bacterial strains, the number of Stenotrophomonas accounts for 49.45% of the total number of bacterial cells in the composite bactericide, and the number of Bacillus subtilis accounts for 27.63% of the total number of bacterial cells in the composite bactericide.

[0043] After fermenting at pH 8, 180 r / min, and 35 °C for 48 h, pump out 10 L of fresh fermentation broth into a sampling bottle.

[0044] In a laminar flow hood, aliquot it into 500 ml bacterial collection bottles with a liquid volume of 400 ml. After freeze-centrifuging at 10,000 rpm for 10 min, evenly smear the bacterial cells onto a glass culture dish. Seal it and place it in a -80 °C refrigerator for pre-freezing for 4 h, and then freeze-dry to obtain the bacterial powder, which is the composite bactericide for preventing and controlling tobacco target spot disease. This composite bactericide includes Achromobacter, Stenotrophomonas, and Bacillus subtilis. The number of Achromobacter accounts for 22.92% of the total number of bacterial cells in the composite bactericide, the number of Stenotrophomonas accounts for 49.45% of the total number of bacterial cells in the composite bactericide, and the number of Bacillus subtilis accounts for 27.63% of the total number of bacterial cells in the composite bactericide.

[0045] Example 2

[0046] It is different from Example 1 in that the number of Achromobacter accounts for 10% of the total number of bacteria in the compound microbial agent, the number of Stenotrophomonas accounts for 60% of the total number of bacteria in the compound microbial agent, and the number of Bacillus subtilis accounts for 30% of the total number of bacteria in the compound microbial agent.

[0047] In the fermentation step, the fermentation conditions are fermentation at pH 7, 170 r / min, and 36 °C for 40 h.

[0048] Others are the same as in Example 1.

[0049] Example 3

[0050] It is different from Example 1 in that the number of Achromobacter accounts for 10% of the total number of bacteria in the compound microbial agent, the number of Stenotrophomonas accounts for 70% of the total number of bacteria in the compound microbial agent, and the number of Bacillus subtilis accounts for 20% of the total number of bacteria in the compound microbial agent.

[0051] In the fermentation step, the fermentation conditions are fermentation at pH 7.5, 160 r / min, and 37 °C for 36 h.

[0052] Example 4

[0053] The bacterial powder of Example 1 is uniformly mixed with auxiliaries such as 2% silica white carrier of the bacterial powder quality, 1% sodium lignosulfonate of the bacterial powder quality, 1% Tween 80 of the bacterial powder quality, 0.2% K2HPO4 of the bacterial powder quality, and 0.01% carboxymethyl cellulose of the bacterial powder quality to obtain a wettable biocontrol powder, that is, a compound microbial agent for controlling tobacco target spot disease.

[0054] Example 5

[0055] In each plot of the experimental tobacco field in Zhangjiajie, 3 rows of tobacco are planted. The middle row is sprayed with the medicament, and the other two rows are used as protection rows. 30 tobacco plants are planted at equal density in each row. Other management is carried out according to local conventional measures and methods.

[0056] Plot setting: As Figure 1 shown, 1 negative control group CK is set; 3 treatment groups Treat, Treat_Mg and Treat_Mn are set, and the wettable biocontrol powders BA, BA_Mg (Mg is 5% MgSO4 of the wettable biocontrol powder quality) and BA_Mn (Mn is 2% MnSO4 of the wettable biocontrol powder quality) are sprayed respectively; 1 positive control group KM is set, which is 4% kasugamycin; each group has 3 replicates, with a total of 15 plots.

[0057] Application rate: In the negative control group CK, 5 L of water was applied to each plot; in the treatment groups Treat, Treat_Mg, and Treat_Mn, 0.45 g of BA, BA_Mg, and BA_Mn powder was dissolved in 5 L of water and applied respectively. At this application rate, the effective viable bacteria count in each treatment group was 10 7 CFU / mL of bacterial suspension; in the positive control group KM, 60 g of kasugamycin was dissolved in 2 L of water and applied.

[0058] Application method: Observe the disease situation in the tobacco field. When sporadic lesions are first observed, start applying the medicine. Use the spraying method to evenly spray both the front and back sides of the tobacco leaves. Apply the medicine once every 7 days for a total of 2 times.

[0059] Disease investigation: Investigate the incidence of target spot disease in the experimental tobacco field 0, 7, and 14 days after spraying the biocontrol powder. Investigate all the leaves of 30 tobacco plants in the middle row of each plot. The disease grading standard and investigation method refer to the "Grading and Investigation Methods for Tobacco Diseases and Pests" in the National Standard of the People's Republic of China GB / T 23222-2008. It is divided into 9 grades according to the severity of the disease. The specific grading situation is shown in Table 1.

[0060] Table 1 Disease grading standard

[0061]

[0062] Data analysis: Use Microsoft Excel to classify and summarize the data obtained from the investigation, and calculate the disease index and relative control effect of target spot disease in the experimental tobacco field.

[0063] The calculation methods of the disease index and relative control effect are as follows:

[0064] Disease index = [∑(number of diseased leaves at each level × value of that disease level) / (total number of investigated leaves × highest disease level value)] × 100%

[0065] Relative control effect (%) = (control disease index - treatment disease index) / control disease index × 100%

[0066] Table 2 Target spot disease index

[0067]

[0068]

[0069] As shown in Table 2, 7 days after the first treatment with wettable biocontrol powder BA, BA_Mg, BA_Mn and 4% kasugamycin, the target spot disease index of treatment groups Treat, Treat_Mg, Treat_Mn and the positive control group Kasumin were all significantly lower than that of the negative control group CK at the same period. The target spot disease index was reduced by 38.8%, 42.07%, 38.24% and 39.34% respectively, while there was no significant difference in the target spot disease index among the treatment groups. 7 days after the second treatment, the target spot disease index of the control group reached 68.33%, while the target spot disease index of the treatment groups and the positive control group were effectively controlled. In particular, the target spot disease index of treatment groups Ttreat and Treat_Mg were significantly lower than those of other treatment groups. This indicates that both biocontrol powders BA and BA_Mg have excellent and stable control effects on target spot disease.

[0070] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. Application of a compound microbial agent in controlling tobacco target spot disease, characterized in that, The composite bacterial agent is composed of Achromobacter sp., Stenotrophomonas sp. and Bacillus subtilis; The Achromobacter is Achromobacter ( Achromobacter sp. ) strain, with the preservation number of CCTCC AB 2013198, and is preserved in the China Center for Type Culture Collection; the Stenotrophomonas is Stenotrophomonas ( Stenotrophomonas sp. ) strain, with the preservation number of CCTCC AB 205694, and is preserved in the China Center for Type Culture Collection; the Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis ) strain, with the preservation number of: CCTCC AB 130001, and is preserved in the China Center for Type Culture Collection.

2. The application according to claim 1, wherein The number of Achromobacter sp. accounts for 10-25% of the total number of bacteria in the composite bacterial agent, the number of Stenotrophomonas sp. accounts for 45-70% of the total number of bacteria in the composite bacterial agent, and the number of Bacillus subtilis accounts for 20-30% of the total number of bacteria in the composite bacterial agent.

3. The application according to claim 1, wherein The number of Achromobacter sp. accounts for 22.92% of the total number of bacteria in the composite bacterial agent, the number of Stenotrophomonas sp. accounts for 49.45% of the total number of bacteria in the composite bacterial agent, and the number of Bacillus subtilis accounts for 27.63% of the total number of bacteria in the composite bacterial agent.

4. The application according to claim 1, wherein The composite bacterial agent further includes additives, and the additives include one or more of silica white carrier accounting for 2% of the mass of the bacteria in the composite bacterial agent, sodium lignosulfonate accounting for 1% of the mass of the bacteria in the composite bacterial agent, Tween 80 accounting for 1% of the mass of the bacteria in the composite bacterial agent, K2HPO4 accounting for 0.2% of the mass of the bacteria in the composite bacterial agent, and carboxymethyl cellulose accounting for 0.01% of the mass of the bacteria in the composite bacterial agent.

5. The application according to claim 1, characterized in that The preparation method of the composite bacterial agent includes the following steps: Ferment the mixed bacterial species corresponding to the composite bacterial agent described in claim 1 to obtain a fermentation broth; Freeze-centrifuge the fermentation broth to separate the mixed bacteria, and freeze-dry the mixed bacteria to obtain the composite bacterial agent.

6. The application according to claim 5, wherein The fermentation conditions in the step of fermenting to obtain the fermentation broth are to ferment the mixed bacterial species at pH 7-8, 160-180 r / min, and 35-37 °C for 36-48 h.

7. The application according to claim 5, characterized in that, The step of freeze-centrifuging the fermentation broth to separate the mixed bacteria includes: Freeze-centrifuge the fermentation broth for 10-20 min.

8. The application according to claim 5, wherein The step of freeze-drying the mixed bacteria includes: Pre-freeze the mixed bacteria at -80 °C for 4-5 h.

9. The application according to claim 4, wherein The preparation method of the composite bacterial agent includes the following steps: Ferment the mixed bacterial species corresponding to the composite bacterial agent described in claim 1 to obtain a fermentation broth; Freeze-centrifuge the fermentation broth to separate the mixed bacteria, freeze-dry the mixed bacteria, and mix the freeze-dried mixed bacteria with the additives to obtain the composite bacterial agent.

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