A composite bacterial preparation for preventing and treating tobacco black shank disease and its preparation method

Abstract

The present invention provides a composite bacterial preparation for preventing and treating tobacco black shank disease and a preparation method thereof. The composite bacterial preparation comprises Bacillus cereus, Pseudomonas fluorescens and cobalt chloride. The Bacillus cereus and Pseudomonas fluorescens are respectively prepared by fermenting Bacillus cereus ATCC 9634 and Pseudomonas fluorescens ATCC 13525. According to a certain ratio of bacterial agent and cobalt chloride, the control effect of the composite bacterial preparation can be improved. The composite bacterial preparation prepared by the present invention is used for preventing and treating tobacco black shank disease, and the control effect can reach more than 95%, and has good application prospects.

Description

Technical Field

[0001] The invention relates to a composite microbial agent capable of preventing and treating tobacco black shank disease and a preparation method thereof. Background Art

[0002] Tobacco black shank, commonly known as rotten waist and black root, is one of the most common and serious diseases in tobacco production. Tobacco black shank is a type of soil-borne fungal disease. The disease is widely occurring in tobacco-growing areas in my country and is quite harmful. In recent years, due to the expansion of the area of ​​continuous cropping tobacco fields and the increase in the number of continuous cropping years, tobacco black shank has tended to become more prevalent in many places. Plants infected with tobacco black shank usually have symptoms of dwarfing, wilting, yellowing of leaves, and necrosis of the roots and stem bases.

[0003] Black shank is caused by tobacco phytophthora, which can occur in both the seedling stage and the field stage. It is more likely to occur when tobacco is planted for a long period of time and when the varieties are over-concentrated. The prevention and control methods for black shank can be divided into soil prevention, biological prevention, chemical prevention, pesticide prevention and comprehensive prevention methods that combine multiple methods.

[0004] Using antagonistic microorganisms to control tobacco plant diseases and reduce the harm of pathogens to crops can reduce the pollution caused by excessive use of chemical pesticides. It has become an important measure for the integrated prevention and control of tobacco plant diseases, especially for typical soil-borne diseases such as tobacco black shank. Antagonistic microorganisms colonize in the planting soil and form a dominant group, which can inhibit the reproduction and growth of tobacco plant pathogens, thereby protecting the tobacco plant roots from infection by pathogens.

[0005] Bacillus and Pseudomonas are bacteria that have antagonistic effects on tobacco black shank pathogens, which mainly dissolve tobacco black shank pathogen hyphae by producing antibiotics. The strains currently used are mainly obtained through natural screening, and in actual use, they generally have low antibiotic production and are difficult to effectively inhibit pathogens.

[0006] In biological control, strains with control effects are mainly found through large-scale screening or accidental discovery, which makes the discovery of corresponding strains very difficult, resulting in slow research progress in biological control. Although biological control can avoid some environmental or pesticide problems, the control effect of biological control is generally lower than that of chemical control. Literature reports that commonly used chemical agents can achieve a control effect of more than 70% on black leg disease, while it is difficult to achieve a control effect of more than 70% with biological control.

[0007] Some researchers have also studied the effects of combining specific microorganisms and chemical drugs in preventing and controlling tobacco diseases. For example, Liu Jianjin and others believe that combining Bacillus amyloliquefaciens YN48 with flumorph·ethylene aluminum and metal frost·mancozeb may have a better control effect. However, the combination of microorganisms and drugs often results in reduced control effects, which may be due to the fact that chemical drugs can easily inhibit the growth of microorganisms or have antagonistic effects with microbial metabolites. For example, Xu Yuru and others found that after adding metal frost·mancozeb to Trichoderma, the control effect dropped significantly.

[0008] In summary, there is an urgent need in this field to address the shortcomings of existing biological control methods and provide a microbial soil improvement agent technology that has high prevention efficiency, improves tobacco growth traits and improves tobacco quality. Summary of the invention

[0009] The technical problem to be solved by the present invention is to overcome the defect that the existing microbial preparations have poor effects in preventing and treating tobacco black shank disease and to improve the disease resistance of tobacco.

[0010] The invention provides a composite bacterial preparation for preventing and treating tobacco black shank disease, which consists of Bacillus Cereus, Pseudomonas fluorescens and cobalt chloride.

[0011] In some embodiments of the present invention, the content of cobalt chloride in the composite bacterial preparation is 10 g / kg.

[0012] In some embodiments of the present invention, the composite bacterial preparation is prepared by mixing powders of Bacillus cereus and Pseudomonas fluorescens in a mass ratio of 3:2.

[0013] In some embodiments of the present invention, the preparation method of the composite bacterial preparation comprises:

[0014] Bacillus cereus ATCC 9634 and Pseudomonas fluorescens ATCC 13525 were used as bacterial strains, and the bacterial strains were activated respectively;

[0015] The bacterial species are cultured to obtain seed solutions of Bacillus cereus and Pseudomonas fluorescens;

[0016] The seed solution is cultured to obtain fermentation liquid of Bacillus cereus and Pseudomonas fluorescens;

[0017] The fermentation liquid is centrifuged to obtain centrifugal cells of Bacillus cereus and Pseudomonas fluorescens;

[0018] The centrifuged bacterial bodies of Bacillus cereus and Pseudomonas fluorescens are spray-dried respectively to obtain Bacillus cereus and Pseudomonas fluorescens bacterial powders;

[0019] The powders of Bacillus cereus and Pseudomonas fluorescens were mixed in a mass ratio of 3:2 and 10 g of cobalt chloride was added per kilogram of powder.

[0020] The present invention also provides a method for preventing and controlling tobacco black shank disease, characterized in that any of the composite bacterial agents is diluted with water and then watered on the roots of tobacco plants.

[0021] In some embodiments of the present invention, the method comprises the following steps:

[0022] (1) After planting tobacco plants, the composite bacterial preparation is diluted with water at a mass ratio of 1:1000 to prepare a bacterial suspension, and the roots of the tobacco plants are watered for the first time;

[0023] (2) Four weeks after the first irrigation, prepare the bacterial suspension again and irrigate the roots of the tobacco plants.

[0024] In some embodiments of the present invention, the single dosage of the bacterial suspension is 100-200 mL per plant.

[0025] The present invention also provides a method for using any of the microbial composite agents, characterized in that the microbial composite agent is diluted with water at a mass ratio of 1:1000-2000 to prepare a bacterial suspension, which is then used to irrigate the roots of tobacco plants.

[0026] According to actual needs, the bacterial suspension of the composite bacterial agent can be used to irrigate the roots of tobacco plants before or after planting. In some embodiments of the present invention, when watering is needed after the tobacco plants are cultivated, the bacterial suspension of the composite bacterial agent is used to irrigate the roots of the tobacco plants to increase the number of active microorganisms colonized at the roots of the tobacco plants. In some embodiments of the present invention, when tobacco seedlings are cultivated in a normal manner, the microbial composite bacterial agent is diluted with water at a mass ratio of 1:1000 and then poured into the roots of the tobacco plants, and the single dosage of the bacterial agent is 100-200mL / plant; according to the disease situation, the bacterial agent can be supplemented after the second week or the fourth week.

[0027] The present invention not only improves the disadvantage of insufficient adaptability of a single biocontrol strain in the environment after compounding Bacillus Cereus, Pseudomonasfluorescens and cobalt chloride, but also can breed beneficial microbial dominant flora at the root of plants, improve soil microecological environment, and enhance plant disease resistance. Field tests have shown that after Bacillus Cereus, Pseudomonasfluorescens and cobalt chloride are mixed and applied in a certain proportion, the control effect of tobacco on black shank disease is greatly improved, and it has an obvious growth-promoting effect on tobacco, and can enhance the ability of tobacco to resist pathogens and other diseases and insect pests. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0029] Suspension concentrate, also known as water suspension concentrate or gel suspension concentrate, is a viscous formulation that can flow by dispersing the water-insoluble or poorly soluble original drug into the aqueous medium under the action of various additives to form a uniform and stable dispersion system. It can replace emulsifiable concentrates containing a large amount of solvents, and can also replace wettable powders containing a large amount of additives, and has the characteristics of no dust hazard, low volatility, no toxic side effects, and high efficiency.

[0030] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0031] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "approximately" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0032] In the following embodiments of the present invention, when the temperature is not particularly limited, it is room temperature. Room temperature refers to the natural room temperature in four seasons without additional cooling or heating treatment, and is generally controlled at 10-30°C, preferably 15-25°C.

[0033] Example 1

[0034] 1. Bacillus cereus fermentation

[0035] Bacillus cereus was purchased from ATCC, USA.

[0036] Seed medium (W / V): peptone 1%, yeast extract 0.5%, sodium chloride 1%, pH 7.0.

[0037] Fermentation medium (W / V): sucrose 1%, peptone 1%, disodium hydrogen phosphate 0.2%, sodium dihydrogen phosphate 0.2%, pH 7.0.

[0038] Fermentation method:

[0039] 1) Activation of bacterial strains: Transfer the preserved bacterial strains to slant culture medium, activate them at 37°C for 24 hours, and set aside.

[0040] 2) Preparation of seed solution: Place 50 ml of seed culture medium in a 250 ml Erlenmeyer flask, add one loop of activated bacteria, and culture at 37°C and 180 rpm for 20 h.

[0041] 3) Fermentation: 100 ml of seed solution was inoculated into a fermenter containing 50 L of fermentation solution at an inoculation rate of 0.2%, and cultured at 37° C. and 180 r / min for 36 h.

[0042] 4) Centrifugal collection of bacteria: After fermentation is completed, place the culture medium in a centrifuge and centrifuge at 10,000 r / min to collect the bacteria.

[0043] 5) Drying of bacterial powder: The bacterial cells collected by centrifugation are placed on a spray dryer to obtain dry bacterial powder.

[0044] 2. Pseudomonas fluorescens fermentation

[0045] Pseudomonas fluorescens was purchased from ATCC, USA.

[0046] Seed medium (W / V): peptone 1%, yeast extract 0.5%, sodium chloride 1%, pH 7.0.

[0047] Fermentation medium (W / V): sucrose 1%, peptone 1%, disodium hydrogen phosphate 0.2%, sodium dihydrogen phosphate 0.2%, pH 7.0.

[0048] Fermentation method:

[0049] 1) Activation of bacterial strains: Transfer the preserved bacterial strains to slant culture medium, activate them at 37°C for 24 hours, and set aside.

[0050] 2) Preparation of seed solution: Place 50 ml of seed culture medium in a 250 ml Erlenmeyer flask, add one loop of activated bacteria, and culture at 37°C and 180 rpm for 20 h.

[0051] 3) Fermentation: 100 ml of seed solution was inoculated into a fermenter containing 50 L of fermentation solution at an inoculation rate of 0.2%, and cultured at 37° C. and 180 r / min for 36 h.

[0052] 4) Centrifugal collection of bacteria: After fermentation is completed, place the culture medium in a centrifuge and centrifuge at 10,000 r / min to collect the bacteria.

[0053] 5) Drying of bacterial powder: The bacterial cells collected by centrifugation are placed on a spray dryer to obtain dry bacterial powder.

[0054] Example 2

[0055] Prepare different compound bacterial agents according to the proportions in the following table:

[0056] Table 1

[0057]

[0058]

[0059] Example 3

[0060] Potted plant protection experiment

[0061] The tobacco seedlings were transplanted into a cultivation pot, and after 40 days, inoculated with tobacco phytophthora. The tobacco seedling pots with black shank were randomly divided into 9 groups, and the composite bacterial preparation configured in Example 2 was diluted with water at a mass ratio of 1:1000 to prepare a bacterial suspension. 100-200 ml of the bacterial suspension was taken for each tobacco plant to irrigate the roots of the plants, and the second irrigation was performed after the fourth week. Statistics were conducted in the 8th week, and the incidence was investigated in accordance with the standard GB / T23222-2008. The results are shown in Table 2 below.

[0062] Table 2

[0063] Group Black shank control efficacy A 67.4％ B 65.2％ C 74.8％ D 90.3％ E 77.5％ F 80.1％ G 88.6％ H 72.7％ I 60.3％

[0064] From the statistical results in Table 2, it can be seen that the composite bacteria prepared by Bacillus cereus and Pseudomonas fluorescens have a certain synergistic effect, which can improve the effect of the composite bacteria in preventing and controlling tobacco black shank disease, but the effect is limited. The composite bacteria prepared by adding cobalt chloride can greatly enhance its control effect. In particular, the composite bacteria preparation of group D has a control effect of more than 90%. Based on this result, we further optimized the ratio of Bacillus cereus and Pseudomonas fluorescens.

[0065] Example 4

[0066] Prepare different compound bacterial agents according to the proportions in the following table:

[0067] Table 3

[0068] Group Bacillus cereus(g) Pseudomonas fluorescens (g) Cobalt chloride (mg) 1 2 8 1 2 3 7 1 3 4 6 1 4 5 5 1 5 6 4 1 6 7 3 1 7 8 2 1

[0069] Referring to the potted plant control effect experiment in Example 3, the control effects of different ratios of Bacillus cereus and Pseudomonas fluorescens were statistically analyzed, and the results are shown in Table 4.

[0070] Table 4

[0071]

[0072]

[0073] As shown in Table 4, under the composite bacteria configuration conditions of Group 5, the synergistic effect was maximized, with a control effect of up to 95%.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composite bacterial preparation for preventing and treating tobacco black shank disease, It is characterized in that By Bacillus cereus ( Bacillus Cereus ), Pseudomonas fluorescens ( Pseudomonas fluorescens ) and cobalt chloride, the preparation method of the composite bacterial preparation comprises: Bacillus cereus ATCC 9634 and Pseudomonas fluorescens ATCC 13525 were used as bacterial strains, and the bacterial strains were activated respectively; The bacterial species are cultured to obtain seed solutions of Bacillus cereus and Pseudomonas fluorescens; The seed solution is cultured to obtain fermentation liquid of Bacillus cereus and Pseudomonas fluorescens; The fermentation liquid is centrifuged to obtain centrifugal cells of Bacillus cereus and Pseudomonas fluorescens; The centrifuged bacterial bodies of Bacillus cereus and Pseudomonas fluorescens are spray-dried respectively to obtain Bacillus cereus and Pseudomonas fluorescens bacterial powders; The powders of Bacillus cereus and Pseudomonas fluorescens were mixed in a mass ratio of 3:2 and 0.1 g of cobalt chloride was added per kilogram of powder.

2. A method for preventing and treating tobacco black shank, It is characterized in that The composite bacterial preparation according to claim 1 is diluted with water and then watered on the roots of tobacco plants.

3. The method according to claim 2, It is characterized in that The method comprises the following steps: (1) After planting tobacco plants, the composite bacterial preparation is diluted with water at a mass ratio of 1:1000 to prepare a bacterial suspension, and the roots of the tobacco plants are watered for the first time; (2) Four weeks after the first irrigation, prepare the bacterial suspension again and irrigate the roots of the tobacco plants.

4. The method according to claim 3, It is characterized in that The single dosage of the bacterial suspension is 100-200 mL per plant.

5. A method for using the composite bacterial preparation according to claim 1, It is characterized in that The composite bacterial preparation is diluted with water at a mass ratio of 1:1000-2000 to prepare a bacterial suspension, which is then used to irrigate the roots of tobacco plants.

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