Method for preparing bacterial powder of bacillus amyloliquefaciens

Abstract

Disclosed in the present invention is a method for preparing bacterial powder of Bacillus amyloliquefaciens. The main component of the present invention is Bacillus amyloliquefaciens C4; and the present invention further comprises kaolin, sodium dodecylbenzenesulfonate, sodium lignosulfonate and humic acid. The components in weight percentages are 3% of Bacillus amyloliquefaciens C4, 20% of kaolin, 6% of sodium dodecylbenzenesulfonate, 5% of sodium lignosulfonate, 6% of humic acid and 60% of water, respectively. The preparation method comprises: (1) activating a strain and preparing a fermentation broth; (2) determining the optimal fermentation condition for the Bacillus amyloliquefaciens C4 strain; and (3) inspecting the quality of wettable powder of the Bacillus amyloliquefaciens C4. After quality inspection, all indexes of the wettable powder of the Bacillus amyloliquefaciens C4 in the present invention meet the national standard. The wettable powder of the Bacillus amyloliquefaciens C4 has the characteristics of low production cost, high active-ingredient content, easy storage, environmentally friendly, etc.; exhibits relatively low toxicity to crops; and, when applied to Solanum tuberosum, also shows a significant prevention effect against bacterial wilt.

Description

A method for preparing bacterial powder of Bacillus amyloliquefaciens Technical Field

[0001] The present invention relates to the field of biological pesticides, and more particularly to bacterial powder of Bacillus amyloliquefaciens and a preparation method and application thereof. Background Art

[0002] Bacillus amyloliquefaciens is a Gram-positive bacterium with strong stress resistance and high antibacterial activity. In industrial production, it is considered a safe and non-toxic microorganism and is one of the main species currently studied in biocontrol studies. Microbial agents are an emerging product with bactericidal, insecticidal, weed-killing, and plant growth-improving effects. Among the many types of microbial agents, wettable powders are the main formulation studied in my country. Compared with other powders, wettable powders have higher active ingredients, lower production costs, and a wide range of applications. Therefore, developing a wettable powder of Bacillus amyloliquefaciens and applying it to agricultural production can reduce pesticide residues, improve soil fertility and fertilizer utilization, promote crop growth, effectively prevent and control crop diseases and pests, and improve the quality of agricultural products.

[0003] The characteristics and advantages of the present invention are that Bacillus amyloliquefaciens is a common beneficial microorganism that can prevent and treat plant diseases and insect pests, has the characteristics of being various and having a fast reproduction speed, and can produce a large amount of substances with antimicrobial activity, immunological activity and antioxidant activity during its growth and metabolism process, and is widely used in multiple fields such as agriculture and animal husbandry. The wettable powder contains the biocontrol bacteria thalline and the substance produced by it that has an antibacterial effect on plant diseases and insect pests, and has the advantages of being stable in activity, convenient in transportation, resistant in storage, easy to produce and commercialized. Therefore, the wettable powder produced by using Bacillus amyloliquefaciens as the thalline makes the method have the advantages of being easy to obtain the thalline, rapid in industrialization, low in production cost, and environmental protection in terms of agricultural production, and provides a theoretical basis for the industrial production of microbial agents.

[0004] In summary, how to provide a bacterial powder of Bacillus amyloliquefaciens is a problem that those skilled in the art urgently need to solve.

[0005] Summary of the Invention

[0006] In view of this, the present invention provides a Bacillus amyloliquefaciens powder, a preparation method and an application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention provides a bacillus amyloliquefaciens bacterial powder. The bacillus amyloliquefaciens C4 fermentation liquid is used as a raw material, 20% of kaolin is used as a carrier, 6% of sodium dodecylbenzenesulfonate is used as a wetting agent, 5% of sodium ligninsulfonate is used as a dispersant, 6% of humic acid is used as a protective agent, 60% of water is added, mixed, dried and crushed to obtain the bacillus amyloliquefaciens bacterial powder.

[0009] A method for preparing bacterial powder of Bacillus amyloliquefaciens comprises the following steps:

[0010] (1) Strain activation and fermentation broth preparation: Bacillus amyloliquefaciens C4 was inoculated onto LB solid medium and cultured at 37°C for 12 h. It was then inoculated into 100 mL of LB liquid medium and cultured with shaking at 37°C and 160 rpm. Fermentation broth was obtained after 12 h.

[0011] (2) Determination of the optimal fermentation conditions for Bacillus amyloliquefaciens C4 strain: An orthogonal experiment with 4 factors (pH, temperature, rotation speed, inoculation size) and 3 levels was set up to determine the optimal fermentation conditions. The viable cell count and active substance content of Bacillus amyloliquefaciens C4 were measured. The Bacillus amyloliquefaciens C4 was fermented under the optimized conditions and set aside.

[0012] (3) Quality testing of Bacillus amyloliquefaciens C4 wettable powder: The fermentation liquid of Bacillus amyloliquefaciens C4 was mixed with the carrier and adjuvant selected in the most suitable amount, placed in a vacuum drying oven, dried at 45°C, and the weight of the sample was tested. When the weight of the sample remained unchanged, the sample was taken out and crushed. After the various quality indicators of the wettable powder were tested and met the standards, the Bacillus amyloliquefaciens C4 wettable powder was obtained.

[0013] Furthermore, in the above step (2), the optimal fermentation conditions of the strain are: pH 7.0, temperature 28°C, rotation speed 200r / min, inoculation size 3%, and fermentation time 13d.

[0014] The present invention also claims protection for the use of the above-mentioned Bacillus amyloliquefaciens powder or the Bacillus amyloliquefaciens powder prepared by the above-mentioned preparation method in the prevention and treatment of potato bacterial wilt.

[0015] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the beneficial effect of improving the disease defense capability of crops. DETAILED DESCRIPTION

[0016] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0017] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0018] Example 1

[0019] Optimization of Fermentation Conditions of Bacillus amyloliquefaciens C4

[0020] The viable cell count and active substance content of Bacillus amyloliquefaciens C4 were determined. The viable cell count was determined by the plate colony count method. The specific detection method refers to GB20287-2006. The active substance content was determined by using high performance liquid chromatography to detect the content of surfactant after optimization of Bacillus amyloliquefaciens C4 fermentation.

[0021] Table 1 Determination of the number of viable bacteria of Bacillus amyloliquefaciens C4 and the content of active substances before and after optimization

[0022] Bacillus amyloliquefaciens produces substances with antimicrobial activity. Its growth and the amount of active substances it produces are affected by culture conditions. Some factors can promote the growth of the strain, while others can inhibit it. The activity and amount of active substances produced by the same strain vary under different culture conditions. The results showed that under the culture conditions of pH 7.0, temperature 28°C, rotation speed 200 r / min, and strain inoculum size 3%, the viable count of Bacillus amyloliquefaciens C4 was 73.33×10 8 CFU / mL, which is the optimal culture condition for Bacillus amyloliquefaciens C4. The surfactin content increased significantly after optimization, reaching 2.45 times that before optimization.

[0023] Example 2

[0024] Wettable powder quality testing

[0025] The fermentation liquid is mixed with the carrier and auxiliary agent with the optimal addition amount screened out, placed in a vacuum drying oven, and dried at a temperature of 45°C. The sample weight is tested. When the sample weight change stabilizes, the sample is taken out and crushed with a grinder. The quality of the prepared wettable powder is tested.

[0026] Table 2 Determination of wettable powder sample indicators

[0027] Wettable powders are prepared by mixing and crushing the active ingredients of a microbial agent with the corresponding excipients, and then diluting them with water when applied. The carriers and adjuvants screened during the preparation process will affect the application effect of the wettable powder, so it is important to select carriers and adjuvants that match the strain. The viable bacterial count and quality indicators of the powder (pH, wettability, suspension rate, water content, fineness, etc.) are also methods for evaluating the quality of the preparation process. The above data show that all indicators of Bacillus amyloliquefaciens C4 wettable powder meet national standards (GB 20287-2006 Agricultural Microbial Agents, GB / T14825-2006 Pesticide Suspension Rate Determination Method, GB / T 5451-2001 Pesticide Wettable Powder Wettability Determination Method).

[0028] Example 3

[0029] Analysis on the biocontrol effect of Bacillus amyloliquefaciens C4 wettable powder

[0030] Potato leaves of similar shape and size were selected. Degreased cotton wool moistened with distilled water was then wrapped around the petiole. The leaves were then placed in a Petri dish containing filter paper, which was moistened with a small amount of sterile distilled water. 30 μL of each of the inoculum at different dilutions (300, 500, and 1000x) and the original bacterial solution was evenly applied to the leaf surface. A control group (CK) treated with the same volume of sterile distilled water was used and incubated in a lighted incubator. After 24 hours, 20 μL of the pathogenic bacteria of potato bacterial wilt was inoculated onto the leaves. The inoculation continued for another 6 days, and the diameter of the lesions was measured.

[0031] Table 3 Preventive effect of different dilution multiples of bacterial agents on potato bacterial wilt

[0032] This experiment used the pathogen of potato bacterial wilt as an indicator bacteria. Potato leaves were treated with Bacillus amyloliquefaciens C4 WP at varying dilutions. The diameter of the lesions on the leaves was measured to verify the biocontrol efficacy of Bacillus amyloliquefaciens C4 WP. The results showed that the WP was most effective against potato bacterial wilt when undiluted. A 500-fold dilution also demonstrated a high preventive effect, reaching 51.05%. However, higher concentrations of the inoculant often reduced its activity. Considering production costs, a 500-fold dilution of Bacillus amyloliquefaciens C4 WP should be used in actual production.

[0033] In summary, the Bacillus amyloliquefaciens C4 wettable powder in the present application has a certain preventive effect on potato bacterial wilt and can be promoted and applied.

[0034] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0035] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A powder of Bacillus amyloliquefaciens, characterized in that The invention comprises the following raw materials in parts by weight: 43% of Bacillus amyloliquefaciens C, 20% of kaolin, 6% of sodium dodecylbenzenesulfonate, 5% of sodium ligninsulfonate, 6% of humic acid and 60% of water.

2. A method for preparing bacterial powder of Bacillus amyloliquefaciens, characterized in that: The specific steps include: (1) Strain activation and fermentation broth preparation; (2) Determination of the optimal fermentation conditions for Bacillus amyloliquefaciens C4 strain; 3. Quality testing of Bacillus amyloliquefaciens C4 wettable powder.

4. The method for preparing a Bacillus amyloliquefaciens powder according to claim 2, wherein In step (2), the optimal fermentation conditions of the strain are: pH 7.0, temperature 28° C., rotation speed 200 r / min, inoculation size 3%, and fermentation time 13 days.

5. Use of the Bacillus amyloliquefaciens powder according to claim 1 or the Bacillus amyloliquefaciens powder prepared by the preparation method according to claims 2-3 in the prevention and treatment of potato bacterial wilt.

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