Biological agent of bacillus amyloliquefaciens and use thereof for preventing and controlling soil-borne disease of solanum tuberosum

Abstract

Provided are a biological agent of Bacillus amyloliquefaciens and the use thereof for preventing and controlling a soil-borne disease of Solanum tuberosum. The main component is Bacillus amyloliquefaciens C4. A preparation method, comprising: (1) activating a strain; (2) fermenting; and (3) collecting supernatant. The Bacillus amyloliquefaciens C4 in the present invention has antibacterial activity, and produces relatively low toxicity to crops. The Bacillus amyloliquefaciens C4 is prepared into the biological agent, which is applied to a soil-borne disease of Solanum tuberosum, and a result shows that the agent has a certain prevention and control effect.

Description

A Bacillus amyloliquefaciens biological preparation and its application in preventing and controlling potato soil-borne diseases Technical Field

[0001] The present invention relates to the fields of microbial technology and biological control, and more particularly to the application of Bacillus amyloliquefaciens C4 in the prevention and control of soil-borne potato diseases. Background Art

[0002] Bacillus amyloliquefaciens is a biocontrol bacterium that secretes antimicrobial substances such as antimicrobial proteins, hydrolases, and lipopeptides. It has excellent control effects on a variety of plant diseases and is widely used in antimicrobial and plant disease control. Potato (Solanum tuberosum) is a plant of the genus Solanum in the Solanaceae family and the fourth largest food crop worldwide. Soilborne potato diseases are caused by pathogens that grow in the soil, breaking through the protective outer skin and attacking the roots or stems. As potato cultivation continues to expand in my country, crop rotation is becoming increasingly common, and pathogenic bacteria are accumulating in the soil. This has led to an increase in soilborne diseases such as dry rot, bacterial wilt, soft rot, and scab, causing devastating damage to potato yield and quality. Currently, chemical control is the primary method for controlling soilborne potato diseases, but this approach poses challenges such as pesticide residues and environmental pollution. Therefore, developing a Bacillus amyloliquefaciens biological agent and applying it to soil-borne diseases of potatoes can not only protect the environment and reduce drug residues, but also reduce costs in the actual production process.

[0003] The characteristics and advantages of the present invention are that Bacillus amyloliquefaciens has the characteristics of rich antibacterial metabolites, strong antibacterial effect, broad antibacterial spectrum, high stability, no toxicity to humans and animals, and environmental friendliness. It can be made into biological preparations and widely used in the process of plant disease prevention and control.

[0004] In summary, how to provide a biological preparation of Bacillus amyloliquefaciens is an urgent problem that needs to be solved by those skilled in the art.

[0005] Summary of the Invention

[0006] In view of this, the present invention provides a Bacillus amyloliquefaciens biological preparation and its application in preventing and controlling soil-borne diseases of potato.

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

[0008] The present invention provides a Bacillus amyloliquefaciens biological preparation. The Bacillus amyloliquefaciens C4 strain is first activated in a liquid culture medium and then cultured with shaking at 37°C and 200 rpm for 48 hours to obtain a fermentation broth. The resulting fermentation broth is centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant is collected to obtain the fermentation supernatant.

[0009] A method for preparing a Bacillus amyloliquefaciens biological preparation comprises the following steps:

[0010] (1) Strain activation: Bacillus amyloliquefaciens C4 stored at 4°C was activated in LB liquid medium and cultured at 37°C and 200 rpm for 24 h;

[0011] (2) Fermentation: The activated Bacillus amyloliquefaciens C4 was inoculated into LB liquid culture medium and cultured at 37°C and 200 rpm for 48 h to obtain a fermentation broth;

[0012] (3) Collecting the supernatant: The fermentation broth obtained in (2) was centrifuged at 4°C and 8000 rpm for 15 min. The collected supernatant was the fermentation supernatant. The fermentation supernatant was sterilized by passing through a 0.22 μm filter membrane.

[0013] Furthermore, in the above step (2), the fermentation conditions of the strain are: temperature 37° C., rotation speed 200 r / min, and fermentation time 48 h.

[0014] The present invention also seeks to protect the use of the above-mentioned Bacillus amyloliquefaciens biological preparation or the Bacillus amyloliquefaciens biological preparation prepared by the above-mentioned preparation method in the prevention and treatment of soil-borne diseases of potatoes.

[0015] It can be seen from the above technical solutions that, compared with the prior art, the beneficial effect achieved by the present invention is: effectively improving the prevention and control effect of potato diseases. 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] Antibacterial activity of C4 fermentation broth and supernatant against major potato pathogens

[0020] The inhibition zone method was used to determine the antibacterial activity of major potato pathogens. 20 mL of LB solid medium was poured onto a sterilized plate. After the medium solidified, 100 μL of pathogenic bacterial culture (Ralstonia solanacearum, Erwinia carotovora ssp. Carotovora, Streptomyces acidiscabies) was evenly spread onto the medium and allowed to dry. 15 μL of Bacillus amyloliquefaciens C4 fermentation broth (or fermentation supernatant) was then injected onto a 5 mm diameter circular filter paper and placed in the center of the solidified plate using sterile tweezers. (To ensure a firm fit between the filter paper and the medium, gently press the filter paper with tweezers.) The plate was incubated at 37°C for 48 hours, and the diameter of the inhibition zone was measured. 15 μL of HO was used as a control. Each group was repeated four times.

[0021] The antibacterial activity of potato pathogens was determined using the plate standoff method: Fuarium sambucinum, a pathogenic fungus, cultured for 7 days on a PDA plate, was beaten into a 5 mm diameter cake and inoculated onto the center of a prepared PDA solid medium. Bacillus amyloliquefaciens C4 fermentation broth (or fermentation supernatant) was then inoculated 2.5 cm from the cake. Four inoculations were performed on each PDA plate, with sterile water used as a blank control. The plates were incubated at 28°C for 7 days, and the colony diameters of the treated and control groups were measured.

[0022] Table 1 Determination of antibacterial activity of C4 fermentation broth and supernatant against major potato pathogens

[0023] No inhibition zones were observed in the control group for three pathogenic bacteria (Erwinia carotovora ssp. Carotovora, Ralstonia solanacearum, and Streptomyces acidiscabies). However, distinct inhibition zones were observed for all three pathogens treated with the fermentation broth and supernatant. The results demonstrated that both the C4 fermentation broth and supernatant exhibited significant inhibitory activity against all three pathogens, with the C4 fermentation broth exhibiting superior inhibition. After seven days of fungal confrontation culture, the pathogenic colonies in the control group (CK) had completely filled the plate. Both the C4 fermentation broth and supernatant exhibited varying degrees of inhibition against Fuarium sambucinum, the pathogen of potato dry rot, with the C4 fermentation broth exhibiting superior inhibition compared to the supernatant.

[0024] Example 2

[0025] Analysis of the biocontrol effect of Bacillus amyloliquefaciens C4 fermentation liquid on soil-borne potato diseases

[0026] Healthy, uniformly sized potato leaves were selected and their petioles were wrapped with soaked cotton wool. Then, 50 μL of Bacillus amyloliquefaciens C4 fermentation broth was evenly applied to the leaf surface. An equal amount of sterile water (H2O) was applied as a control. The leaves were incubated at 20°C under a 16 / 8 hour light / dark cycle for 24 hours. 20 μL of pathogenic bacterial culture (Ralstonia solanacearum, Erwinia carotovora ssp. Carotovora, Streptomyces acidiscabies) was applied to the potato leaves. A 5 mm thick cake of the pathogenic fungus Fuarium sambucinum was inoculated at the leaf tip and incubated at 20°C under a 16 / 8 hour light / dark cycle for 9 days, maintaining moisture. The diameter (cm) of the plaque was measured at 3, 5, 7, and 9 days.

[0027] Table 2 Biocontrol effect of Bacillus amyloliquefaciens C4 fermentation broth on major potato pathogens

[0028] After inoculation of potato leaves with major soilborne pathogens, the diameter of lesions gradually increased with treatment time, and the diameter of lesions on leaves inoculated with the CK treatment was significantly greater than that on leaves treated with the C4 fermentation solution. On the seventh day after treatment, the diameter of lesions on leaves inoculated with Ralstonia solanacearum after CK treatment was 2.2 times that of lesions on leaves inoculated with the C4 fermentation solution. On the third, fifth, and seventh days after inoculation with Erwinia carotovora ssp. Carotovora, the diameter of lesions on leaves reached over 2.2 times that of lesions on leaves inoculated with the C4 fermentation solution. On the ninth day after treatment, the diameter was 1.85 times that of lesions on leaves inoculated with the C4 fermentation solution. On the seventh day after inoculation with the pathogen Streptomyces acidiscabies, the diameter of lesions on leaves inoculated with the CK treatment was over 1.94 times that of lesions on leaves inoculated with the C4 fermentation solution. Nine days after inoculation with Fuarium sambucinum, the pathogen responsible for potato dry rot, the diameter of the lesions on CK leaves was 1.88 times greater than that on leaves treated with C4 fermentation broth. These results demonstrate that Bacillus amyloliquefaciens C4 fermentation broth can enhance potato disease resistance and demonstrate a certain biocontrol efficacy, providing a theoretical basis for the prevention and control of potato diseases.

[0029] In summary, the Bacillus amyloliquefaciens biological preparation in this application has a certain prevention and control effect on potato soil-borne diseases.

[0030] 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.

[0031] 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 Bacillus amyloliquefaciens biological preparation, characterized in that Mainly includes Bacillus amyloliquefaciens C4.

2. A Bacillus amyloliquefaciens biological preparation, characterized in that The specific steps include: (1) Strain activation; (2) fermentation; (3) Collect the supernatant.

3. The method for preparing a Bacillus amyloliquefaciens biological preparation according to claim 2, characterized in that: In step (2), the fermentation conditions of the strain are: temperature 37° C., rotation speed 200 r / min, and fermentation time 48 h.

4. Use of the Bacillus amyloliquefaciens biological preparation according to claim 1 or the Bacillus amyloliquefaciens biological preparation prepared by the preparation method according to claim 2 in the prevention and control of soil-borne diseases of potatoes.

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