Purification method of buckwheat white mold pathogenic bacteria

Abstract

The invention discloses a purification method of buckwheat white mold pathogenic bacteria, which belongs to the technical field of biology, and comprises the following steps: S1, inoculating buckwheat white mold pathogenic bacteria spores on a PDA solid plate culture medium by using a sterilized brush or a single cell picking method, and culturing for 10 days; s2, punching the culture medium obtained in the step S1 by using a fungus cake puncher to obtain a fungus cake; s3, the fungus cakes obtained in the step S2 do circular motion on the edge of a new PDA culture medium to rub back and forth, so that white mold spores on the fungus cakes are rubbed to the culture medium, and culture is conducted. When the purification method disclosed by the invention is used for culturing the buckwheat white mold pathogenic bacteria, a large number of target strains after purification culture can be obtained about 10 days after a new PDA culture medium is inoculated with a bacterial cake.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a method for purifying the pathogen of white mold in buckwheat. Background Technology

[0002] Buckwheat white mold is Teratoramularia persicariae This is an important fungal disease caused by [a fungal name - likely a fungal infection], widely distributed in buckwheat growing areas of my country. In severe cases, the incidence rate can reach 100%. This disease primarily infects buckwheat leaves, causing white mold on the underside of the leaves, premature aging, and slow plant growth, leading to a significant decrease in yield and quality, resulting in serious economic losses for buckwheat production.

[0003] The traditional method for purifying bacterial strains involves first culturing the strain on a culture medium, then using a mycelium cake punch to create holes in the medium, and finally transferring the resulting mycelium cakes to a new culture medium for further cultivation to obtain the purified strain. While this method allows for rapid cultivation of most strains, the inventors discovered that when culturing the pathogen of white mold on buckwheat, the method resulted in slow strain growth. After 30 days of cultivation on a new culture medium, the mycelium cakes only covered about 1 / 5 of the plate, indicating low purification efficiency. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the present invention proposes a purification method for the pathogen of white mold in buckwheat.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: A method for purifying the pathogen causing white mold in buckwheat includes the following steps: S1. Use a sterilized brush or single-cell picking method to inoculate buckwheat white mold pathogen spores onto PDA solid plate medium and culture for 7-10 days. S2, Use a mushroom cake puncher to punch holes in the culture medium obtained in step S1 to obtain mushroom cakes; S3. The mycelium cake obtained in step S2 is rubbed back and forth in a circular motion on the edge of a new PDA medium to transfer the white mold spores on the mycelium cake onto the medium for cultivation.

[0006] Furthermore, the formula for the PDA culture medium is: 200g potato, 20g glucose, 20g agar, 1L water, natural pH.

[0007] Furthermore, in step S3, the culture conditions are: room temperature, with light and darkness alternating every 12 hours.

[0008] Furthermore, in step S2, the diameter of the mushroom cake is 5 mm.

[0009] Through the above technical solution, the present invention can achieve at least the following beneficial effects: 1. When culturing the buckwheat white mold pathogen using the purification method described in this invention, a large number of purified target strains can be obtained in about 10 days after the mycelial cake is inoculated into a new PDA medium. 2. Purifying the obtained strain requires studying its biological characteristics (C source, N source, temperature, pH, etc.), and calculating the growth area. Using a punch to obtain a mycelial cake, placing it in the middle of the culture medium to allow the strain to grow, the area covered by the mycelial cake will cause errors when calculating the area. However, in this application, the strain is scraped along the edge of the culture medium, and it can only grow inward. When calculating the area, the large circle is subtracted from the small circle that has not grown in the middle, and the error is smaller. 3. Traditional inoculation methods using inoculation needles and loops are inefficient, and the strains cannot grow in a specific shape, making it difficult to calculate the area. The same applies to coating sticks. In this invention, the diameter of the mycelium cake is fixed, and it is rubbed back and forth along the edge of the petri dish. The width formed by the circumferential movement is the diameter of the mycelium cake. The inoculation method is simple and uniform, and the inoculation width is easy to control. Attached Figure Description

[0010] Figure 1 This is a picture of the mycelium obtained by drilling holes and then culturing it on fresh PDA medium for 10 days according to the purification method of Example 1 of this invention. Figure 2 This is a photograph of a mycelium obtained by drilling holes and then culturing it on a new PDA medium for 10 days, following the conventional method described in the background art. Detailed Implementation

[0011] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0012] Example 1 Preparation of PDA medium The PDA culture medium comprises the following ingredients by weight: 190-210 parts potato, 18-22 parts agar powder, and 18-22 parts glucose. In this embodiment, the specific preparation method is as follows: Weigh 2000g of potatoes, wash and peel them, cut them into small pieces, boil them until soft, filter through gauze, add water to 10000ml, add 200g of agar while hot, and after the agar is fully dissolved, add 200g of glucose, stir well, pour into plates, and dispense. Seal with sealing film and newspaper, then autoclave at 121℃ for 20 minutes to obtain the PDA culture medium.

[0013] Example 2 A method for purifying the pathogen causing white mold in buckwheat includes the following steps: S1. Use a sterilized brush or single-cell picking method to inoculate buckwheat white mold pathogen spores onto PDA solid plate medium and culture for 10 days. S2, use a mycelium cake puncher to punch holes in the culture medium obtained in step S1 to obtain 5mm mycelium cakes; S3. The mycelial cake obtained in step S2 is rubbed back and forth in a circular motion along the edge of a new PDA medium, causing the white mold spores on the cake to be transferred to the medium for cultivation. The cultivation period is 10 days. A picture of the specimen after 10 days of cultivation is attached. Figure 1 As shown.

[0014] Comparative Example 1 A method for purifying the pathogen causing white mold in buckwheat includes the following steps: S1. Use a sterilized brush or single-cell picking method to inoculate buckwheat white mold pathogen spores onto PDA solid plate medium and culture for 10 days. S2, use a mycelium cake puncher to punch holes in the culture medium obtained in step S1 to obtain 5mm mycelium cakes; S3. Incubate the mycelial cakes obtained in step S2 in the center of a fresh PDA medium for 20 days. A picture of the final product after 20 days of incubation is attached. Figure 2 As shown.

[0015] Example 3 The diameter of the buckwheat white mold pathogen colonies in Example 2 and Comparative Example 1 was measured using a measuring tool, and the colony area was calculated.

[0016] Area (S) = πr 2 Measurement Figure 1 and Figure 2 The colony diameter in the culture plate is 8.5 cm.

[0017] Figure 1 The width of the colony loop is 1.1 cm (measured at the narrowest point). Figure 2 The colony diameter was 1.9 cm (measured at the point of maximum diameter). This was calculated. Figure 1 The area is 2.46πcm 2 , Figure 2 The area is 0.90πcm 2 Therefore, it can be seen that the method of the present invention can significantly improve the purification and culture efficiency of the buckwheat white mold pathogen.

[0018] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for purifying the pathogen of white mold in buckwheat, characterized in that: Includes the following steps: S1. Use a sterilized brush or single-cell picking method to inoculate buckwheat white mold pathogen spores onto PDA solid plate medium and culture for 10 days. S2, Use a mushroom cake puncher to punch holes in the culture medium obtained in step S1 to obtain mushroom cakes; S3. The mycelium cake obtained in step S2 is rubbed back and forth in a circular motion on the edge of a new PDA medium to transfer the white mold spores on the mycelium cake onto the medium for cultivation.

2. The purification method of the buckwheat white mold pathogen according to claim 1, characterized in that: The PDA culture medium contains the following components in parts by weight: 190-210 parts potato, 18-22 parts agar powder, and 18-22 parts glucose.

3. The method for purifying the buckwheat white mold pathogen according to claim 1, characterized in that: In steps S1 and S3, the culture conditions for the strain are: room temperature, with light and darkness alternating every 12 hours.

4. The purification method of the buckwheat white mold pathogen according to claim 1, characterized in that: In step S2, the diameter of the mushroom cake is 5 mm.

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