Method for separating and culturing pathogenic bacteria by directly picking sporocarp
By observing with a stereoscopic dissecting microscope and directly picking spores, combined with antibiotic culture medium and moisturizing culture, the problems of low efficiency and low purity of pathogen isolation in traditional methods are solved, and efficient and convenient pathogen isolation is achieved, which is suitable for indoor and outdoor environments.
Patent Information
- Application Number
- CN202510751633.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-09-16
AI Technical Summary
Traditional methods are difficult to accurately capture the spore-forming structure of ascomycetes, resulting in low efficiency and low purity of pathogen isolation. The operation is complex and is greatly affected by environmental and technical factors.
The surface of the diseased tissue was observed through a stereoscopic dissecting microscope, and the spores of the ascomycetes were directly picked and cultured in antibiotic MEA medium. Combined with the moisturizing culture strategy, spore germination and fruiting body formation were promoted, avoiding the complicated mycelium culture steps.
It improves the accuracy and purity of pathogen isolation, shortens the separation cycle, expands the scope of application, lowers the technical threshold, and enables more people to participate in separation and identification work, with a separation success rate of over 90%.
Smart Images
Figure CN120648565A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of agricultural disease prevention and control, and specifically to a method for directly picking fruiting bodies to isolate and culture pathogens. Background Art
[0002] The isolation and identification of plant pathogens is fundamental to plant pathology research and a key component of agricultural disease prevention and control. Isolating pathogens provides a deeper understanding of their biological characteristics, pathogenic mechanisms, and transmission methods, providing a scientific basis for developing effective prevention and control strategies. Pathogen isolation is also a crucial technical tool for disease diagnosis, epidemic investigation, and early warning.
[0003] In the process of isolating plant pathogens, traditional methods often rely on directly isolating mycelium from lesion tissue. However, this method has limitations when dealing with certain specific types of pathogens. In particular, for pathogens that can produce fruiting bodies, such as Ascomycetes, traditional methods (tissue separation methods) have difficulty accurately capturing spore-producing structures, resulting in low separation efficiency and low purity; methods that can accurately obtain spore-producing structures (single spore separation) also have certain limitations due to the complex operation process. In addition, traditional methods may also be affected by various factors such as environmental factors and operating techniques, further increasing the difficulty and uncertainty of separation.
[0004] Given the limitations of the above two isolation methods, it is particularly important to develop new, more efficient and accurate pathogen isolation technologies. Therefore, the method of directly picking fruiting bodies for pathogen isolation came into being in this context. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for directly picking fruiting bodies for pathogen isolation and cultivation. This technology effectively avoids the shortcomings of traditional methods by directly operating the spore-producing structure, improves separation efficiency and purity, and provides new ideas and methods for the isolation and identification of plant pathogens to solve the problems raised in the above-mentioned background technology.
[0006] In order to achieve the above object, the present invention adopts the following technical solution: a method for directly picking fruiting bodies for pathogen isolation and culture, comprising the following steps:
[0007] S1. Surface disinfection: Use an alcohol cotton ball to gently wipe the surface of the diseased tissue to remove surface bacteria and contaminants, providing a basis for subsequent aseptic operation;
[0008] S2. Observation and Picking: On a clean bench, carefully observe the surface of the diseased tissue using a stereoscopic dissecting microscope to locate and locate the ascomycete structures that produce the fruiting bodies. Then, carefully and precisely pick the spores using a dissecting needle or forceps.
[0009] S3. Inoculation and culture: Place the selected spores directly into MEA medium containing antibiotics and gently spread the spores on the surface of the medium with an inoculation loop to ensure even distribution of the spores. Then, incubate the medium at a constant temperature of 25°C to promote spore germination.
[0010] S4. Moisture-keeping culture: For samples where spore-forming structures have not yet formed, a moisturizing culture strategy is adopted to promote the formation of fruiting bodies. After the fruiting bodies mature, they are separated according to the above steps;
[0011] S5. Transfer culture: After the spores germinate, in order to avoid the influence of antibiotics on the growth of pathogens, they need to be transferred to a culture medium without antibiotics for further culture.
[0012] Preferably, the step S1 is performed by using 75% alcohol cotton balls for disinfection.
[0013] Preferably, the dissecting needle or tweezers in step S2 need to be sterilized by burning with an alcohol lamp and then cooled before picking.
[0014] Preferably, the antibiotic in step S3 is a mixture of penicillin and streptomycin.
[0015] Preferably, the MEA culture medium in step S3 contains 3% malt extract culture medium.
[0016] Preferably, the 3% malt extract culture medium comprises: 30 g malt extract powder, 20 g agar, and 1000 mL distilled water.
[0017] Preferably, in step S3, the culture medium is cultured at a constant temperature of 25° C. for 1-3 days.
[0018] Preferably, the specific steps of the moisturizing culture strategy are: placing the diseased tissue in a culture dish covered with filter paper, and promoting the formation of fruiting bodies by controlling the humidity and temperature conditions.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The present invention has high accuracy: compared with the tissue separation method, the advantage of this technology is that it can be observed through a stereoscopic dissecting microscope, and can accurately identify and locate the spore-producing structure, ensuring that the isolated pathogens are pure and highly active.
[0021] The present invention is highly efficient: it directly operates on the spore-producing structure, omitting the complicated mycelium culture and purification steps in the traditional method, and greatly shortening the separation cycle.
[0022] The invention has wide applicability: it is not only applicable to mature spore-producing ascomycetes, but also promotes the formation of fruiting bodies of immature samples through moisturizing culture, thereby expanding the application scope of the technology; it is not limited to being carried out in a laboratory (indoor) environment, but can also complete the isolation of pathogens under outdoor conditions. Outdoor separation does not require the use of a dissecting mirror. After direct surface disinfection, the fruiting body can be directly picked out and placed on the inclined surface of a 2ml centrifuge tube (cryotube) added with antibiotics prepared in advance, covered with a lid, and placed in a cryotube box, which can be taken back to the laboratory for observation and purification. Under sunny conditions, the outdoor separation success rate is generally above 90%, the separation success rate is high, and it is convenient and fast.
[0023] The present invention is easy to operate: it requires no complex equipment and can be mastered by general laboratory personnel after simple training. This lowers the technical barrier to entry, enabling more researchers and grassroots technicians to participate in the isolation and identification of pathogens. Furthermore, this ease of operation means the isolation process is faster and more efficient, enabling the acquisition of large quantities of pure pathogen samples in a short period of time, providing strong support for subsequent research and applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The present invention provides a flow chart of a method for directly picking fruiting bodies for pathogen isolation and culture. DETAILED DESCRIPTION
[0025] In order to further explain the technical solution of the present invention, specific embodiments are described in detail below.
[0026] Example 1
[0027] See also Figure 1 The present invention provides a method for directly picking fruiting bodies to isolate and culture pathogens, comprising the following steps:
[0028] S1. Surface disinfection: Use a 75% alcohol cotton ball to gently wipe the surface of the diseased tissue to remove surface bacteria and contaminants, providing a basis for subsequent aseptic operation;
[0029] S2. Observation and Selection: On a clean bench, carefully observe the surface of the diseased tissue using a stereoscopic dissecting microscope to locate and locate the ascomycete structures that produce the fruiting bodies. Then, disinfect the dissecting needle or tweezers by burning them with an alcohol lamp, then cool them, and carefully and precisely pick the spores.
[0030] S3. Inoculation and culture: Place the selected spores directly into MEA medium containing antibiotics and gently spread the spores on the surface of the medium with an inoculation loop to ensure even distribution of the spores. Then, incubate the medium at a constant temperature of 25°C for 1-3 days to promote spore germination.
[0031] S4. Moisture-keeping culture: For samples where spore-forming structures have not yet formed, a moisturizing culture strategy is adopted. The diseased tissue is placed in a culture dish covered with filter paper. By controlling the humidity and temperature conditions, the formation of fruiting bodies is promoted. After the fruiting bodies mature, they are separated according to the above steps.
[0032] S5. Transfer culture: After spores germinate, they need to be transferred to an antibiotic-free culture medium for continued culture to avoid the effects of antibiotics on the growth of pathogens. This step ensures that the pathogens grow and reproduce in a more natural environment, which is beneficial for subsequent research and application.
[0033] Wherein, the antibiotics in step S3 are a mixture of penicillin and streptomycin.
[0034] The MEA culture medium in step S3 is composed of 3% malt extract culture medium, which includes 30 g of malt extract powder, 20 g of agar, and 1000 mL of distilled water.
[0035] The outdoor separation method specifically includes the following steps:
[0036] S10. When collecting in the field, the type of pathogen was preliminarily determined by visual observation, and samples with obvious fruiting bodies on the lesions were selected;
[0037] S20. Use a 75% alcohol cotton ball to wipe the dissecting needle or tweezers for simple handling, or use a windproof lighter to burn the pointed tweezers. After the tweezers cool, pick the fruiting body directly under a magnifying glass.
[0038] S30, re-implant the MEA culture medium in a frozen tube containing antibiotics (penicillin and streptomycin mixture) prepared in advance;
[0039] S40. Cover the tube cap and culture. Take it back to the laboratory to observe and inspect the culture. Purify and preserve the successfully separated samples.
[0040] Example 2
[0041] The present invention provides a method for directly picking fruiting bodies to isolate and culture pathogens, comprising the following steps:
[0042] S1. Surface disinfection: Use a 75% alcohol cotton ball to gently wipe the surface of the diseased tissue to remove surface bacteria and contaminants, providing a basis for subsequent aseptic operation;
[0043] S2. Observation and Selection: On a clean bench, carefully observe the surface of the diseased tissue using a stereoscopic dissecting microscope to locate and locate the ascomycete structures that produce the fruiting bodies. Then, disinfect the dissecting needle or tweezers by burning them with an alcohol lamp, then cool them, and carefully and precisely pick the spores.
[0044] S3. Inoculation and culture: Place the selected spores directly into MEA medium containing antibiotics and gently spread the spores on the surface of the medium with an inoculation loop to ensure even distribution of the spores. Then, incubate the medium at a constant temperature of 25°C for 1-3 days to promote spore germination.
[0045] S4. Moisture-keeping culture: For samples where spore-forming structures have not yet formed, a moisturizing culture strategy is adopted. The diseased tissue is placed in a culture dish covered with filter paper. By controlling the humidity and temperature conditions, the formation of fruiting bodies is promoted. After the fruiting bodies mature, they are separated according to the above steps.
[0046] S5. Transfer culture: After spores germinate, they need to be transferred to an antibiotic-free culture medium for continued culture to avoid the effects of antibiotics on the growth of pathogens. This step ensures that the pathogens grow and reproduce in a more natural environment, which is beneficial for subsequent research and application.
[0047] Wherein, the antibiotics in step S3 are a mixture of penicillin and streptomycin.
[0048] The MEA culture medium in step S3 is composed of 3% malt extract culture medium, which includes 30 g of malt extract powder, 20 g of agar, and 1000 mL of distilled water.
[0049] The indoor separation method specifically includes the following steps:
[0050] S100. Wipe the surface of the diseased tissue with a 75% alcohol cotton ball to remove surface bacteria and contaminants, providing a basis for subsequent aseptic operation;
[0051] S200: Place the diseased tissue under a stereoscopic dissecting microscope on a clean bench to observe the spore-forming structure. Place a dissecting needle or forceps on an alcohol lamp to burn and cool, then directly pick out the spores and place them in MEA medium containing a double antibiotic (a mixture of penicillin and streptomycin).
[0052] S300, gently smear the surface of the culture medium with an inoculating loop, incubate at room temperature at 25°C for 1-3 days, and after spores germinate, transfer to a medium without antibiotics for further cultivation;
[0053] S400. For samples where spore-bearing structures have not yet formed, place the diseased tissue on a culture dish covered with filter paper and culture it in a moisturizing manner for 1-2 days to promote the formation of fruiting bodies, and then use the same method to separate them.
[0054] The present invention provides a method for directly extracting fruiting bodies for pathogen isolation and culture. The method offers high accuracy compared to tissue isolation methods in that it can be observed through a stereoscopic dissecting microscope, enabling accurate identification and localization of spore-forming structures, ensuring the purity and high activity of isolated pathogens. The method also offers high efficiency by directly targeting spore-forming structures, eliminating the complex mycelial culture and purification steps required in traditional methods and significantly shortening the isolation cycle. The method also offers broad applicability, being applicable not only to mature spore-forming ascomycetes but also promoting fruiting body formation in immature samples through moisturizing culture, thus expanding the scope of application. The method is not limited to laboratory (indoor) environments but can also be used to isolate pathogens outdoors. The method is also convenient to operate, requiring no complex instrumentation and equipment, and can be mastered by general laboratory personnel after simple training. This lowers the technical barrier to entry, enabling more researchers and grassroots technicians to participate in the isolation and identification of pathogens. Furthermore, the ease of operation means that the isolation process is faster and more efficient, enabling the acquisition of large quantities of pure pathogen samples in a short period of time, providing strong support for subsequent research and application.
[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A method for directly picking fruiting bodies for pathogen isolation and culture, characterized in that: The steps include: S1. Surface disinfection: Use an alcohol cotton ball to gently wipe the surface of the diseased tissue to remove surface bacteria and contaminants, providing a basis for subsequent aseptic operation; S2. Observation and Picking: On a clean bench, carefully observe the surface of the diseased tissue using a stereoscopic dissecting microscope to locate and locate the ascomycete structure that produces the fruiting body. Then, use a dissecting needle or forceps to pick out the spores. S3. Inoculation and culture: Place the selected spores directly into MEA medium containing antibiotics and gently spread the spores on the surface of the medium with an inoculation loop to ensure even distribution of the spores. Then, incubate the medium at a constant temperature of 25°C to promote spore germination. S4. Moisture-keeping culture: For samples where spore-forming structures have not yet formed, a moisturizing culture strategy is adopted to promote the formation of fruiting bodies. After the fruiting bodies mature, they are separated according to the above steps; S5. Transfer culture: After the spores germinate, in order to avoid the influence of antibiotics on the growth of pathogens, they need to be transferred to a culture medium without antibiotics for further culture.
2. The method for directly picking fruiting bodies for pathogen isolation and culture according to claim 1, wherein: In step S1, 75% alcohol cotton balls are used for disinfection.
3. The method for directly picking fruiting bodies for pathogen isolation and culture according to claim 1, wherein: The dissecting needle or tweezers in step S2 need to be sterilized by burning with an alcohol lamp and then cooled before picking.
4. The method for directly picking fruiting bodies for pathogen isolation and culture according to claim 1, wherein: The antibiotics in step S3 are a mixture of penicillin and streptomycin.
5. The method for directly picking fruiting bodies for pathogen isolation and culture according to claim 1, wherein: The MEA culture medium in step S3 contains 3% malt extract culture medium.
6. A method for directly picking fruiting bodies for pathogen isolation and culture according to claim 5, characterized in that: The 3% malt extract culture medium comprises: 30 g malt extract powder, 20 g agar, and 1000 mL distilled water.
7. The method for directly picking fruiting bodies for pathogen isolation and culture according to claim 1, characterized in that: In step S3, the culture medium is placed at a constant temperature of 25° C. for 1-3 days.
8. The method for directly picking fruiting bodies for pathogen isolation and culture according to claim 1, wherein: The specific steps of the moisturizing culture strategy are: placing the diseased tissue in a culture dish covered with filter paper, and promoting the formation of fruiting bodies by controlling the humidity and temperature conditions.