Single spore isolation method of alternaria alternata
By using an alcohol lamp flame to prepare a glass needle and then using a microscope to pick up single spores, the problem of expensive and complex equipment for isolating Alternaria alternata spores has been solved. This method achieves efficient and low-cost single spore isolation, making it suitable for use in grassroots laboratories and field applications.
Patent Information
- Application Number
- CN202511825007.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-01-20
AI Technical Summary
Existing methods for isolating single spores of Alternaria have problems such as expensive equipment, cumbersome operation, low success rate and high contamination rate, making it difficult to meet the needs of rapid isolation in grassroots laboratories and fields.
The glass needle was prepared using an alcohol lamp flame, and single spores were picked up under a microscope and inoculated into the culture medium. This simplified the operation steps, reduced equipment dependence, improved the success rate of single spore isolation, and reduced costs.
It achieves efficient and low-cost single-spore isolation with a success rate of ≥95% and a contamination rate of <10%, making it suitable for both grassroots laboratories and field settings, and significantly improving ease of operation and environmental adaptability.
Smart Images
Figure CN121362643A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of mycology and biotechnology, and in particular to a single spore isolation method of Alternaria alternata. BACKGROUND
[0002] Alternaria alternata is a kind of widely distributed plant pathogenic fungi. In agriculture, some strains of Alternaria alternata can cause serious diseases of crops, such as tomato early blight, onion purple spot disease, cruciferous vegetable black spot disease, etc., affecting crop yield and quality. The single spore isolation technology of Alternaria alternata is the basis for strain purification, identification and genetic property research.
[0003] The traditional single spore isolation method of Alternaria alternata has many limitations.
[0004] For example, in the operation process of the conventional plate dilution method, it is difficult to effectively disperse single spores due to the aggregation of Alternaria alternata spores, resulting in a low probability of obtaining single spores, and the operation is complicated and time-consuming. In addition, some single spore isolation methods with the aid of micro-manipulators have high precision, but the instruments and equipment are expensive and require high technical requirements for operators, which is not conducive to large-scale popularization and application. Moreover, the existing methods are easily contaminated by external bacteria during single spore isolation, which affects the purity and accuracy of single spore isolation. These problems to some extent limit the efficient development of Alternaria alternata related research and application, and there is an urgent need for a more efficient, simple, accurate and low-cost single spore isolation method of Alternaria alternata.
[0005] Therefore, in view of the device limitations and efficiency problems in the operation of the existing technology, it is a technical problem that needs to be solved by the person skilled in the art to provide a method for preparing and sterilizing a glass needle by alcohol lamp flame synchronously without professional equipment, and then performing single spore isolation. SUMMARY
[0006] Therefore, the present application provides a single spore isolation method of Alternaria alternata.
[0007] To solve the above technical problems, the present application adopts the following technical solutions:
[0008] A single spore isolation method of Alternaria alternata, comprising the following steps:
[0009] Step 1, treatment of diseased tissue:
[0010] Collecting diseased tissue infected by Alternaria alternata and culturing Alternaria alternata;
[0011] Step 2, preparation of culture medium:
[0012] Preparing a culture medium for the growth of Alternaria alternata;
[0013] Step 3, preparation of glass needle:
[0014] Burn the end of the clean glass capillary tube and stretch it into a sharp needle shape;
[0015] Step 4, picking up single spores under a microscope:
[0016] Under a microscope, use a glass needle to pick up single Alternaria alternata spores from the moist culture of the diseased tissue;
[0017] Step 5, single spore inoculation culture:
[0018] Transfer the picked single spores to the culture medium for culture to obtain a pure culture as the basis for purification and identification.
[0019] Preferably, in step 1, the Alternaria alternata culture is a moist culture with a humidity of 70-80%.
[0020] Preferably, in step 2, the prepared culture medium is PDA medium with ingredients of potato dextrose broth 24 g / L, agar 20 g / L, and distilled water. After being prepared in proportion, it is sterilized at 121℃ for 20 min, poured into a culture dish under sterile conditions, and cooled for standby use.
[0021] Preferably, the potato dextrose broth 24 g / L includes potato infusion powder 4 g / L and glucose 20 g / L.
[0022] Preferably, in step 3, the material of the glass capillary tube is borosilicate glass with an inner diameter of 1-1.5 mm.
[0023] Preferably, the glass capillary tube is placed in the outer flame of an alcohol lamp for melting and stretched with tweezers to form a single spore separation glass needle with a specific shape and size.
[0024] Preferably, the tweezers are sharp-tipped tweezers.
[0025] Preferably, in step 4, the outer diameter of the tip of the glass needle is 120-450 μm to facilitate the absorption of single Alternaria alternata spores.
[0026] Preferably, in step 4, the microscope is a body microscope with a magnification of 10-50 times.
[0027] Preferably, in step 5, the culture is carried out at 25-28℃ for 2-3 days to obtain a pure culture.
[0028] The present application has the following technical effects compared with the prior art:
[0029] (1) Extremely low cost: the present application only needs an alcohol lamp, tweezers, and a glass capillary tube (total cost <50 yuan);
[0030] (2) Convenient operation: the glass needle is prepared and sterilized synchronously, and the single operation is less than 2 minutes;
[0031] (3) High success rate: the success rate of single spore picking is greater than or equal to 70% and the pollution rate is less than 10% under the experience operation of the application;
[0032] (4) Wide applicability: the application is suitable for field rapid separation and basic laboratory application. BRIEF DESCRIPTION OF DRAWINGS
[0033] Figure 1 It is a schematic diagram of a moisturizing culture device (disease tissue + filter paper + sterile water) of the application;
[0034] Fig. 2 is a glass needle preparation flow chart of the application ((a). melting→ (b). stretching→ (c). shaping);
[0035] Figure 3 It is a single spore picking operation schematic diagram under a body microscope of the application;
[0036] Fig. 4 is a single spore inoculation operation schematic diagram (a) and a single spore culture device schematic diagram (b) of the application. DETAILED DESCRIPTION
[0037] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.
[0038] Example 1: Single spore separation of alternaria alternata
[0039] a. Disease tissue treatment:
[0040] Two layers of sterile filter paper (Φ 90 mm) are clamped with tweezers, the filter paper is placed in a beaker containing distilled water to be soaked, and the soaked filter paper is taken out without dripping water;
[0041] A sterile culture dish (90 mm×15 mm) is taken, the soaked filter paper is laid in the dish, and the filter paper is pressed flat;
[0042] A wheat leaf spot disease leaf is taken, and several (more than 10) 5 mm×5 mm disease-healthy interface tissue blocks are cut with a sterile scalpel. The disease tissue is clamped with tweezers and evenly distributed on the culture dish with sterile filter paper, and cultured at 25 ℃ in the dark for 2-4 days.
[0043] b. Preparation of culture medium:
[0044] PDA medium was prepared with the following ingredients: potato dextrose broth 24 g / L (including potato infusion powder 4 g / L, glucose 20 g / L), agar 20 g / L, distilled water;
[0045] After being proportionally configured, high-temperature sterilization at 121 DEG C for 20 min was performed, and then the medium was poured into a culture dish (20 ml / dish) under sterile conditions, and after cooling, the film was sealed for standby use.
[0046] c. Glass needle preparation:
[0047] The tip tweezers and borosilicate glass capillary (I.D. 1.1-1.2 mm; Wall 0.2±0.02 mm; Length 75 mm) were simultaneously placed in the center of the outer flame of an alcohol lamp for heating, and the capillary was heated for 8-12 s to red or melted;
[0048] The tweezers clamped the melted end of the glass capillary, and maintained this shape.
[0049] The glass capillary and tweezers were moved to the air outside the outer flame; during the process of cooling and setting of the glass capillary and tweezers in the air, a pulling force was quickly applied to the clamped part of the tweezers, and the end of the glass capillary was stretched into a glass needle with an outer diameter of 0.15-0.45 mm and a length of 20-40 mm.
[0050] Then, the excess part of the glass needle was clamped or knocked off using the tip of the tweezers, and the length was reserved to 10-20 mm, which removed the part of the glass needle that was too thin and soft at the tip and was not easy to operate during the single spore picking process (the glass needle was too long, too thin and too soft to be operated, and other spores were easily sucked during the single spore picking process).
[0051] d. Microscopic picking of single spores:
[0052] Under a body microscope (40x), a single Alternaria alternata spore was identified and electrostatically adsorbed and picked from the moistened culture of the diseased tissue.
[0053] e. Inoculation and culture of single spore isolated strains:
[0054] After the single spore was adsorbed by the glass needle, it was inoculated into PDA medium, and cultured at 28 DEG C in the dark for 2-3 days to form a single spore colony.
[0055] Comparative experiment design:
[0056] Experimental method:
[0057] The same fungal spore sample was treated by using a traditional method (dilution separation method) and the method of the present application, respectively;
[0058] The operation cost, operation time, single spore picking success rate and culture contamination rate of the two methods were compared.
[0059] The experimental conditions are shown in Table 1:
[0060] Table 1:
[0061]
[0062] The same fungus species, the same environmental conditions, the same batch of culture medium and the same experimental equipment are used for the experiment.
[0063] The traditional method requires aseptic operation throughout the experiment, while the method of the present application only requires aseptic conditions for the preparation of the culture medium.
[0064] Experimental data table:
[0065] Technical effect comparison:
[0066] 1. Simplified operation steps
[0067] Traditional method: requires steps such as spore suspension preparation, gradient dilution, culture plating, single spore confirmation and multiple purification, which is complex and relies on multiple aseptic operations.
[0068] The method of the present application: only needs to directly pick single spores after moist culture of the diseased tissue, and the main operation steps are simplified to 2 steps (moist culture and single spore picking), without gradient dilution and repeated purification.
[0069] 2. Improved experimental efficiency
[0070] Traditional method: total time consumption is 5-7 days (including multiple purification cycles).
[0071] The method of the present application: the total time consumption is shortened to 2-4 days (moist culture for 2-4 days + single spore culture for 2-3 days), and the experimental period is compressed by more than 40%.
[0072] 3. Significantly improved single spore separation success rate
[0073] Traditional method: single spore separation success rate is 65-70%, which relies on multiple aseptic operations and purification.
[0074] The method of the present application: through precise picking with a glass needle, the single spore separation success rate is ≥95%, and the error rate is reduced to
[0075] 5% or less.
[0076] 4. Reduced experimental cost
[0077] Traditional method: single experiment cost is 30-50 yuan (requires multiple culture media and aseptic consumables).
[0078] The method of the present application: single experiment cost is <30 yuan (only requires filter paper, glass capillary and single culture medium), and the cost is reduced by 40-50%.
[0079] 5. Environmental adaptability is enhanced
[0080] Traditional method: The whole process needs a sterile environment (clean bench, sterilization equipment).
[0081] The method of the present application: Only the preparation of the culture medium needs sterile conditions, and other steps (such as moist culture and single spore picking) can be completed in a general experimental environment, significantly reducing the dependence on equipment.
[0082] Conclusion:
[0083] The present application has the following advantages:
[0084] 1. High efficiency: The success rate of single spore isolation is increased to ≥95%, and the experimental period is shortened to 60% of the traditional method.
[0085] 2. Low cost: The cost of a single experiment is reduced by 40-50%, and the total cost of materials is less than 30 yuan.
[0086] 3. Operation convenience: No professional equipment (such as a micromanipulator) is needed, and it is suitable for basic laboratories and field scenes.
[0087] 4. Environmental compatibility: Breakthrough the limitation of sterile environment, reduce the threshold of experimental conditions.
[0088] The method of the present application is significantly superior to the traditional dilution separation method in terms of operation efficiency, cost control and success rate, and is especially suitable for basic laboratories with limited equipment and rapid field separation scenes, providing efficient and economical technical support for the genetic research and disease control of Alternaria alternata.
[0089] The above is only the preferred embodiment of the present application, and does not limit the technical scope of the present application, so any slight modification, equivalent change and modification made according to the technical essence of the present application are still within the scope of the technical solution of the present application.
Claims
1. A method for isolating a single spore of Alternaria alternata, characterized by, The method comprises the following steps: Step 1, treatment of the diseased tissue: Collecting the diseased tissue infected by Alternaria alternata and culturing the Alternaria alternata; Step 2, preparation of the culture medium: Preparing the culture medium for the growth of the Alternaria alternata; Step 3, preparation of the glass needle: Melting the end of a clean glass capillary tube and stretching it into a sharp needle; Step 4, picking up single spores under a microscope: Under a microscope, picking up single spores of the Alternaria alternata from the moistened cultured diseased tissue by using the glass needle; Step 5, inoculation of single spores: Transferring the picked single spores to the culture medium for culturing, so as to obtain a pure culture as a basis for purification and identification.
2. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, In the step 1, the culturing of the Alternaria alternata is moistened culturing, and the humidity is 70-80%.
3. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, In the step 2, the prepared culture medium is PDA medium, and the ingredients are potato glucose broth 24 g / L, agar 20 g / L and distilled water. After being prepared in proportion, the medium is sterilized at 121℃ for 20 min, poured into a culture dish under sterile conditions, and cooled for standby.
4. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, The potato glucose broth 24 g / L comprises potato powder 4 g / L and glucose 20 g / L.
5. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, In the step 3, the material of the glass capillary tube is borosilicate glass, and the inner diameter is 1-1.5 mm.
6. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, The glass capillary tube is melted in the outer flame of an alcohol lamp and stretched by using tweezers to form a single-spore separation glass needle with a specific shape and size.
7. The method of claim 6, wherein the single spore isolation of Alternaria alternata is characterized by, The tweezers are sharp-tipped tweezers.
8. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, In the step 4, the outer diameter of the tip of the glass needle is 120-450 μm, so as to facilitate the picking up of single spores of the Alternaria alternata.
9. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, In the step 4, the microscope is a body microscope, and the magnification is 10-50 times.
10. The method of claim 1, wherein the single spore isolation of Alternaria alternata is characterized by, In the step 5, the pure culture is obtained by culturing at 25-28℃ for 2-3 days.