Method for long-term effective preservation of vermiform conidium fungi

Abstract

The invention relates to a method for effectively preserving vermicular conidium fungi for a long time. The method comprises the following steps: preparing air vents of a cryopreservation tube, preparing a sorghum grain culture, preparing the vermicular conidium fungi, inoculating the sorghum grain culture, and finally storing the sorghum grains with bacteria in the cryopreservation tube. The vermicular conidium fungus preservation method has the advantages of being long in preservation period, high in survival rate, low in cost, easy to operate and the like, the strain storing and taking operation process can be simplified, and the experiment efficiency can be improved; the dependence on complex equipment is reduced; strain variation and pollution in the preservation process are avoided, the high survival rate of the vermicular conidium fungi is maintained for a long time, and the method is suitable for being used in a laboratory and provides help for scientific research work of corn.

Description

Technical Field

[0001] This invention relates to the field of microbial strain technology, specifically to a long-term preservation method that can maintain the survival rate of vermicular conidial fungi and reduce their contamination rate. Background Technology

[0002] Demodex fungi are a class of filamentous deuteromycetes whose conidia have multiple septa and resemble worms. They comprise six genera of asexual fungi: *Hemangiosporium*, *Curvulina*, *Hemangiosporium endostrum*, *Hemangiosporium convexum*, *Hemangiosporium longum*, and *Hemangiosporium oostrum*. These fungi are important plant pathogens that can infect a variety of plants, especially gramineous crops such as corn, wheat, rice, sorghum, and sugarcane. Therefore, research on demodex fungi is crucial for controlling their diseases and breeding disease-resistant crop varieties, and the stable preservation of pathogen strains is the core foundation for conducting related research.

[0003] Currently, commonly used methods for preserving fungal strains in laboratories include: slant agar cryopreservation, glycerol cryopreservation, freeze-drying, and liquid nitrogen preservation. Slant agar cryopreservation is only suitable for short-term preservation (re-subculturing is required within six months), and multiple subculturings can easily lead to strain mutations and high contamination rates, failing to meet the needs of long-term preservation. Glycerol cryopreservation results in low survival rates for Demodex conidial fungi, with survival rates continuously decreasing over time, and it requires ultra-low temperature equipment, leading to relatively high preservation costs. Freeze-drying and liquid nitrogen preservation, while allowing for long-term preservation, involve complex procedures and require significant equipment investment. Furthermore, laboratory research often requires batch preservation of strains for immediate use, and existing methods cannot simultaneously meet the demands of "long-term preservation, high survival rate, low cost, and ease of operation," causing significant inconvenience to research on Demodex conidial fungi.

[0004] Therefore, developing a novel strain preservation method specifically for vermicular conidial fungi is of irreplaceable significance in effectively breaking through the current obstacles to the preservation of this type of fungal strain. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing fungal strain preservation methods and provide a method for long-term preservation of vermicular conidial fungi.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for long-term and effective preservation of vermicular conidial fungi includes the preparation of aeration pores in cryovials, preparation of sorghum grain culture, preparation of vermicular conidial fungi, inoculation of sorghum grain culture with fungi, and storage of fungal-infected sorghum grains in cryovials. The specific method is as follows:

[0008] (1) Preparation of air vents in cryopreservation tubes: Select cryopreservation tubes with screw caps and make air vents in the center of the caps; then use a breathable membrane to cover the air vents on the outside of the caps; sterilize the prepared cryopreservation tubes by high pressure and moist heat, dry them after sterilization, and place them in a sterile environment for later use.

[0009] (2) Preparation of sorghum grain culture: Select plump, mold-free sorghum grains, soak them in water to fully absorb water, then heat and boil until the sorghum grains soften. Drain excess water, fill 1 / 3 to 1 / 2 of the Erlenmeyer flask with the sorghum grains, and place them in a high-pressure moist heat sterilization. After sterilization, take them out and place them in a sterile environment to cool to room temperature for later use.

[0010] (3) Preparation of vermicular conidial fungi: Select the target vermicular conidial fungi strain that has been isolated and purified by single spore, transfer it to PDA plate, and culture it in the dark at 25℃ for 5-7 days until the plate is covered with uniform mycelium for later use.

[0011] (4) Inoculation of sorghum grain culture: On a sterile operating table, pour the prepared sterilized sorghum grains into a sterile petri dish and spread them into a uniform thin layer; use a sterile inoculation knife to cut the cultured mycelium into mycelial blocks, and take the mycelial blocks to inoculate evenly onto the thin layer of sorghum grains in the petri dish; place the petri dish after inoculation at 25°C in the dark for 10 days until the surface of the sorghum grains is completely covered with mycelium; continue to incubate for 2-3 days, and wait for the sorghum grains to dry naturally to obtain the sorghum grains with mycelium;

[0012] (5) Preservation: On a sterile operating table, transfer the dried sorghum grains containing bacteria into cryovials, tighten the screw cap of the cryovials, and place the cryovials into a cryopreservation box for storage.

[0013] In step (1) of the method, when preparing the vent hole of the cryopreservation tube, a 5mL cryopreservation tube with a screw cap is selected, and a vent hole with a diameter of 5mm is opened in the center of the cap; a vent membrane with a diameter of 1.8cm is used and pasted on the vent hole on the outside of the cap to ensure that the vent membrane seals the vent hole; the prepared cryopreservation tube is placed at 121℃ and sterilized by high pressure moist heat for 20min.

[0014] In step (2) of the method, the sorghum grains are soaked in water for 1 hour, boiled for 30 minutes, and sterilized under high pressure at 121°C for 60 minutes.

[0015] In step (4) of the method, the thickness of the uniform thin layer is 1 cm; the size of the mycelium block is 1 cm × 1 cm, and 5 to 10 mycelium blocks are evenly inoculated onto the thin layer of sorghum grains in the petri dish.

[0016] In step (5) of the method, the bacteria-infected sorghum grains are filled to 2 / 3 of the volume of the cryopreservation tube, and the cryopreservation temperature is 15~25℃.

[0017] The beneficial effects of this invention are:

[0018] (1) The method for preserving vermicular conidial fungi of the present invention, compared with the existing methods, can simplify the operation process of fungal storage and retrieval, improve experimental efficiency, reduce dependence on complex equipment, avoid strain variation and contamination during preservation, and achieve a high survival rate of vermicular conidial fungi for a long time.

[0019] (2) The preservation method of the present invention is simple to operate, low in cost, and has a high survival rate of strains. The survival rate of strains reaches 97.5% after two years and the contamination rate of strains is only 6.0%. The survival rate of strains in the fourth year reaches 86.0% and the contamination rate is 17%. This preservation method makes it convenient to obtain strains and is a long-term effective method for preserving vermicular conidial fungi, which is very suitable for laboratory use.

[0020] (3) The preservation method of the present invention is applicable not only to the preservation of Helicobacter zebrina var. zebrina, but also to the preservation of other dermoid conidial fungi. Attached Figure Description

[0021] Figure 1 This is a photograph of the fabrication of the breathable cryopreservation tube of the present invention and a sample containing *Sorghum sacchariformis*.

[0022] In the diagram: a: ​​front of the breathable membrane; b: back of the breathable membrane; c: perforation of the cryovial cap; d: film attached to the cryovial cap; e: sorghum grains inoculated with bacteria; f: bacteria-carrying sorghum grains; g: bacteria-carrying sorghum grains stored in cryovials. Detailed Implementation

[0023] Example 1: A method for long-term effective preservation of *Helicobacter zebrina*, the pathogen causing small leaf spot in corn.

[0024] The process includes steps such as preparing the vent holes for cryovials, preparing sorghum grain cultures, preparing Helicobacter pylori in corn, inoculating the sorghum grain cultures with the bacteria, and storing the inoculated sorghum grains in cryovials. See [link to relevant documentation]. Figure 1 .

[0025] The specific method is as follows:

[0026] (1) Preparation of the vent hole of the cryopreservation tube: Select a 5ml cryopreservation tube with a screw cap, make a 5mm diameter hole in the center of the cap, and attach a 1.8cm diameter breathable membrane (such as a 0.22μm sterile breathable membrane). Place the prepared cryopreservation tube under high pressure and wet heat sterilization at 121℃ for 20min. After sterilization, dry it and place it in a sterile environment for later use.

[0027] (2) Preparation of sorghum grain culture: Select plump, mold-free sorghum grains, soak them in water for 1 hour, then heat and boil for 30 minutes until the sorghum grains soften but do not break. Drain excess water, fill the Erlenmeyer flasks to 1 / 3 to 1 / 2 full, and sterilize them under high pressure at 121℃ for 60 minutes. Cool and set aside for later use.

[0028] (3) Preparation of Helicobacter zedoaria: 200 strains of Helicobacter zedoaria, the pathogen of corn leaf spot, which was isolated and purified by single spore in 2020, were selected and transferred to PDA plates; they were cultured in the dark at 25℃ for 5 to 7 days until the plates were covered with uniform mycelium for later use.

[0029] (4) Inoculation of sorghum grain culture: On a sterile operating table, pour the sterilized sorghum grains from the Erlenmeyer flask into a petri dish and spread them to form a uniform thin layer with a thickness of 1 cm. Divide 200 *Helicobacter spp.* into mycelial blocks (1 cm × 1 cm in size). Take 5-10 mycelial blocks and inoculate them evenly onto the sorghum grains in the petri dish. Incubate the inoculated petri dish in the dark at 25°C for about 10 days until the sorghum grains are covered with mycelium. Continue to incubate for 2-3 days until the sorghum grains slowly lose water and dry out, and obtain the sorghum grains with bacteria.

[0030] (5) Storing bacteria-carrying sorghum grains into cryovials: On a sterile operating table, transfer bacteria-carrying sorghum grains into the prepared cryovials, fill the cryovials to 2 / 3 of their volume, tighten the screw caps of the cryovials, and mark each strain number. Tighten the screw caps to ensure that the breathable membrane is undamaged and well-sealed.

[0031] (6) Storage: Place the cryopreservation tube containing the bacteria-infected sorghum grains into a cryopreservation box and store it in a dry, clean, and contaminant-free indoor environment at a temperature of 15~25℃.

[0032] Example 2: Preservation of Curvularia crescentis, the fungus that causes Curvularia leaf spot disease in maize

[0033] Using the method of Example 1, 200 strains of Curvularia crescentis, the leaf spot disease of maize, isolated and purified by single spore in 2020, were preserved. In the first, second, third and fourth years of preservation, the strains were activated on PDA plates and cultured in the dark at 25°C for 5 to 7 days. The survival rate and contamination rate of the strains were then counted.

[0034] The results showed that the survival rates of the 200 strains after 1 year, 2 years, 3 years and 4 years of preservation were 100%, 100%, 98% and 96.5%, respectively, and the cumulative contamination rate of the strains over 4 years was 13.5%, indicating that the strain preservation method of the present invention is suitable for the preservation of Curvularia crescentis, the leaf spot disease of maize.

[0035] Example 3: Comparison of Preservation Methods

[0036] Comparison of the method of the present invention with the slant-plane cryopreservation method and the glycerol cryopreservation method

[0037] For ease of comparison, the corn spores from step (3) of Example 1 were preserved using the following methods: 200 corn spore strains were preserved using the slant culture low-temperature preservation method and the glycerol cryopreservation method, respectively.

[0038] 1. Slant culture preservation method: First, prepare PDA medium, dispense it into test tubes, sterilize and cool to solidify to form slant culture. Under aseptic conditions, inoculate the purified corn slant culture medium blocks onto the slant culture medium and incubate at 25-28℃ for 3-5 days until obvious colonies are formed. Seal the tubes with sterile stoppers, label them with the strain name and inoculation date, and store them at 4℃. Subculture every 3-6 months.

[0039] 2. Glycerol cryopreservation method: First, prepare a 50% sterile glycerol solution, autoclave it for later use, culture the target strain for 3-7 days, collect the mycelium, and prepare a 10% concentration solution. 6 -10 8 For a bacterial suspension of CFU / mL, mix the bacterial suspension with sterile glycerol solution at a 1:1 volume ratio, label the strain name and storage date, refrigerate at -20℃ for 2-4 hours, and then transfer to -80℃ for storage.

[0040] Two hundred strains preserved by different methods were activated onto PDA plates at 1, 2, 3 and 4 years after preservation, respectively, and cultured in the dark at 25°C for 5-7 days. The survival rate and contamination rate of the strains were counted, and the results are shown in Table 1.

[0041] Table 1 Comparison of several preservation methods

[0042]

[0043] The slant culture method is mainly suitable for short-term preservation of strains. It requires subculturing the strain every six months. The survival rate of the strain is high, reaching over 90% after 3 years. However, it requires multiple subculturings, which is labor-intensive and time-consuming. It is also prone to strain mutation, which increases the contamination rate. After 4 years, the contamination rate of the strain reaches 32%.

[0044] Glycerol cryopreservation is mainly suitable for medium-term preservation of strains. It is simple to operate, but requires ultra-low temperature equipment, which limits the experiment and is slightly more expensive. The survival rate of *Helicobacter zebrina* preserved by this method is relatively high in the first year (94.5%) and the second year (87.0%). After that, the survival rate of the strain decreases with the extension of preservation time, and the survival rate drops to 58.5% in the fourth year.

[0045] As can be seen, the method of this invention can preserve strains for a long time, is simple to operate, has low cost, and a high survival rate of strains. The survival rate of strains can reach 97.5% after two years, with a contamination rate of only 6.0%. After four years, the survival rate of strains reaches 86.0%, with a contamination rate of only 17%. This preservation method makes it convenient to access strains in daily use, is very suitable for laboratory use, and can provide strong support for maize research.

Claims

1. A method for long-term effective preservation of myceliogenus spore fungi, characterized by, The method comprises the steps of preparing a gas-permeable hole of a freezing tube, preparing a sorghum grain culture, preparing a myceliophthora fungus, inoculating the sorghum grain culture, and storing the sorghum grain culture in the freezing tube. (1) Preparation of a gas-permeable hole of a freezing tube: a screw cap is selected, and a gas-permeable hole is formed in the center of the cap; a gas-permeable film is pasted on the gas-permeable hole on the outside of the cap; the prepared freezing tube is subjected to high-pressure moist heat sterilization, dried after sterilization, and placed in a sterile environment for standby; (2) Preparation of a sorghum grain culture: full and non-molded sorghum grains are soaked in water, fully soaked, heated and boiled until the sorghum grains become soft, and the excess water is drained; the sorghum grains are filled in 1 / 3-1 / 2 of a triangular flask, and subjected to high-pressure moist heat sterilization; after sterilization, the sorghum grains are taken out, cooled to room temperature, and standby; (3) Preparation of a myceliophthora fungus: a target myceliophthora fungus strain separated and single-spore purified is transferred to a PDA plate, and cultured at 25 DEG C in the dark for 5-7 days; the plate is full of uniform mycelium, and standby; (4) Inoculation of the sorghum grain culture: in a sterile operation table, the prepared sterilized sorghum grains are poured into a sterile culture dish, and spread into a uniform thin layer; the cultured mycelium is cut into blocks with a sterile inoculation knife, and the blocks are inoculated into the thin layer of sorghum grains in the culture dish; the culture dish after inoculation is cultured at 25 DEG C in the dark for 10 days, until the surface of the sorghum grains is fully covered with mycelium; the culture is further cultured for 2-3 days, until the sorghum grains are naturally dried, and the sorghum grains with mycelium are obtained; (5) Preservation: the dried sorghum grains with mycelium are transferred into a freezing tube in a sterile operation table, the screw cap of the freezing tube is tightly screwed, and the freezing tube is placed in a freezing box for storage.

2. The method of claim 1, wherein, In step (1), a 5 mL freezing tube with a screw cap is selected, and a gas-permeable hole with a diameter of 5 mm is formed in the center of the cap; a gas-permeable film with a diameter of 1.8 cm is pasted on the gas-permeable hole on the outside of the cap, so as to ensure that the gas-permeable film seals the gas-permeable hole; the prepared freezing tube is subjected to high-pressure moist heat sterilization at 121 DEG C for 20 min.

3. The method of claim 1, wherein, In step (2), the sorghum grains are soaked in water for 1 h, heated and boiled for 30 min, and subjected to high-pressure moist heat sterilization at 121 DEG C for 60 min.

4. The method of claim 1, wherein, In step (4), the thickness of the uniform thin layer is 1 cm; the size of the blocks is 1 cm x 1 cm, and 5-10 blocks are inoculated into the thin layer of sorghum grains in the culture dish.

5. The method of claim 1, wherein, In step (5), the sorghum grains with mycelium are filled in 2 / 3 of the volume of the freezing tube, and the storage temperature of the freezing tube is 15-25 DEG C.

Images