Method for preserving rust fungi

Abstract

The invention discloses a rust fungus preservation method which comprises the following steps: carrying out spore production treatment on rust fungus, collecting rust fungus spores obtained after spore production treatment, and carrying out drying treatment to obtain dried spores; and transferring the dried spores into a preservation tube, carrying out vacuum treatment on the preservation tube, and carrying out tube sealing treatment to complete rust fungus preservation. The method has the advantages that long-term preservation and activity maintenance of rust fungi are realized, and the fungi after being preserved are easy to transport, take and preserve.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganism preservation, and particularly relates to a rust fungus preservation method. BACKGROUND

[0002] Rust fungi are an important class of plant pathogenic microorganisms, and their preservation is of great significance for plant pathology research, disease control and biodiversity protection. At present, the preservation methods of rust fungi mainly include suspension preservation, ultra-low temperature preservation (-80℃) and liquid nitrogen preservation. However, the existing technologies have different degrees of shortcomings. For example, the suspension preservation method reduces the difficulty and cost of urediniospore preservation, although it realizes the "instant" of urediniospore in a short period of time, it is suitable for short-term preservation, and the preservation time is 1-12 months, which is not conducive to long-term preservation and research. The ultra-low temperature preservation method (-80℃) can prolong the preservation period of spores to a certain extent, and usually can reach 1-2 years. However, this method has strong dependence on equipment, high requirements for long-term stability of temperature environment, and the problem of gradual reduction of spore activity with the prolongation of preservation time still exists, which is difficult to meet the actual needs of long-term preservation. The liquid nitrogen preservation method can realize long-term preservation of rust fungi, but the preservation cost is high, the operation is complex, and it needs to be supplemented with liquid nitrogen regularly, which is expensive. Therefore, there is an urgent need for a rust fungus preservation method which is simple in operation, low in cost and can maintain the activity of rust fungi for a long time. SUMMARY

[0003] The present application aims to solve the above technical problems.

[0004] In order to achieve the above-mentioned purpose, the present application provides a rust fungus preservation method, which specifically comprises the following steps: subjecting rust fungi to sporulation treatment, collecting urediniospores obtained after the sporulation treatment, and drying the urediniospores to obtain dried spores; transferring the dried spores to a preservation tube, and performing vacuum treatment on the preservation tube while performing tube sealing treatment to complete the preservation of the rust fungi.

[0005] The method provided by the present application is simple in steps, can save a large amount of cost compared with the liquid nitrogen preservation method of the prior art, can prolong the preservation time compared with the suspension preservation method of the prior art, and the activity of the rust fungi after culture and activation after preservation can also be maintained at a very high level.

[0006] Preferably, the spore production treatment refers to inoculating the rust fungus on the susceptible wheat seedlings by smearing method, after inoculation, the wheat seedlings are moved into a humidification frame, sprayed with clean water to make a layer of uniform droplets adhere to the surface of the leaves, and then sealed in a humidification box. After 18-24 hours of humidification culture in a dark environment at 9-13℃, the wheat seedlings are moved to a greenhouse for culture, and the culture temperature is controlled at 14±3℃, the light illumination is 10-14 hours per day, and the light intensity is 8000-10000Lx. When the leaf shows spots, the central leaf is removed, a glass cover is added for isolation, and after the uredospores mature, the leaves are gently shaken with an inoculation needle to make the spores fall into the glass cover. Then the wheat seedlings are removed, the glass cover is gently tapped to concentrate the spores, and then the spores are loaded into a finger-shaped tube. Preferably, this convenient method is used to culture spores, which greatly reduces the cost compared with culture medium culture, and is simple and easy to operate.

[0007] Preferably, the drying treatment is to place the spores in a sealed dryer, put drying agent in the dryer, and dry at 1-10℃. This simple drying method is easy to operate and does not adversely affect the rust spores. Combined with other steps, it can prolong the storage time and maintain high activity.

[0008] Further, the moisture of the dried spores is not reduced. The drying time is generally 5-10 days. The present application uses a spore production treatment method and combines the structure of the preservation tube to greatly shorten the drying time to 5-10 days. This is because: during the spore production treatment process, the microorganism will redistribute and adjust the water and other substances in the cell to adapt to the upcoming drying process, and the cell structure will change, such as thickening or modification of the cell wall and cell membrane, and the metabolic activity of the microorganism will be significantly reduced, and it will no longer synthesize a large amount of substances and energy metabolism as in the vegetative growth stage. This means that the utilization and consumption of water by the cell is reduced, and the water in the cell is in a relatively static state rather than being consumed or transformed by constantly participating in various metabolic reactions. This relatively static water state is more easily removed during the drying process, as it does not need to overcome the resistance to drying caused by the dynamic changes in water due to metabolic activity, thereby shortening the drying time.

[0009] In combination with the structure of the preservation tube of the present application, the present application uses a reduced-diameter tube segment to allow water to diffuse out at an appropriate rate while ensuring that the microorganism is not contaminated by the outside environment. These structures enable the water in the tube to quickly diffuse out to the outside under the action of the external drying environment. Compared with ordinary containers, it is more conducive to the removal of water, avoiding the accumulation of water in the tube, thereby accelerating the drying process of the microorganism and greatly shortening the drying time.

[0010] Further, the preservation tube comprises a first tube segment, a second tube segment, and a reduced-diameter tube segment between the first tube segment and the second tube segment.

[0011] Further, the outer diameter of the reduced diameter pipe section is smaller than the first pipe section and the second pipe section;

[0012] Further, the reduced diameter pipe section is connected to the first pipe section and the second pipe section respectively;

[0013] Further, the second pipe section is provided with a pipe mouth at one end away from the first pipe section;

[0014] Further, the length of the second pipe section is smaller than the length of the first pipe section. The dried spores are transferred to the first pipe section of the preservation tube. Preferably, the vacuum treatment is performed by connecting the preservation tube to a negative pressure device;

[0015] Further, the negative pressure device comprises:

[0016] a negative pressure device;

[0017] a support, the support having a vertical pipe and a plurality of lugs, the vertical pipe being connected to the negative pressure device, the vertical pipe having a cavity communicating with the negative pressure device, each of the lugs being provided on the vertical pipe, each of the lugs being arranged along the length direction of the vertical pipe in sequence, and each of the lugs being connected with a coupling sleeve;

[0018] Further, the vacuum treatment is performed by detachably connecting the second pipe section of the preservation tube to the corresponding coupling sleeve to communicate the cavity of the vertical pipe.

[0019] By using the above-mentioned vacuum treatment device, the preservation tube can be subjected to vacuum treatment when the negative pressure device is running. The preservation tube has a reduced diameter pipe section, which can be easily and quickly heat-sealed at the first pipe section under the action of flame. The length of the second pipe section of the preservation tube is smaller than that of the first pipe section, which does not affect the plug-in cooperation between the second pipe section and the coupling sleeve.

[0020] Preferably, the pipe sealing treatment is performed by burning the preservation tube in vacuum state by using flame;

[0021] Further, the temperature of the flame is 1900-2100°C;

[0022] Further, the burning time is 1-3s.

[0023] Preferably, the preservation tube is stored at-20 to 4°C. In summary, by using the above-mentioned technical solutions, the present application has the following beneficial effects:

[0024] The long-term preservation and activity maintenance of rust fungi are realized. The fungus in the preservation tube is easy to use, transport, take and store. Experimental results show that the preservation time of rust fungi can be extended by more than five years.

[0025] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings. Attached Figure Description

[0026] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0027] Figure 1 A schematic diagram is shown illustrating the preservation of spores in a preservation tube using the method of the present invention.

[0028] Figure 2 A schematic diagram of the apparatus structure for vacuum treatment of rust fungi provided in Embodiment 1 is shown;

[0029] Figure 3 Show Figure 1 Enlarged view of section A in the middle;

[0030] Figure 4 A schematic diagram showing the combined structure of the tee and pressure sensor in the rust vacuum treatment device;

[0031] Figure 5 An exploded view showing the assembly structure of the tee and pressure sensor in the rust vacuum treatment apparatus;

[0032] Figure 6 A three-dimensional structural diagram of the preservation tube in the apparatus for vacuum treatment of rust bacteria is shown.

[0033] In the diagram: 1. Negative pressure equipment; 2. Support; 211. First vertical pipe; 212. Second vertical pipe; 213. T-pipe; 2131. Third pipe body; 21311. Second flange ring; 21311a. Conical surface; 21312. Insert; 22. Protruding nozzle; 23. Connecting sleeve; 24. Clamp; 241. Fastener; 2411. First baffle; 2412. Second baffle; 242. Bolt; 3. Storage pipe; 31. First pipe section; 32. Second pipe section; 33. Reduced diameter pipe section; 4. Pressure sensor; 41. Interface pipe; 411. First flange ring; 5. Sealing ring.

[0034] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0035] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments with reference to the drawings in the embodiments of the present application. The following embodiments are used to explain the present application, but are not used to limit the scope of the present application.

[0036] In the description of the present application, it should be noted that the terms "upper", "lower", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or components referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0037] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection" should be understood broadly, for example, it can be fixed connection, or detachable connection, or integrally connected; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0038] Embodiment one: the rust fungus preservation method provided by the present application comprises:

[0039] After spore production treatment of the rust fungus, the rust fungus spores obtained after spore production treatment are collected and dried to obtain dried spores;

[0040] When the preservation tube is subjected to vacuum treatment, the vacuum treatment is performed for 15 minutes, the air pressure is reduced to 5 mbar or less, and the tube sealing treatment is performed, the rust fungus preservation is completed, and the preservation is as shown in Figure 6 .

[0041] Specifically, the spore production treatment refers to inoculating the rust fungus on the susceptible wheat seedlings by smearing method, after inoculation, the wheat seedlings are moved into a humidification frame, sprayed with clean water to make the leaf surface adhere to a layer of uniform mist droplets, closed in a humidification box, and humidified and cultured in a 9℃ dark environment for 18 hours, then the wheat seedlings are moved to a greenhouse for culture, the culture temperature is controlled at 14℃, the light is on for 10 hours per day, and the light intensity is 8000Lx. When the leaf shows spots, the central leaf is removed, a glass cover is isolated, after the uredospores mature, the leaf is gently shaken with an inoculation needle to make the strain fall into the glass cover, the wheat seedlings are removed, and the strain is concentrated in a finger-shaped tube by gently tapping the glass cover.

[0042] The drying process involves placing the spores in a sealed desiccant container at a temperature of 1°C. Commonly used desiccants can be used, such as silica gel, molecular sieves, calcium chloride, phosphorus pentoxide, calcium sulfate, etc. After drying, the spores will no longer lose moisture, and the drying time is 7 days.

[0043] Specifically, the present invention uses a negative pressure device and a support structure when performing vacuum treatment on the storage tube. Both of these structures are products independently developed by the inventor.

[0044] Specifically, the storage tube 3, negative pressure device 1, and support 2 used in this invention are as follows: Figures 1-5 As shown, the support 2 has a vertical tube and multiple protrusions 22. The vertical tube is connected to the negative pressure device 1 and has a cavity communicating with the negative pressure device 1. Each of the protrusions 22 is disposed on the vertical tube and is arranged sequentially along the length of the vertical tube. Each protrusion 22 is connected to a connecting sleeve 23. The storage tube 3 has a first tube section 31, a second tube section 32, and a reduced-diameter tube section 33. The reduced-diameter tube section 33 is located between the first tube section 31 and the second tube section 32. The outer diameter of the reduced-diameter tube section is smaller than that of the first tube section 31 and the second tube section 32. The reduced-diameter tube section 33 is connected to the first tube section 31 and the second tube section 32 respectively. The second tube section 32 has a pipe opening at one end away from the first tube section 31. The length of the second tube section 32 is smaller than that of the first tube section 31. The second tube section 32 of each storage tube 3 is detachably connected to the corresponding connecting sleeve 23 to communicate with the cavity of the vertical tube.

[0045] The support 2 of this application has a vertical tube that extends along the height direction, occupying little indoor floor space. Multiple protrusions 22 can be installed on the vertical tube, which can facilitate the arrangement of a large number of connecting sleeves 23 to simultaneously perform vacuum treatment on multiple storage tubes 3. The connecting sleeve 23 can be a rubber tube, which is sleeved on the outside of the protrusions 22. When the storage tube 3 and the connecting sleeve 23 are connected, the connecting sleeve 23 is sleeved on the second section 32 of the storage tube 3. The opening of the second section 32 is located inside the connecting sleeve 23, so that the cavity of the storage tube 3 and the vertical tube is sealed and connected.

[0046] Rust fungi can be placed inside the first pipe section 31. During the operation of the negative pressure equipment 1, a vacuum treatment can be performed on the preservation tube 3. The preservation tube 3 has a reduced diameter section 33, which can be easily and quickly heat-sealed under flame conditions to seal the first pipe section 31. Figure 1 As shown, the second segment 32 of the preservation tube 3 is shorter than the first segment 31, which does not affect the insertion and mating of the second segment 32 and the connecting sleeve 23, and also shortens the length of the preservation tube 3, saving costs. The preservation and processing component of this application realizes the long-term preservation and activity maintenance of rust fungi. The fungi after the preservation tube 3 are easy to transport, handle and preserve. Experiments have shown that it can extend the preservation time of rust fungi by more than five years.

[0047] In some possible implementations, the length ratio of the first pipe segment 31, the reduced-diameter pipe segment 33, and the second pipe segment 32 is (4.5–6):1:(2–3.5). The ratio of the outer diameter of the reduced-diameter pipe segment 33 to the outer diameter of the first pipe segment 31 or the second pipe segment 32 is 1:(1.5–3).

[0048] The storage tube 3 can be a glass tube. Compared to a regular ampere tube, the storage tube 3 of this application has been optimized in design. Through the aforementioned dimensional ratio constraints, the inner diameter of the reduced-diameter section 33 of the storage tube 3 is smaller, and the extension length of the reduced-diameter section 33 is longer, allowing for easy and rapid heat fusion sealing of the first section 31 under flame conditions. Figure 1 As shown, this allows for shorter processing time in preservation tube 3 and less damage to the fungi, enabling long-term preservation and high activity maintenance of rust fungi.

[0049] In some possible implementations, such as Figure 1 As shown, the support 2 includes at least two rows of protruding nozzles 22, with each row of nozzles 22 arranged sequentially at intervals around the circumference of the vertical tube, and each nozzle 22 in each row arranged sequentially at intervals along the length of the vertical tube. The structural design of the vertical tube in this application allows a large number of nozzles 22 to be arranged on a single vertical tube, enabling simultaneous vacuuming of multiple storage tubes 3.

[0050] In some possible implementations, such as Figure 1 As shown, the rust preservation and processing assembly includes a pressure sensor 4, which is connected to the vertical pipe and communicates with the cavity. The pressure sensor 4 is used to monitor the air pressure in the cavity. By setting the pressure sensor 4, the air pressure in the cavity inside the vertical pipe can be monitored in real time to ensure that the air pressure inside the vertical pipe reaches the set value. The negative pressure device, also known as a vacuum device, is used to evacuate air, so that the air pressure inside the vertical pipe is maintained at a low value. Usually, when the air pressure monitored by the pressure sensor 4 reaches below 5 mbar, the experimenter can perform flame preservation treatment on the preservation tube 3. By burning the reduced diameter section 33 of the preservation tube 3 with a high-temperature flame, the locking section is heat-melted and sealed, such as... Figure 1 As shown, the first pipe section 31 and the second pipe section 32 eventually separate, and the second pipe section 32 will be connected to the connecting sleeve 23.

[0051] In some possible implementations, such as Figure 1 and Figure 2As shown, the vertical pipe includes a first vertical pipe 211, a second vertical pipe 212, and a three-way pipe 213. The first vertical pipe 211 is connected to the negative pressure device 1. The second vertical pipe 212 is provided with the protruding nozzle 22. The three-way pipe 213 has a first pipe body, a second pipe body, and a third pipe body 2131. The three-way pipe 213 is located between the first vertical pipe 211 and the second vertical pipe 212. The first pipe body is connected to the first vertical pipe 211, the second pipe body is connected to the second vertical pipe 212, and the air pressure sensor 4 is connected to the third pipe body 2131. In order to achieve accurate and effective monitoring of the air pressure in the cavity of the vertical pipe, this embodiment of the application provides a three-way pipe 213 to connect the air pressure sensor 4 to the vertical pipe.

[0052] In some possible implementation schemes, combined Figures 1 to 4 As shown, the bracket 2 includes a clamp 24, the air pressure sensor 4 has an interface tube 41, the end of the interface tube 41 is provided with a first flange ring 411, the third tube body 2131 is provided with a second flange ring 21311, and the clamp 24 is sleeved on the first flange ring 411 and the second flange ring 21311 to connect and fix the interface tube 41 and the third tube body 2131.

[0053] Specifically, the rust preservation and processing component includes a sealing ring 5. A sleeve 21312 is provided on the third tube 2131, protruding from the second flange ring 21311. The sealing ring 5 is fitted onto the sleeve 21312. When the third tube 2131 and the interface tube 41 are connected, the sleeve 21312 is inserted into the interface tube 41, and the sealing ring 5 is compressed and positioned between the first flange ring 411 and the second flange ring 21311. The sealing ring 5 serves to seal the gap between the third tube 2131 and the interface tube 41.

[0054] In some possible implementations, such as Figure 4 As shown, the second flange ring 21311 has a tapered surface 21311a on the side opposite to the first flange ring 411. The clamp 24 has a first baffle 2411 and a second baffle 2412. The first baffle 2411 fits against the side of the first flange ring 411 opposite to the second flange ring 21311, and the second baffle 2412 is fitted onto the tapered surface 21311a. By providing the tapered surface 21311a on the second flange ring 21311, when the second baffle 2412 is fitted onto the tapered surface 21311a, a compressive force can be effectively applied to the tapered surface 21311a, which is beneficial for the first flange ring 411 and the second flange ring 21311 to approach and clamp together to form the sealing ring 5.

[0055] The clamp 24 includes two fasteners 241, one end of which is hinged together, and the other ends of which are detachably connected. Each fastener 241 is provided with a first baffle 2411 and a second baffle 2412. When the two fasteners 241 of the clamp 24 are connected, the first baffles 2411 on the two fasteners 241 form an annulus and are located on the same plane, and the second baffles 2412 on the two fasteners 241 form an annulus and are located on the same plane. The free ends of the two fasteners 241 can be connected and fixed by bolts 242.

[0056] It should be noted that the clamp connection between the interface pipe and the third pipe can be applied to the connection between the first pipe and the first vertical pipe, as well as the connection between the second pipe and the second vertical pipe.

[0057] Vacuum sealing: After drying, the spore sample is placed in the preservation tube 3 of this application, and the air inside the tube is removed by the negative pressure device 1. After the predetermined vacuum degree is reached, the tube opening is sealed.

[0058] In this step, the dried rust fungus is transferred to the preservation tube 3, and the preservation tube 3 is vacuumed using the negative pressure device 1 to reduce the pressure inside the tube to 5 mbar. Then, the preservation tube 3 is sealed with a flame.

[0059] Sealing and preservation: After completing vacuum preservation tube 3, the sample can be stored for a long time under refrigeration conditions, and the activity of the sporophyte can be restored by reculturing.

[0060] In this step, the rust fungus after sealing is stored at 4℃ or -20℃ for a long period of time.

[0061] The rust fungus was dried before being sealed into the first section 31 of the preservation tube 3. During the preservation process in tube 3, vacuum treatment was performed, significantly extending the preservation time of the rust fungus; experiments showed that the preservation time could reach more than 5 years. The activity loss rate of the sealed rust fungus was better than that of existing technologies. In the sealing test, the preservation and processing component of this application was simple to operate, and the fungus after preservation tube 3 was easy to transport, handle, and preserve. The preservation and processing component of this application has a simple structure, low cost, and significantly reduced cost compared to liquid nitrogen preservation.

[0062] Furthermore, the vacuum treatment involves detachably connecting the second section of the storage tube to a corresponding connecting sleeve to connect the cavity of the vertical tube.

[0063] The sealing process involves burning the vacuum-sealed storage tube with a flame; the flame temperature is 1900°C; and the burning time is 1 second. The storage tube is then stored at -20°C.

[0064] The vacuum preservation tube is formed by placing the dried spore sample in the preservation tube 3 of this application, and removing the air from the tube by the negative pressure device 1 to achieve a predetermined vacuum level before sealing the tube opening.

[0065] In this step, the dried rust fungus is transferred to the preservation tube 3, and the preservation tube 3 is vacuumed using the negative pressure device 1 to reduce the pressure inside the tube to 5 mbar. Then, the preservation tube 3 is sealed with a flame.

[0066] The preservation method used in this embodiment is simple to operate, has a lower cost than liquid nitrogen preservation, and a longer preservation time than suspension preservation, which can preserve the rust fungus for more than 5 years. After preservation, the rust fungus can still have high activity after being cultured and activated. Therefore, it has great value for industrial application and is suitable for large-scale promotion and application.

[0067] Example 2: A method for preserving rust fungi provided by the present invention, comprising:

[0068] After the rust fungus is subjected to sporulation treatment, the rust fungus spores obtained after the sporulation treatment are collected and dried to obtain dried spores.

[0069] After drying, the spores are transferred to preservation tubes. When vacuuming the preservation tubes, the tubes are evacuated for 25 minutes. When the pressure drops to below 5 mbar, the tubes are sealed to complete the preservation of the rust fungus.

[0070] The sporulation treatment involves inoculating susceptible wheat seedlings with rust fungus using a smear method. After inoculation, the seedlings are transferred to a humidified box, sprayed with water to coat the leaf surface with a uniform layer of droplets, and then sealed in a dark environment at 12°C for 24 hours. Afterward, the seedlings are transferred to a greenhouse for further cultivation, with the temperature controlled at 11°C, light exposure for 14 hours / day, and a light intensity of 10000 Lx. When spots appear on the leaves, the central leaves are removed, and the seedlings are isolated with a glass cover. Once the urediniospores mature, the leaves are gently shaken with an inoculation needle to disperse the inoculum into the glass cover. The seedlings are then removed, and the glass cover is gently tapped to collect the inoculum, which is then placed into finger-shaped tubes.

[0071] The drying process involves placing the spores in a sealed desiccant container at a temperature of 10°C. Commonly used desiccants can be used, such as silica gel, molecular sieves, calcium chloride, phosphorus pentoxide, calcium sulfate, etc. After drying, the spores will no longer lose moisture, and the drying time is 10 days.

[0072] The device described in this application enables long-term preservation and activity maintenance of rust fungi. The fungi in the preservation tube 3 are easy to transport, handle and preserve. Experiments have shown that the preservation time of rust fungi can be extended to more than five years.

[0073] Example 3: A method for preserving rust fungi provided by the present invention, comprising:

[0074] After the rust fungus is subjected to sporulation treatment, the rust fungus spores obtained after the sporulation treatment are collected and dried to obtain dried spores.

[0075] After drying, the spores are transferred to preservation tubes. When vacuuming the preservation tubes, the tubes are evacuated for 20 minutes. When the pressure drops to below 5 mbar, the tubes are sealed to complete the preservation of the rust fungus.

[0076] The sporulation treatment involves inoculating susceptible wheat seedlings with rust fungus using a smear method. After inoculation, the seedlings are transferred to a humidified box, sprayed with water to coat the leaf surface with a uniform layer of droplets, and then sealed in a dark environment at 10°C for 20 hours. Afterward, the seedlings are transferred to a greenhouse for further cultivation, with the temperature controlled at 17°C, light exposure 12 hours / day, and light intensity at 9000 Lx. When spots appear on the leaves, the central leaves are removed, and the seedlings are isolated with a glass cover. Once the urediniospores mature, the leaves are gently shaken with an inoculation needle to disperse the inoculum into the glass cover. The seedlings are then removed, and the glass cover is gently tapped to collect the inoculum, which is then placed into finger-shaped tubes.

[0077] The drying process involves placing the spores in a sealed desiccant container at a temperature of 5°C. Commonly used desiccants can be used, such as silica gel, molecular sieves, calcium chloride, phosphorus pentoxide, calcium sulfate, etc. After drying, the spores will no longer lose moisture, and the drying time is 10 days.

[0078] It should be noted that the drying temperature and storage temperature used in the method of the present invention are not limited to the examples listed in the above embodiments. For example, drying temperatures of 2℃, 3℃, 4℃, 5℃, and 6℃ are all acceptable, and storage temperatures of -20℃, -15℃, -10℃, -5℃, 0℃, and 4℃ are also acceptable. Combining the overall steps of the present invention for storage will not damage the storage time of the rust fungus or its activity after storage and activation.

[0079] The following are some experiments to illustrate the beneficial effects of using the method of the present invention to preserve rust fungi:

[0080] Activation method of wheat stripe rust fungus urediniospores:

[0081] 1. Hydration treatment

[0082] Objective: This step aims to hydrate dried wheat stripe rust urediniospores to restore their biological activity, enhance their germination rate and pathogenicity, and provide a guarantee for subsequent inoculation experiments.

[0083] The specific steps are as follows:

[0084] ① Take an appropriate amount of dried and preserved wheat stripe rust urediniospores and place them in a sealed container (such as a plastic box, petri dish, or glass humidifier). Add an appropriate amount of sterile water to the bottom of the container beforehand as a source of humidity, and the amount of water should be controlled within a range that does not directly contact the spores to create a saturated water vapor environment.

[0085] ② Place the sealed container containing the spores at a low temperature of 4°C for 8–16 hours to complete the hydration treatment, with 12 hours being the preferred treatment time. During this period, the spores are only exposed to saturated water vapor and do not come into direct contact with liquid water to prevent premature germination or reduced activity.

[0086] ③ If the experimental schedule is tight, an alternative solution of hydration at room temperature (20-25℃) for 2-4 hours can be used, but the hydration effect is slightly inferior to that of treatment at 4℃, and the spore activation level is relatively low.

[0087] 2. Activation and inoculation treatment

[0088] ① Apply the hydrated urediniospores evenly to the leaf surface of the susceptible wheat variety “Mingxian 169” seedlings, using an inoculation needle to gently spread the spores, ensuring even inoculation without damaging the leaf epidermis.

[0089] ② After inoculation, place the wheat seedlings in a dark environment at 9-13℃ for high humidity treatment for 24 hours to promote spore germination and infection.

[0090] ③ After the moisturizing treatment, transplant the seedlings to a greenhouse for continued cultivation. The greenhouse conditions are: temperature 14±3℃, light duration 10~14 hours / day, and light intensity 8000–10000Lx.

[0091] ④ After cultivation and observation, obvious uredinia (lesions) are usually visible at the inoculation site 10 to 14 days after inoculation, indicating that the urediniospores have recovered well and the pathogenicity of the inoculated urediniospores remains normal.

[0092] This processing procedure can effectively improve the viability recovery rate and inoculation efficiency of dried and preserved urediniospores, and is suitable for long-term preservation of rust fungi and their resuscitation use in subsequent biological research.

[0093] Period of preservation Number of inoculated leaves Number of diseased leaves Disease incidence (%) Fresh zoospores 30 30 100 Preserved for 1 month (2025 preservation) 30 30 100 Preserved for 1 year (2024 preservation) 30 29 96.67 Preserved for 2 years (2023 preservation) 30 29 96.67 Preserved for 3 years (2022 preservation) 30 28 93.33 Preserved for 4 years (2021 preservation) 30 28 93.33 Preserved for 5 years (2020 preservation) 30 28 93.33 Preserved for 6 years (2019 preservation) 30 27 90.00

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preserving rust fungi, comprising: After the rust fungus is subjected to sporulation treatment, the rust fungus spores obtained after the sporulation treatment are collected and dried to obtain dried spores. The dried spores were transferred to preservation tubes, which were then vacuum-treated for 15-25 minutes. When the pressure dropped to below 5 mbar, the tubes were sealed to complete the preservation of the rust fungus.

2. According to the preservation method of claim 1, the sporulation treatment involves inoculating rust fungi into susceptible wheat seedlings, culturing them in a dark environment with moisture, then transferring them to a greenhouse for further cultivation, and collecting the fungal inoculum when spots appear on the wheat leaves; Preferably, the dark environment moist culture involves sealing the inoculated wheat seedlings in a moist culture box and culturing them in a dark environment at 9-13℃ for 18-24 hours. Preferably, the cultivation temperature in the greenhouse is controlled at 14±3℃, the light intensity is 10-14 hours / day, and the light intensity is 8000-10000Lx. Preferably, the collection of inoculum when wheat leaves show spots is achieved by gently shaking the leaves with an inoculation needle to scatter and collect the inoculum.

3. The preservation method according to claim 1, wherein the drying process involves placing the spores in a sealed desiccator containing a desiccant at a drying temperature of 1-10°C.

4. The preservation method according to claim 1, wherein the dried spores are spores in a state where the moisture content no longer decreases during the drying process.

5. The preservation method according to claim 1, wherein the preservation tube comprises a first tube segment, a second tube segment, and a reduced-diameter tube segment between the first tube segment and the second tube segment; Furthermore, the outer diameter of the reduced-diameter pipe section is smaller than that of the first pipe section and the second pipe section; Furthermore, the reduced-diameter pipe section is connected to the first pipe section and the second pipe section respectively; Furthermore, the second pipe section has a pipe opening at the end opposite to the first pipe section; Furthermore, the length of the second pipe segment is less than the length of the first pipe segment; Furthermore, the length ratio of the first pipe segment, the reduced-diameter pipe segment, and the second pipe segment is (4.5-6):1:(2-3.5); Furthermore, the ratio of the outer diameter of the reduced-diameter pipe section to that of the first or second pipe section is 1:(1.5 to 3).

6. The preservation method according to claim 5, wherein the dried spores are transferred to the first segment of the preservation tube.

7. The preservation method according to claim 6, wherein the vacuum treatment is performed by connecting the preservation tube to a negative pressure device; Furthermore, the negative pressure device includes: Negative pressure equipment; The bracket has a vertical tube and multiple protrusions. The vertical tube is connected to the negative pressure device and has a cavity communicating with the negative pressure device. Each of the protrusions is disposed on the vertical tube and is arranged sequentially along the length of the vertical tube. Each protrusion is connected to a connecting sleeve. Furthermore, the vacuum treatment involves detachably connecting the second section of the storage tube to the corresponding connecting sleeve to connect the cavity of the vertical tube; Furthermore, the bracket includes at least two rows of protruding nozzles, with each row of nozzles arranged sequentially at intervals around the circumference of the vertical tube, and each nozzle in each row arranged sequentially at intervals along the length of the vertical tube.

8. The preservation method as described in claim 7, characterized in that, The negative pressure device also includes a pressure sensor, which is connected to the vertical pipe and communicates with the cavity. The pressure sensor is used to monitor the pressure in the cavity. Furthermore, the vertical pipe includes a first vertical pipe, a second vertical pipe, and a tee pipe; Furthermore, the first vertical pipe is connected to the negative pressure device, and the second vertical pipe is provided with the protruding nozzle; Furthermore, the tee pipe has a first pipe body, a second pipe body, and a third pipe body; Furthermore, the tee pipe is located between the first vertical pipe and the second vertical pipe, with the first pipe body connected to the first vertical pipe and the second pipe body connected to the second vertical pipe; Furthermore, the pressure sensor is connected to the third tube. Furthermore, the bracket includes a clamp; the pressure sensor has an interface tube, and a first flange ring is provided at the end of the interface tube; a second flange ring is provided on the third tube body; the clamp is sleeved on the first flange ring and the second flange ring to connect and fix the interface tube and the third tube body. Furthermore, it also includes a sealing ring; a plug is provided on the third pipe body, and the plug protrudes from the second flange ring; The sealing ring is fitted onto the insert; with the third tube and the interface tube connected, the insert is inserted into the interface tube, and the sealing ring is compressed and disposed between the first flange ring and the second flange ring; Furthermore, the second flange ring has a tapered surface on the side opposite to the first flange ring; the clamp has a first baffle and a second baffle, the first baffle is attached to the side of the first flange ring opposite to the second flange ring, and the second baffle is sleeved on the tapered surface; Furthermore, the clamp includes two fasteners; one end of the two fasteners is hinged together, and the other end of the two fasteners is detachably connected; each fastener is provided with a first baffle and a second baffle.

9. The preservation method according to claim 1, wherein the sealing process is performed by burning the preservation tube under vacuum with a flame; The further flame temperature is 1900-2100℃; Furthermore, the burning time is 1-3 seconds.

10. The preservation method as described in claim 1, wherein the preservation tube is stored at a temperature of -20 to 4°C.

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