Inoculation method convenient for wheat stripe rust single spore isolation

Abstract

The invention provides an inoculation method convenient for wheat stripe rust single spore isolation, and belongs to the technical field of wheat stripe rust single spore isolation. Uniformly mixing the summer spore powder and the talcum powder according to a preset proportion, and adding the mixture into the L-shaped glass bent pipe; wax layers of bred wheat seedlings are removed, and the wheat seedlings are placed on a rotating table after being sprayed with water mist; the rotating table device is started to rotate uniformly; under the action of pressure, uniformly spraying the mixed bacterial powder in the L-shaped glass bent pipe to wheat seedling leaves for inoculation; wheat seedlings are subjected to isolated culture after being moisturized, and then single spore isolation is carried out. According to the method, the ratio of the summer spore powder to the talcum powder is accurately controlled, and an improved bacterium spraying device is adopted, so that the inoculation success rate and the spore distribution uniformity are remarkably improved, the separation effect is better, and single spores are easier to obtain.

Description

Technical Field

[0001] This invention relates to the field of single-spore isolation technology of wheat rust fungi, and specifically to an inoculation method for wheat stripe rust fungi that facilitates efficient single-spore isolation. Background Technology

[0002] Artificial inoculation of wheat stripe rust is a fundamental technique in wheat stripe rust research and resistance identification, widely used for the identification of standards and races of wheat stripe rust fungus, and analysis of virulence variation. To further purify the strain, single-spore isolation is usually required. However, currently used single-spore isolation methods have significant shortcomings: methods such as glass fiber inoculation, microscopic agar plate inoculation, and water agar plate inoculation are complex, time-consuming, and prone to contamination, making them unsuitable for large-scale application. Microscopic manipulation requires precise techniques and is difficult to perform, hindering batch isolation. The dilution plate method is suitable for single-spore isolation of common fungi, but cannot meet the requirements for single-spore isolation of obligate parasites such as wheat stripe rust fungus. Existing technologies struggle to achieve efficient single-spore isolation of wheat stripe rust fungus, affecting the accurate analysis of virulence variation and limiting the accuracy and consistency of resistance research.

[0003] In response to this situation, the present invention provides an improved inoculation method to simplify the operation process, improve inoculation efficiency, and effectively increase the success rate of single spore isolation, providing reliable technical support for the epidemic monitoring of wheat stripe rust and the analysis of the virulence variation of wheat stripe rust fungus. Summary of the Invention

[0004] This invention proposes an improved method for spore inoculation and single-spore isolation of wheat stripe rust. Through an innovative spore sedimentation device and optimized spraying process, the uniformity and success rate of inoculation are significantly improved, effectively reducing spore overlap and facilitating single-spore isolation. The device uses a rotating platform to evenly spray spores onto wheat leaves. Combined with precisely controlled mixing ratios of spores and talc powder and spraying parameters, the consistency and reliability of experimental results are enhanced. This improved inoculation and isolation method has broad application prospects in wheat stripe rust resistance studies, epidemiological studies, and fungal genetics research, providing an efficient and reliable experimental tool that significantly improves experimental efficiency and result accuracy.

[0005] The purpose of this invention is to provide an inoculation method that facilitates the isolation of single spores of wheat stripe rust, achieving efficient isolation of single spores through precise control of the inoculation process.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solutions are proposed:

[0007] This invention provides an inoculation method for facilitating the isolation of single spores of wheat stripe rust, comprising the following steps:

[0008] S1. Provide a spore settling device: The spore settling device includes: a box, the box having a rotating platform for holding wheat seedlings to be inoculated; a motor is provided below the rotating platform to drive the rotating platform to rotate;

[0009] A spraying device is provided above the rotating platform. The spraying device includes a support pipe and an L-shaped bend. The L-shaped bend includes a connecting section and a spraying section that are perpendicular to each other. One end of the support pipe is connected to an external air compressor and fixed to the top of the box. The other end of the support pipe opens in the horizontal direction. The connecting section of the L-shaped bend is detachably fixed to the other end of the support pipe by a clamp. The outlet direction of the spraying section of the L-shaped bend is vertically upward.

[0010] The diameter of the joint between the connecting section and the ejection section of the L-shaped bend is reduced, and the diameter of the outlet of the ejection section of the L-shaped bend is reduced.

[0011] S2. Remove the detachable L-shaped bend from the spraying device, add fungal powder made of urediniospore powder and talc powder in a mass ratio of 1:400-600 to the connecting section, and then fix the bend to the support tube; after removing the waxy layer from the cultivated wheat seedlings, spray a layer of water mist and place them on the rotating platform.

[0012] The device can be configured with multiple L-shaped bends to meet the batch inoculation needs of different bacterial strains, thereby improving the flexibility and efficiency of experiments.

[0013] S3. Start the air compressor and motor. The rotary table will start to rotate. When the air compressor reaches the working pressure, open the air compressor switch valve so that the air pressure pushes the bacterial powder out of the spray section outlet and settles onto the surface of the wheat seedling leaves for a period of time.

[0014] S4. After inoculation, the wheat seedlings were kept moist and then placed in a greenhouse for further isolated culture. After spotting, single spores were isolated.

[0015] The following is a detailed explanation:

[0016] S1, Spore sedimentation device:

[0017] The container is a closed structure to ensure environmental control during the inoculation process and reduce external contamination. It is equipped with a transparent observation door for easy real-time monitoring by operators, improving work efficiency. A stable rotating platform, driven by a motor, is installed at the bottom of the container to support the wheat seedlings to be inoculated. The platform rotates evenly, ensuring the inoculum is evenly distributed on the wheat seedling leaves during inoculation, thereby increasing the success rate.

[0018] The spraying device includes a support pipe and an L-shaped bend installed at the top of the chamber. The L-shaped bend is connected to the support pipe and is used to spray the bacterial powder onto the wheat seedlings on the rotating platform below. The L-shaped bend is made of transparent glass, ensuring that the flow of the bacterial powder can be observed during operation. The support pipe is fixed to the chamber through holes at the top, ensuring the stability of the bend during spraying and allowing air from the air compressor to reach the L-shaped bend through the support pipe. The material and structure of the support pipe ensure that it will not deform during spraying and it can be made of PVC. The L-shaped bend is connected to the support pipe by a fixing clamp; the material and design of the clamp should have sufficient strength and durability.

[0019] An air compressor is located outside the enclosure to provide the air pressure required for the inoculation process. A pressure gauge is mounted on the air compressor to display the compressor's operating pressure in real time. This pressure gauge ensures that operators can monitor and adjust the operating pressure of the inoculation device, guaranteeing stability during the inoculation process.

[0020] In the inoculation process of wheat stripe rust, the structural design of the spraying device directly affects the inoculation and separation effects. The L-shaped bend designed in this invention has one end for storing the mycelium and the other end for spraying. Under the pressure of the airflow, the mycelium powder at the storage end can be smoothly transported to the spray nozzle. The right-angle bend of the bend and the spray nozzle both employ a constriction design to increase the resistance when the mycelium powder passes through, achieving a flow-limiting effect and ensuring uniform spraying of the mycelium powder, avoiding accumulation and uneven inoculation caused by excessive spraying. Compared with traditional U-shaped or V-shaped bends, the L-shaped bend structure of this invention significantly improves inoculation efficiency and success rate, resulting in a more uniform inoculation process and facilitating the acquisition of single spores. It is an ideal choice for wheat stripe rust isolation and inoculation devices.

[0021] Step S2:

[0022] In some implementations, the cultivation of wheat seedlings includes: selecting flowerpots with a diameter of 10cm and a height of 10cm, filling them with soil rich in organic matter, placing them in a seedling bed, sowing 20-30 healthy and plump wheat varieties of the "Mingxian 169" in each pot, and cultivating them in a greenhouse until the first leaf unfolds for use.

[0023] In some implementations, the waxy layer on the leaf is gently wiped off from top to bottom with clean, water-dipped fingers, and then a sprayer is used to coat the leaf surface with a uniform layer of droplets.

[0024] Step S3:

[0025] In some implementations, the rotational speed of the rotary table is 20 r / min.

[0026] In some implementations, the air compressor operates at a pressure of 0.2 MPa.

[0027] In some implementations, the settling time of each inoculation powder is 10-15 minutes.

[0028] Step S4:

[0029] In some implementations, moisturizing culture includes: after inoculation, transferring wheat seedlings into a moisturizing frame, spraying the leaves with a layer of water mist, sealing the moisturizing frame, and moisturizing in a dark environment at 9-12°C for 24 hours.

[0030] In some implementations, single-spore isolation includes: when the leaves develop mottled patterns but have not yet produced spore masses, retaining one leaf infected with a single spore lesion per pot, transferring it to a new pot for continued cultivation until spores are produced and the inoculum is collected.

[0031] Beneficial effects:

[0032] This invention utilizes a self-made spore sedimentation device and a spraying device with a specific structure. By precisely controlling the ratio of urediniospore powder to talc powder and inoculation parameters, it significantly improves the inoculation effect, resulting in a high inoculation success rate, good spore distribution uniformity, and a high single spore isolation success rate. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the spore sedimentation device;

[0034] Figure 2 This is an enlarged view of the L-shaped bend.

[0035] Diagram: 1-Box body; 2-Rotating table; 31-Support pipe; 32-L-shaped bend; 321-Connecting section; 322-Ejection section; 33-Clamp; 4-Air compressor. Detailed Implementation

[0036] To enable those skilled in the art to more clearly understand the purpose, technical solution, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and examples.

[0037] It should be understood that the specific examples described herein are for illustrative purposes only and are not intended to limit the scope of the invention. All embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] A spore settling device for wheat stripe rust urediniospores, such as Figure 1 , Figure 2As shown, the system includes: a box 1 (inoculation room), with a transparent glass door at the front; a rotating platform 2 (driven by a motor) installed inside the box 1 for placing wheat seedlings; a spraying device above the rotating platform 2, including a PVC support pipe 31 and a glass L-shaped bend 32; the L-shaped bend 32 includes a perpendicular connecting section 321 and a spraying section 322 (the diameter decreases at the L-shaped bend); one end of the support pipe 31 is connected to an air compressor and fixed to the top of the box 1 through a top hole, and the other end is horizontally open and connected to the connecting section 321 of the L-shaped bend 32 by a clamp 33; the outlet direction of the spraying section 322 of the L-shaped bend 32 is upward (the diameter decreases at the outlet), used to spray the inoculum onto the wheat seedlings on the rotating platform 2 below (the outlet of the spraying section 322 of the L-shaped bend 32 and the center of the rotating platform can be located in the same vertical direction).

[0040] Example 2

[0041] The method for isolating wheat stripe rust urediniospores using the spore sedimentation device of Example 1 includes the following steps:

[0042] 1. Seedling raising:

[0043] Select flowerpots with a diameter of 10cm and a height of 10cm, fill them with soil rich in organic matter, and place them in the seedling bed. Water will be absorbed through the bottom to moisten the soil. Sow 20-30 healthy and plump wheat variety "Mingxian 169" seeds in each pot and cultivate them in a greenhouse. Once the wheat seedlings have fully unfolded their first leaf, they are ready for use.

[0044] 2. Preparation of bacterial powder:

[0045] Mix 2.5 mg of fresh wheat rust fungus urediniospore powder with 1-1.5 g of talc powder (dilution ratio 400-600 times) and put it into a 9 mm diameter glass test tube. After thorough mixing, add it to an L-shaped bend tube for later use (the detachable L-shaped bend tube can be removed from the spraying device, added from the connecting section, and then the bend tube can be fixed back to the support tube).

[0046] 3. Preparations before vaccination:

[0047] Use clean fingers dipped in water to gently wipe away the waxy layer from top to bottom on the leaf. Then use a sprayer to coat the leaf surface with a uniform layer of droplets in preparation for inoculation.

[0048] 4. Vaccination procedure:

[0049] After spraying water mist inside the inoculation chamber (box), close the door and allow the water mist to settle. Place the water-mist-sprayed wheat seedlings on a rotating platform, start the air compressor and the rotating platform, and set the speed to 20 r / min. When the compressor pressure reaches 0.2 MPa, open the valve to allow the inoculum powder to be sprayed upwards through the L-shaped bend, and then allowed to settle evenly onto the wheat seedling leaves through free fall. Each settling time should be controlled to 10-15 minutes. After inoculation, close the rotating platform and remove the wheat seedlings.

[0050] 5. Moisture-controlled culture:

[0051] After inoculation, the wheat seedlings were transferred into a moisture-retaining frame, the leaves were sprayed with a layer of water mist, the frame was sealed, and the seedlings were kept in the dark at 9-12℃ for 24 hours. After that, the seedlings were transferred to a greenhouse for isolation and cultivation.

[0052] 6. Single-spore isolation:

[0053] When the inoculated leaves develop mottled patterns but have not yet formed spore masses, perform the following steps: Carefully observe the inoculated wheat seedlings, remove uninfected leaves and leaves with more than two spore masses, retaining only leaves with one infection point. Keep only one plant per pot with a leaf and only one infection point. Transfer the remaining suitable plants to another pot. Cover each pot with a glass dome to prevent cross-contamination and external environmental influences. Place the covered pots in a greenhouse, maintaining suitable temperature and humidity, and continue cultivation until spore masses form. After spore masses form, number each plant and collect single spores from each leaf using test tubes to obtain a single-spore strain. The spores produced by each leaf constitute one single-spore strain.

[0054] Test case

[0055] 1. Infection rate

[0056] Definition: Infection rate is the ratio of the number of leaves with infected spots to the total number of leaves.

[0057] This experiment compared different shapes of spraying devices (L-shaped, U-shaped, V-shaped bends, and L-shaped bends (without reduced diameter)) and different powder formulations. The results are shown in Table 1. To ensure the effectiveness of single-spore isolation, the infection rate should be controlled between 10% and 30%.

[0058] Table 1 Infection rate results (%)

[0059]

[0060] Note: Other test conditions: air compressor operating pressure 0.2MPa, settling time 10 minutes.

[0061] 2. Uniformity of spore distribution and separation effect of individual spore clusters:

[0062] Observe the spore distribution on each leaf, ensuring that only one monospore cluster remains on each leaf for subsequent separation. A high proportion of leaves with only one monospore cluster indicates a good device performance. Evaluation criteria:

[0063] Advantages: The number of diseased leaves is moderate (e.g., 2-5 leaves per pot), and there is basically only one spore mass on the diseased leaf, with only one infection point on the spore mass, resulting in a high single spore production rate.

[0064] Medium: The number of diseased leaves is relatively small (e.g., 0-1 leaves per pot), there is only one spore mass on the diseased leaf, and only one infection point on the spore mass, but the number of diseased leaves is small and the single spore production rate is low.

[0065] Poor: There are many diseased leaves (e.g., more than 6 leaves per pot), and there are two or more spore masses on the diseased leaves, or one spore mass has two or more overlapping infection points. There are few leaves with only one infection point on one spore mass, resulting in low isolation quality; or there are no diseased leaves, resulting in low single spore production rate.

[0066] This experiment used different shaped spraying devices (L-shaped, U-shaped, V-shaped bends and L-shaped bends (without reduced diameter)) and different bacterial powder ratios for comparison. The separation results are shown in Table 2.

[0067] Table 2 Spore distribution and isolation effect

[0068]

[0069] Note: Other test conditions: air compressor operating pressure 0.2MPa, settling time 10 minutes.

[0070] Analysis: An appropriate amount of inoculum powder can improve the infection rate, but excessive amounts can lead to multispore masses and overlapping infections on leaves, which is detrimental to single-spore isolation. Compared to other designs, the L-shaped curved tube exhibits superior performance in inoculation uniformity and single-spore isolation; ratios 2 and 3 are recommended as standards.

[0071] In addition, the effects of different pressures and settling times on infection rate and isolation efficiency were studied, and the results are shown in Tables 3 and 4.

[0072] Table 3 Effects of Pressure

[0073] Test number pressure Infection rate (%) Spore distribution and isolation effect 1 0.05 10 middle 2 0.1 20 middle 3 0.15 30 middle 4 0.2 30 excellent 5 0.25 40 Difference 6 0.3 50 Difference

[0074] Note: Other test conditions: dilution factor 400.

[0075] Table 4. Effect of Settlement Time

[0076] Test number Settlement time (minutes) Infection rate (%) Spore distribution and isolation effect 1 5 5 middle 2 10 30 excellent 3 15 30 excellent 4 20 30 middle 5 25 30 middle

[0077] Note: Other test conditions: dilution factor 400.

[0078] In summary, the experimental results show that the L-shaped bend design of this invention is significantly superior to the traditional U-shaped and V-shaped bend designs in terms of inoculation uniformity, infection rate, and single-spore isolation efficiency. It is recommended to use the bacterial powder concentrations at ratios 2 and 3, and to control the air compressor operating pressure at 0.2 MPa and the settling time at 10-15 minutes to achieve optimal inoculation effect and single-spore isolation efficiency.

[0079] Those skilled in the art will readily understand that the above description is merely a general embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An inoculation method for facilitating the isolation of single spores of wheat stripe rust, characterized in that, Includes the following steps: S1. Provide a spore settling device: The spore settling device includes: a box, the box having a rotating platform for holding wheat seedlings to be inoculated; a motor is provided below the rotating platform to drive the rotating platform to rotate; A spraying device is provided above the rotating platform. The spraying device includes a support pipe and an L-shaped bend. The L-shaped bend includes a connecting section and a spraying section that are perpendicular to each other. One end of the support pipe is connected to an external air compressor and fixed to the top of the box. The other end of the support pipe opens in the horizontal direction. The connecting section of the L-shaped bend is detachably fixed to the other end of the support pipe by a clamp. The outlet direction of the spraying section of the L-shaped bend is vertically upward. The diameter of the joint between the connecting section and the ejection section of the L-shaped bend is reduced, and the diameter of the outlet of the ejection section of the L-shaped bend is reduced. S2. Remove the L-shaped bend from the spraying device, add bacterial powder through the opening of the connecting section, and then fix it back onto the support tube. The bacterial powder is obtained by mixing urediniospore powder and talc powder in a mass ratio of 1:400-600. After removing the waxy layer from the cultivated wheat seedlings, spray a layer of water mist and place them on the rotating platform. S3. Start the air compressor and motor. The rotary table will start to rotate evenly. When the air compressor reaches the working pressure, open the air compressor switch valve so that the air pressure pushes the bacterial powder out from the spray section outlet and settles onto the surface of the wheat seedling leaves. The inoculation continues for a period of time. S4. After inoculation, the wheat seedlings are kept moist and then placed in a greenhouse for isolated culture. After spotting, single spores are isolated.

2. The method according to claim 1, characterized in that, The enclosure has a transparent observation door.

3. The method according to claim 1, characterized in that, The support tube is made of PVC.

4. The method according to claim 1, characterized in that, The L-shaped bend is made of glass.

5. The method according to claim 1, characterized in that, In step S2, the wheat seedlings are obtained by the following method: select flower pots with a diameter of 10cm and a height of 10cm, fill them with soil rich in organic matter, sow 20-30 healthy and plump wheat seeds in each pot, and cultivate them in a greenhouse until the first leaf unfolds.

6. The method according to claim 1, characterized in that, In step S2, use clean fingers dipped in water to gently wipe away the wax layer on the leaf from top to bottom, and then use a sprayer to coat the leaf surface with a uniform layer of mist droplets.

7. The method according to claim 1, characterized in that, In step S3, the rotational speed of the rotary table is 20 r / min.

8. The method according to claim 1, characterized in that, In step S3, the working pressure of the air compressor is 0.2 MPa.

9. The method according to claim 1, characterized in that, In step S3, the settling time of each inoculation powder is 10-15 minutes.

10. The method according to claim 1, characterized in that, In step S4, the moisturizing culture includes: after inoculation, the wheat seedlings are transferred into a moisturizing frame, then sprayed with water, the moisturizing frame is sealed, and moisturized in a dark environment at 9-12℃ for 24 hours; Single spore isolation involves: when leaves develop mottled patterns but have not yet produced spore masses, retaining one leaf infected with a single spore spot from each pot, transferring it to a new pot for continued cultivation until spores are produced and the inoculum is collected.

Images