A device and method for inducing rice bacterial leaf blight

Abstract

The present application relates to the technical field of plant pathology and agricultural science research, more particularly to a rice seedling blight inducing device and method, wherein the device comprises a box body, a box cover, a first measuring cup and a second measuring cup, the box cover is detachably connected to the top of the box body, the box body is a transparent structure, the box cover is provided with air holes, the bottom of the box body is provided with a drainage hole, the side wall of the box body is provided with a scale, the capacity of the first measuring cup is greater than that of the second measuring cup, and the first measuring cup can be inverted to cover the second measuring cup to form a closed germination cavity. The present application has the advantages of simple structure and facilitating operation standardization; through the sandwich type matrix structure of "bottom moisture layer + seed layer + top covering layer", combined with the two-stage culture mode of indoor constant temperature disease induction and outdoor natural adversity induction, the disease incidence is significantly improved, and disease identification is facilitated.

Description

Technical Field

[0001] This invention relates to the technical field of plant pathology and agricultural science research, and more specifically, to a device and method for inducing rice bakanae disease. Background Technology

[0002] Rice bakanae disease is a typical seed-borne disease caused by Fusarium oxysporum. In research on disease resistance breeding and fungicide efficacy evaluation, there is an urgent need to establish a stable, efficient, and ecologically sound system for inducing and identifying the disease. Existing artificial induction techniques for the disease mainly fall into two categories: one is the indoor pure culture inoculation method, and the other is the field natural disease identification method.

[0003] Traditional indoor inoculation methods often rely on strains that have undergone multiple purification and culture processes. These strains are prone to mutations in pathogenicity and sensitivity to pesticides during long-term three-dimensional culture, leading to significant differences in their infectivity compared to the actual state of the original infected seeds in the field. This results in a weak correlation between experimental results and actual control effectiveness. While identification methods relying on natural disease occurrence in the field can reflect the true disease situation, they are greatly affected by environmental fluctuations, resulting in low and unstable disease incidence, long identification cycles, and poor reproducibility, making it difficult to meet the efficiency and reliability requirements of high-throughput, precision scientific research.

[0004] Existing technology CN105284450A discloses a method for rapidly inducing rice blast disease using a box-type seedling cultivation system. This method employs a closed box-type microenvironment to cultivate rice seedlings and inoculates them with rice blast fungus during the needle-like bud stage, achieving rapid induction of the disease at the seedling stage. While this method has certain advantages in controlling environmental conditions and shortening the cycle, it primarily targets rice blast and relies on artificially prepared nutrient solutions and spore suspensions for inoculation. The operation steps are relatively cumbersome, and it does not address targeted induction designs for seed-borne diseases such as bakanae disease. In particular, it has significant shortcomings in simulating the natural disease process of infected seeds in the field, increasing the incidence rate, and facilitating identification. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a device and method for inducing rice bakanae disease, which has a simple structure, standardized operation, can significantly improve the incidence of rice bakanae disease and facilitate disease identification.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: to provide a rice seedling disease induction device, including a box body, a box lid, a first measuring cup and a second measuring cup, the box lid being detachably connected to the top of the box body, the box body being a transparent structure, the box lid having an air hole, the bottom of the box body having a drainage hole, the side wall of the box body having a scale, the capacity of the first measuring cup being greater than the capacity of the second measuring cup, and the first measuring cup being able to be inverted to cover the second measuring cup to form a sealed germination chamber.

[0007] This invention relates to a rice bakanae disease induction device. The transparent box facilitates observation of seedling growth and disease development, and the scale on the side wall allows for direct observation of plant growth. In use, rice seeds are soaked, drained, and placed in a second measuring cup. A first measuring cup is used to cover the second measuring cup, creating a simple germination chamber for seed germination. Vermiculite is first measured and spread evenly at the bottom of the box using the first measuring cup. Then, water is measured using the second measuring cup and poured over the vermiculite layer, allowing excess water to drain out through the drainage hole to prevent waterlogging. The germinated seeds are then spread evenly on the moist vermiculite surface, and dry vermiculite is measured and covered over the seeds using the second measuring cup, forming a sandwich-like substrate structure. This two-stage culture—indoor induction followed by outdoor natural stress induction—creates conditions highly favorable for pathogen infection and disease development, raising the incidence rate to a significantly high level required for scientific research. This invention has a simple structure, facilitates standardized operation, significantly improves the incidence rate of rice bakanae disease, and facilitates disease identification.

[0008] Furthermore, the bottom of the box is provided with support legs.

[0009] Furthermore, the lid has air holes at both ends.

[0010] Furthermore, the box is rectangular in shape, and its internal dimensions are 20cm in length, 10cm in width, and 18cm in height.

[0011] Furthermore, the first measuring cup is a 400ml measuring cup, and the second measuring cup is a 200ml measuring cup.

[0012] Furthermore, the scale includes a negative scale area located at the lower part of the box body and a positive scale area located at the upper part of the box body, wherein the negative scale area is used to guide the laying thickness of the subbase.

[0013] The present invention also provides a method for inducing rice bakanae disease, applied to the above-mentioned rice bakanae disease inducing device, comprising the following steps: S1: Select susceptible rice seeds carrying Bakanae disease, soak them in water, then remove and drain them; place the drained rice seeds in the second measuring cup, cover the second measuring cup with the first measuring cup to form a sealed germination chamber, and germinate them in the dark. S2: Place the box on a level surface, use the first measuring cup to measure the vermiculite, and spread it evenly on the bottom of the box; S3: Use the second measuring cup to measure clean water and pour it evenly into the vermiculite layer. Excess water will flow out from the drain hole. S4: Spread the germinated seeds evenly on the moist vermiculite surface; S5: Use the second measuring cup to measure the dried vermiculite and evenly cover the seed surface; S6: Cover the top of the box with the lid and place the box in a well-lit incubation room or artificial climate chamber for incubation. S7: When the rice seedlings grow to the point of touching the inner wall of the box, move the box outdoors, remove the box, and expose the rice seedlings to a sunny and rain-free outdoor environment for continued cultivation. S8: When the healthy plants that are not infected with the disease rise above the edge of the box, check and count the number of diseased plants.

[0014] The rice bakanae disease induction method of this invention utilizes a sandwich matrix structure of "bottom moisturizing layer + seed layer + top covering layer" combined with a two-stage culture mode of indoor constant temperature induction and outdoor natural stress induction. By utilizing the natural stress pressures such as outdoor sunlight and temperature fluctuations, it promotes the full development and phenotypic differentiation of the disease, providing a stable and suitable microenvironment for pathogen infection, significantly increasing the incidence rate and meeting the needs of high-throughput scientific research. In the later natural light and temperature induction stage, healthy plants and diseased plants show a clear contrast in terms of plant height, growth vigor, and leaf color, which facilitates rapid visual identification and statistical analysis, reducing the misjudgment rate.

[0015] Preferably, in step S1, 15g of rice seeds that are susceptible to and carry Bakanae disease are selected, and the soaking time is 24h and the germination time is 24h.

[0016] Preferably, in step S6, the culture time is 7 days.

[0017] Preferably, in step S7, the culture time is 7-14 days.

[0018] Compared with existing technologies, the beneficial effects of this invention are as follows: It has a simple structure and facilitates standardized operation; through a sandwich-type substrate structure of "bottom moisturizing layer + seed layer + top covering layer," combined with a two-stage culture mode of indoor constant temperature induction and outdoor natural stress induction, it utilizes natural stress pressures such as outdoor sunlight and temperature fluctuations to promote the full development and phenotypic differentiation of diseases, providing a stable and suitable microenvironment for pathogen infection, significantly increasing the incidence rate and meeting the needs of high-throughput scientific research; in the later natural light and temperature induction stage, healthy plants and diseased plants show a clear contrast in plant height, growth vigor, and leaf color, facilitating disease identification and reducing the misjudgment rate. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the rice bakanae disease induction device in an embodiment of the present invention; Figure 2 This is a flowchart of the rice bakanae disease induction method in an embodiment of the present invention; Figure 3 This is a comparison diagram of diseased seedlings infected with bakanae disease induced in Embodiment 3 of the present invention and healthy seedlings; Figure 4This is a comparison diagram of the "Zhongzu 53" bakanae disease diseased plants induced by different methods in Embodiment 3 of the present invention; Figure 5 This is a comparison diagram of the "Zhong'an No. 2" seedlings infected with bakanae disease induced by different methods in Embodiment 3 of the present invention; Figure 6 This is a comparison chart of the incidence rates of bakanae disease induced by different methods in Embodiment 3 of the present invention; Figure 7 This is a diagram illustrating the control effect of the fungicide coating agent in Embodiment 4 of the present invention on the "Zhongzu 53" bakanae disease. Figure 8 This is a diagram illustrating the control effect of the fungicide coating agent in Embodiment 4 of the present invention on the bakanae disease of "Zhong'an No. 2" seedlings; Figure 9 This is a comparison chart of the incidence rate of bakanae disease under different drug treatments in Example 4 of the present invention.

[0020] In the attached diagram: 1-box body; 101-drain hole; 102-scale; 103-support leg; 2-box lid; 201-vent; 3-first measuring cup; 4-second measuring cup. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments. The accompanying drawings are for illustrative purposes only, representing schematic diagrams rather than actual physical objects, and should not be construed as limiting the scope of this patent. To better illustrate the embodiments of the present invention, some components in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0022] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0023] Example 1 A device for inducing rice bakanae disease, such as Figure 1As shown, the device includes a box body 1, a lid 2, a first measuring cup 3, and a second measuring cup 4. The lid 2 is detachably connected to the top of the box body 1. The box body 1 is a transparent structure. The lid 2 has an air hole 201. The bottom of the box body 1 has a drainage hole 101. The side wall of the box body 1 has a scale 102. The capacity of the first measuring cup 3 is greater than the capacity of the second measuring cup 4. The first measuring cup 3 can be inverted to cover the second measuring cup 4 to form a sealed germination chamber.

[0024] The aforementioned rice bakanae disease induction device features a transparent box 1 for easy observation of seedling growth and disease development. The scale 102 on the side wall of box 1 allows for direct observation of plant growth. In use, rice seeds are soaked, drained, and placed in the second measuring cup 4. The first measuring cup 3 covers the second measuring cup 4, creating a simple germination chamber for seed germination. Vermiculite is first measured and spread evenly at the bottom of the box using the first measuring cup 3. Then, water is measured using the second measuring cup 4 to irrigate the vermiculite layer, with excess water draining through the drainage hole 101 to prevent waterlogging. The germinated seeds are then spread evenly on the moist vermiculite surface, and dry vermiculite is measured and covered with the seeds using the second measuring cup 4, forming a sandwich-like substrate structure. This two-stage culture process—indoor induction followed by outdoor natural stress induction—creates conditions highly conducive to pathogen infection and disease development, raising the incidence rate to a significantly high level required for scientific research. The device in this embodiment has a simple structure, facilitates standardized operation, can significantly reduce the incidence of rice bakanae disease, and is easy to identify.

[0025] The bottom of the box 1 is provided with support feet 103. Specifically, the bottom of the box 1 is provided with 9 drainage holes 101 evenly distributed, with a hole diameter of 2mm. The four corners of the bottom of the box 1 are provided with support feet 103 with a height of 1cm, which can raise the box 1 and isolate the box 1 from the table surface to ensure smooth drainage.

[0026] Both ends of the lid 2 are provided with air holes 201. Specifically, two air holes 201 with a diameter of 2mm are provided on each side of the lid 2 for gas exchange and subsequent humidity regulation. The top of the lid 2 may be provided with a handle for easy closing or removal of the lid 2.

[0027] Box 1 is rectangular in shape, with internal dimensions of 20cm long, 10cm wide, and 18cm high. Box 1 is made of transparent PP material with a thickness of 1mm. The scale 102 includes a negative scale area at the bottom of box 1 and a positive scale area at the top. The negative scale area guides the thickness of the substrate layer. Specifically, the scale 102 on the outside of box 1 ranges from -3cm to 15cm from bottom to top, with each scale 102 spaced 1cm apart. Bakanae disease-infected plants exhibit typical symptoms such as etiolation, weakness, and yellowing. They are typically taller than healthy plants, usually exceeding 18cm in height. When the cultured plant touches the inner wall of the box lid 2, it indicates the presence of early-stage disease, prompting the operator to transfer box 1 from indoor induction culture to outdoor natural stress induction culture. Similarly, it facilitates direct inspection and counting of diseased plants, allowing for the calculation of the disease incidence rate.

[0028] The first measuring cup 3 is a 400ml quantitative measuring cup, and the second measuring cup 4 is a 200ml quantitative measuring cup. During inoculation and culture, use the 400ml first measuring cup 3 to measure vermiculite and spread it evenly on the bottom of the container, reaching a height of -1cm from mark 102 on the container body. Then, use the 200ml second measuring cup 4 to measure water and pour it over the vermiculite layer. Spread the germinated seeds evenly on the moist vermiculite layer, and then use the 200ml second measuring cup 4 to measure dry vermiculite and evenly cover the seed surface. Precisely quantifying the amount of vermiculite and water ensures consistent experimental conditions. It should be noted that the first measuring cup 3 can also be set with a 200ml mark. If the second measuring cup 4 is damaged, lost, or contains germinated seeds, water can also be measured using the first measuring cup 3 to pour water over the vermiculite layer.

[0029] The device in this embodiment has a simple structure and can be made of inexpensive plastic. It integrates cultivation, drainage, and quantitative support functions, and is equipped with a quantitative container to realize the quantification and standardization of key steps such as substrate laying and watering. This greatly reduces human error, reduces operational variables, and improves experimental repeatability. It is suitable for promotion and use in various breeding units, research institutes, and high-efficiency laboratories.

[0030] Example 2 A method for inducing rice bakanae disease, applied to the rice bakanae disease inducing device of Example 1, wherein the internal dimensions of the transparent box 1 are 20cm long, 10cm wide, and 18cm high; the first measuring cup 3 is a 400ml quantitative measuring cup; and the second measuring cup 4 is a 200ml quantitative measuring cup. Figure 2 As shown, the method includes the following steps: Select 15g of susceptible rice seeds carrying bakanae disease and incubate them at 28-30°C. After soaking the rice seeds in water for 24 hours, remove them and drain the water. Place the drained rice seeds in the second measuring cup 4, and cover the second measuring cup 4 with the first measuring cup 3 upside down to form a sealed germination chamber. Germinate the seeds at 28-30°C. Germination in darkness for 24 hours; Place box 1 on a horizontal platform, use the first measuring cup 3 to measure vermiculite, and spread it evenly on the bottom of the box up to the mark 102, which is -1cm. Use the second measuring cup 4 to measure out clean water and pour it evenly into the vermiculite layer. Excess water will flow out from the drain hole 101. Spread the germinated seeds evenly on the surface of the moist vermiculite; Use the second measuring cup 4 to measure the dried vermiculite and evenly cover the seed surface with a thickness of about 0.5cm; Cover box 1 with lid 2 and place box 1 in a place with light and a temperature of 30-32 degrees Celsius. Cultured in a culture room or artificial climate chamber for 7 days; When the rice seedlings grow to the point of contacting the inner wall of the lid 2, transfer the container 1 to a temperature of 20-38 degrees Celsius. Outdoors, remove the lid 2 and expose the rice seedlings to a sunny and rain-free outdoor environment for 7-14 days. Once the healthy, uninfected plants have grown above the edge of the box, inspect and count the number of diseased plants.

[0031] The aforementioned method for inducing rice bakanae disease utilizes a sandwich-like substrate structure consisting of a bottom moisturizing layer, a seed layer, and a top covering layer. This, combined with a two-stage cultivation model—indoor constant-temperature induction and outdoor natural stress induction—takes advantage of natural stress factors such as sunlight and temperature fluctuations to promote the full development and phenotypic differentiation of the disease. This provides a stable and suitable microenvironment for pathogen infection, significantly increasing the incidence rate and meeting the needs of high-throughput research. The use of a dedicated quantitative measuring cup enables the quantification and standardization of key steps such as substrate preparation and watering, greatly reducing human error and improving experimental reproducibility. In the later natural light and temperature induction stage, healthy plants and diseased plants show a clear contrast in plant height, growth vigor, and leaf color, facilitating rapid visual identification and statistical analysis, thus reducing the misjudgment rate.

[0032] Example 3 Using the apparatus of Example 1 and the method of Example 2, seedling blight was induced in known susceptible rice varieties “Zhongzu 53” and “Zhong’an No. 2”.

[0033] When the rice seedlings grew to the point of touching the inner wall of the lid 2, a small number of plants appeared pale yellow and were excessively elongated. The seedlings were then moved outdoors, the lid 2 was removed, and the plants were kept in a cool, dry place under natural light and temperature (25-35°C). Cultured for 10-14 days under sunny conditions.

[0034] The diseased plants induced by this embodiment of seedling blight were compared with healthy plants, such as... Figure 3As shown, from left to right, the plants are: Group 53 healthy plants, Zhong'an No. 2 healthy plants, Group 53 diseased plants, and Zhong'an No. 2 diseased plants. The healthy plants averaged 22cm in height with dark green leaves; the diseased plants were leggy, reaching 25-28cm in height, but were extremely thin, weak, and yellow. The disease incidence rate was 97%.

[0035] During the same period, different methods were used to conduct disease-inducing tests on "Zhongzu 53" and "Zhong'an No. 2", such as... Figure 4 As shown, from left to right, these are plants of "Zhongzu 53" induced to develop the disease using methods one, two, three, and four, respectively; Figure 5 As shown, from left to right, the plants of "Zhong'an No. 2" induced with disease using methods one, two, three, and four are shown. Method three represents the plants induced using the method described in this embodiment. The disease incidence rate of the plants grown using different induction methods was statistically analyzed. Figure 6 As shown, the induction method of this embodiment can significantly increase the incidence of bakanae disease.

[0036] Example 4 The induction method described in Example 3 was used to evaluate the control effect of a certain fungicide coating agent on bakanae disease.

[0037] Experimental group: The seeds were coated with the fungicide at the recommended concentration and dosage, and then the induction method of Example 3 was used.

[0038] Control group: The seeds were coated with water and then induced using the method described in Example 3.

[0039] like Figure 7 As shown, from left to right, these are disease-inducing plants of "Zhongzu 53" coated with water and coated with different concentrations of fungicide; Figure 8 As shown, from left to right, these are disease-inducing plants of "Zhong'an No. 2" coated with water and coated with different concentrations of fungicide; Figure 9 As shown, the incidence rate in the control group was over 97%, while the incidence rate in the experimental group was less than 10%, demonstrating significant preventive efficacy. This indicates that the present invention can be effectively used for the precise evaluation of drug efficacy.

[0040] In the specific implementation of the above embodiments, the technical features can be combined in any non-contradictory way. For the sake of brevity, not all possible combinations of the above technical features are described. However, as long as the combination of these technical features is not contradictory, it should be considered to be within the scope of this specification.

[0041] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A device for inducing rice bakanae disease, characterized in that, The box includes a box body (1), a box lid (2), a first measuring cup (3) and a second measuring cup (4). The box lid (2) is detachably connected to the top of the box body (1). The box body (1) is a transparent structure. The box lid (2) has an air hole (201). The bottom of the box body (1) has a drainage hole (101). The side wall of the box body (1) has a scale (102). The capacity of the first measuring cup (3) is greater than the capacity of the second measuring cup (4). The first measuring cup (3) can be inverted to cover the second measuring cup (4) to form a sealed germination chamber.

2. The rice bakanae disease inducing device according to claim 1, characterized in that, The bottom of the box (1) is provided with support feet (103).

3. The rice bakanae disease inducing device according to claim 1, characterized in that, The lid (2) has air holes (201) at both ends.

4. The rice bakanae disease inducing device according to claim 1, characterized in that, The box (1) is rectangular in shape, and the internal dimensions of the box (1) are 20cm long, 10cm wide and 18cm high.

5. The rice bakanae disease inducing device according to claim 4, characterized in that, The first measuring cup (3) is a 400ml measuring cup, and the second measuring cup (4) is a 200ml measuring cup.

6. The rice bakanae disease inducing device according to claim 1, characterized in that, The scale (102) includes a negative scale area located at the lower part of the box body (1) and a positive scale area located at the upper part of the box body (1). The negative scale area is used to guide the laying thickness of the subbase substrate.

7. A method for inducing rice bakanae disease, applied to the rice bakanae disease inducing device according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Select susceptible rice seeds carrying Bakanae disease, soak them in water and then drain them; place the drained rice seeds in the second measuring cup (4), and cover the second measuring cup (4) with the first measuring cup (3) to form a closed germination chamber, and germinate them in the dark. S2: Place the box (1) on a horizontal platform, use the first measuring cup (3) to measure the vermiculite, and spread it evenly on the bottom of the box; S3: Use the second measuring cup (4) to measure clean water and pour it evenly into the vermiculite layer. Excess water will flow out from the drain hole (101). S4: Spread the germinated seeds evenly on the moist vermiculite surface; S5: Use the second measuring cup (4) to measure the dried vermiculite and evenly cover the seed surface; S6: Cover the top of the box (1) with the lid (2) and place the box (1) in a lighted incubation room or artificial climate chamber for incubation; S7: When the rice seedlings grow to the point of contacting the inner wall of the box cover (2), transfer the box (1) to the outdoors, remove the box cover (2), and expose the rice seedlings to the outdoor environment with sunlight and no rain to continue cultivation. S8: When the healthy plants that are not infected with the disease are higher than the edge of the box (1), check and count the number of diseased plants.

8. The method for inducing rice bakanae disease according to claim 7, characterized in that, In step S1, 15g of rice seeds that are susceptible to and carry Bakanae disease are selected, soaked for 24 hours, and germinated for 24 hours.

9. The method for inducing rice bakanae disease according to claim 7, characterized in that, In step S6, the culture time is 7 days.

10. The method for inducing rice bakanae disease according to claim 7, characterized in that, In step S7, the culture time is 7-14 days.

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