Method for rapidly and stably identifying rice blast resistance indoors and application

Abstract

The invention relates to a method for rapidly and stably identifying rice blast resistance indoors and application, and belongs to the technical field of plant disease prevention and control. The invention provides a plum culture medium which takes water as a solvent and comprises 30-60 mL / L of prune juice, 3-10 g / L of alpha-lactose, 0.5-2 g / L of yeast extract and 10-20 g / L of agar powder. The pH value of the plum culture medium is 6.5 to 7.5. According to the inoculation method disclosed by the invention, the inoculation success rate and the scab appearing degree of the pyricularia oryzae are remarkably improved by optimizing the key steps of seed soaking and germination acceleration, strain activation and propagation, conidium suspension preparation, spray inoculation and the like, and the inoculation method has the advantages of low cost, simplicity and convenience in operation, high standardization degree, good inoculation effect, small batch difference and high repeatability.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to a method and application for rapid and stable indoor identification of rice blast resistance. Background Technology

[0002] Rice blast is a serious fungal disease of rice caused by Magnaphalthe oryzae, resulting in significant yield and economic losses. Among rice cultivation management and chemical control measures, breeding new resistant rice varieties is considered a safe, economical, and effective way to control the occurrence and spread of rice blast.

[0003] Plant pathogen inoculation is a crucial step in crop disease experiments and the breeding of disease-resistant crop varieties. Currently, indoor inoculation methods for rice blast fungus suffer from problems such as long strain propagation cycles, unstable activation efficiency, cumbersome conidium suspension preparation, low conidium collection efficiency, and insufficient uniformity in spray inoculation. These issues lead to significant variations in inoculation results and make it difficult to simulate real field disease conditions. Therefore, there is an urgent need to provide a standardized, efficient, and reproducible indoor inoculation method for rice blast fungus. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for rapid and stable indoor identification of rice blast resistance, in order to solve the problems of complex operation, low efficiency, insufficient inoculation uniformity, and large differences in inoculation effect in the existing indoor inoculation methods for rice blast pathogen.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The present invention provides a plum culture medium, wherein the plum culture medium uses water as a solvent and comprises the following components at the following concentrations: 30-60 mL / L plum juice, 3-10 g / L α-lactose, 0.5-2 g / L yeast extract, and 10-20 g / L agar powder;

[0007] The pH of the plum culture medium is 6.5–7.5.

[0008] The present invention also provides the application of the plum culture medium in the cultivation of rice blast fungus.

[0009] The present invention also provides a method for indoor inoculation of rice with rice blast fungus, comprising the following steps:

[0010] (1) After culturing rice blast fungus on plum medium for 15-20 days, add Tween-20 solution and filter to obtain conidial suspension;

[0011] (2) Cover the rice plants with a bottomless cola bottle, with the bottle opening facing upwards, and inoculate the rice plants with conidial suspension from the mouth of the cola bottle until the rice leaves are covered with mist but do not drip.

[0012] Preferably, the culture temperature in step (1) is 25–30°C;

[0013] In the conidial suspension described in step (1), the concentration of conidia is 0.5–2 × 10⁻⁶. 6 per mL.

[0014] Preferably, the mass concentration of the Tween-20 solution in step (1) is 0.05% to 0.2%.

[0015] Preferably, the rice plants inoculated in step (2) are at the three-leaf-one-heart stage.

[0016] Preferably, the inoculation method in step (2) is spray inoculation, and the inoculation pressure is 0.5 to 1 MPa.

[0017] The present invention also provides the application of the method in the selection and / or identification of rice blast-resistant plants.

[0018] The present invention has the following technical effects and advantages:

[0019] This invention utilizes a cola bottle combined with an air compressor to form a closed, small spray chamber, thereby significantly reducing inoculation costs; by activating and culturing rice blast fungus strains using plum culture medium, the viability and quantity of conidia can be improved, thus ensuring stable inoculation results; by adopting standardized temperature-controlled germination and precise water and fertilizer control measures, the disease resistance uniformity of rice plants can be improved, greatly reducing the interference of environmental conditions on inoculation results.

[0020] The inoculation method of this invention significantly improves the inoculation success rate and lesion manifestation of rice blast fungus by optimizing key steps such as seed soaking and germination, strain activation and propagation, conidium suspension preparation, and spray inoculation. It has the advantages of low cost, simple operation, high standardization, good inoculation effect, small batch-to-batch variation, and high repeatability. It can be used for rice variety disease resistance identification, pathogen pathogenicity analysis, rice blast control research, and large-scale screening of high-quality rice germplasm materials resistant to disease. Attached Figure Description

[0021] Figure 1 A field photo showing the method of inoculating rice with rice blast fungus indoors. Detailed Implementation

[0022] This invention provides a plum culture medium, which uses water as a solvent and comprises the following components at the following concentrations: 30-60 mL / L of plum juice, preferably 40 mL / L; 3-10 g / L of α-lactose, preferably 5 g / L; 0.5-2 g / L of yeast extract, preferably 1 g / L; and 10-20 g / L of agar powder, preferably 15 g / L.

[0023] The pH of the plum culture medium is 6.5 to 7.5, preferably 7.0.

[0024] The present invention also provides the application of the plum culture medium in the cultivation of rice blast fungus.

[0025] The present invention also provides a method for indoor inoculation of rice with rice blast fungus, comprising the following steps:

[0026] (1) After culturing the rice blast fungus on plum medium for 15-20 days, preferably 17 days, add Tween-20 solution and filter to obtain a conidial suspension.

[0027] (2) Cover the rice plants with a bottomless cola bottle, with the bottle opening facing upwards, and inoculate the rice plants with conidial suspension from the mouth of the cola bottle until the rice leaves are covered with mist but do not drip.

[0028] In this invention, the culture temperature in step (1) is 25-30°C, preferably 28°C;

[0029] In the conidial suspension described in step (1), the concentration of conidia is 0.5–2 × 10⁻⁶. 6 Cells / mL, preferably 1×10⁻⁶ 6 per mL.

[0030] In this invention, the mass concentration of the Tween-20 solution in step (1) is 0.05% to 0.2%, preferably 0.1%.

[0031] In this invention, the method for cultivating rice plants in step (2) includes the following steps:

[0032] (a) Soak rice seeds completely in water for 18-36 hours, preferably 24 hours, then adjust to a semi-submerged state for germination, changing the water every 8-14 hours, preferably 12 hours; germinate for 1.5-2.5 days, preferably 2 days, to obtain seeds with white sprouts;

[0033] (b) Sow the white seeds into the mixed soil and cultivate them to obtain rice seedlings. When the rice seedlings are in the one-leaf-one-heart stage, add water to a depth of 1-3 cm, preferably 2 cm. Apply nitrogen fertilizer to the rice seedlings 2-5 days before inoculation, preferably 3 days before inoculation to obtain rice plants.

[0034] In this invention, the temperature of the water in step (a) is 30-40°C, preferably 34°C;

[0035] The semi-submerged state refers to the state in which rice seeds are half submerged in water and half exposed to air.

[0036] In this invention, the mixed soil in step (b) includes peat and crushed soil, wherein the mass ratio of peat to crushed soil is 1:1 to 3, preferably 1:2;

[0037] The soil moisture content of the mixed soil is 90% to 100%, preferably 95%;

[0038] The concentration of the nitrogen fertilizer applied is 0.5% to 1%, preferably 0.8%.

[0039] In this invention, the rice plants inoculated in step (2) are at the three-leaf-one-heart stage.

[0040] In this invention, the inoculation method in step (2) is spray inoculation, and the inoculation pressure is 0.5 to 1 MPa, preferably 0.7 MPa.

[0041] In this invention, step (2) is followed by cultivating rice plants inoculated with conidial suspension;

[0042] The cultivation steps are as follows: rice plants inoculated with conidial suspension are dark-treated for 12-36 hours, preferably 24 hours, and then cultivated at 20-28℃ for 5-10 days, preferably 7 days at 25℃.

[0043] In this invention, the relative humidity for cultivation is 80% to 95%, preferably 90%; the light duration for cultivation is 8 to 14 hours per day, preferably 12 hours per day.

[0044] The present invention also provides the application of the method in the selection and / or identification of rice blast-resistant plants.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] In the test materials of this invention, the rice blast fungus strain GUY11 was obtained from Hunan Agricultural University, the Xiangzaoxian 45 rice variety was obtained from Hunan Jinse Nongfeng Seed Industry Co., Ltd., and the CO39 rice variety was obtained from Hunan Agricultural University.

[0047] In the equipment of this invention, the 120L-4X550W air compressor was purchased from Taizhou Aotus Industry and Trade Co., Ltd.

[0048] Example 1: Preparation of plum culture medium

[0049] Take 40 mL of plum juice, 5 g of α-lactose, 1 g of yeast extract, and 15 g of agar powder and dilute to 1 L of water. Adjust the pH to 7.0 and sterilize at 121 °C for 2 h to obtain plum culture medium.

[0050] Example 2: Preparation of plum culture medium

[0051] Take 30 mL of prune juice, 10 g of α-lactose, 0.5 g of yeast extract, and 10 g of agar powder and dilute to 1 L of water. Adjust the pH to 6.5 and sterilize at 121 °C for 2 h to obtain prune culture medium.

[0052] Example 3: Preparation of plum culture medium

[0053] Take 60 mL of plum juice, 3 g of α-lactose, 2 g of yeast extract, and 20 g of agar powder and dilute to 1 L of water. Adjust the pH to 7.5 and sterilize at 121℃ for 2 h to obtain plum culture medium.

[0054] Example 4: Method for indoor inoculation of rice with rice blast fungus

[0055] (1) Rice germination: Rice seeds were placed at -4℃ to break dormancy, soaked in 75% alcohol for 2 minutes, and then disinfected by shaking with 5% sodium hypochlorite for 25 minutes. After that, they were soaked in 34℃ warm water for 24 hours. Then, they were adjusted to a semi-submerged state with half of them submerged in water and half exposed to air for germination. The warm water was changed every 12 hours during the process. After 2 days of germination, 95% of the seeds showed white sprouting.

[0056] (2) Rice plant cultivation: Mix peat and crushed soil evenly at a mass ratio of 1:2 and water to obtain a mixed soil with organic matter and humic acid content >55%, pH 6.5 and soil moisture content of 95%; sow the sprouted seeds into the mixed soil and cultivate rice seedlings; when the rice seedlings are in the one-leaf-one-heart stage, add water to a depth of 2cm, and apply nitrogen fertilizer to the rice seedlings to a concentration of 0.8% 3 days before inoculation, and continue to cultivate to obtain rice plants in the three-leaf-one-heart stage;

[0057] (3) Preparation of conidium suspension: The rice blast fungus strain GUY11 was inoculated into the plum culture medium prepared in Example 1 and cultured upside down at 28℃ for 7 days to obtain activated mycelia. The activated mycelia were then inoculated into a new plum culture medium and cultured upside down for 10 days. Conidia were induced under black light for 5 days, and then 0.1% Tween-20 solution was added. The mixture was filtered to obtain a conidium suspension with a conidium concentration of 1×10⁻⁶. 6 cells / mL;

[0058] (4) Inoculation: Cover the rice plants at the three-leaf-one-heart stage with a bottomless cola bottle, mouth facing upwards. Use a 120L-4X550W air compressor at 0.7MPa to spray the inoculation conidium suspension onto the rice plants through the cola bottle opening. Inoculate until the rice leaves are covered with a mist without dripping. After inoculation, tighten the bottle cap to prevent the spread of conidia. Figure 1 As shown;

[0059] (5) Rice cultivation: After rice plants inoculated with conidial suspension were kept moist and dark for 24 hours, they were cultivated for 7 days at 25℃ and 90% relative humidity, with a light duration of 12 hours / day.

[0060] Comparative Example 1: Spray inoculation method for rice blast fungus in inoculation pool

[0061] Rice germination, rice plant cultivation, conidium suspension preparation, and rice cultivation were carried out according to the method described in Example 4. The difference was that, in this comparative example, the inoculation method was as follows: a semi-closed inoculation pool was constructed, rice plants at the three-leaf-one-heart stage were transferred to the semi-closed inoculation pool, and sterile sprayers were used to directly spray the rice plants for inoculation.

[0062] Experiment Example 1: Inoculation of rice blast fungus

[0063] Using the methods described in Example 4 and Comparative Example 1, rice plants of the Xiangzaoxian 45 variety and the susceptible CO39 variety were inoculated with the rice blast fungus strain GUY11. After inoculation with the conidial suspension of the rice blast fungus, each rice plant was cultivated for 7 days. The disease incidence and the average number of lesions on the leaves of each rice plant were measured. The results are shown in Table 1.

[0064] Table 1. Inoculation results of rice blast fungus.

[0065]

[0066] The results showed that, compared with the spray inoculation method in the inoculation pool described in Comparative Example 1, the inoculation method of the present invention can significantly improve the inoculation success rate and the degree of lesion manifestation of rice blast fungus, and has good repeatability.

[0067] As can be seen from the above embodiments, the present invention provides a method and application for rapid and stable indoor identification of rice blast resistance. The inoculation method of the present invention can significantly improve the inoculation success rate and lesion manifestation of rice blast fungus, and has the characteristics of low cost, simple operation, good inoculation effect, and high repeatability.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A plum culture medium, characterized in that, The plum culture medium uses water as a solvent and includes the following components at the following concentrations: 30-60 mL / L plum juice, 3-10 g / L α-lactose, 0.5-2 g / L yeast extract, and 10-20 g / L agar powder; The pH of the plum culture medium is 6.5–7.

5.

2. The application of the plum culture medium according to claim 1 in the cultivation of rice blast fungus.

3. A method for indoor inoculating rice with rice blast fungus, characterized in that, Includes the following steps: (1) After culturing rice blast fungus on plum medium for 15-20 days, add Tween-20 solution and filter to obtain conidial suspension; (2) Cover the rice plants with a bottomless cola bottle, with the bottle opening facing upwards, and inoculate the rice plants with conidial suspension from the bottle opening until the rice leaves are covered with mist but do not drip.

4. The method according to claim 3, characterized in that, The culture temperature in step (1) is 25–30°C; In the conidial suspension described in step (1), the concentration of conidia is 0.5–2 × 10⁻⁶. 6 per mL.

5. The method according to claim 4, characterized in that, The mass concentration of the Tween-20 solution in step (1) is 0.05% to 0.2%.

6. The method according to claim 5, characterized in that, The rice plants in step (2) are inoculated at the three-leaf-one-heart stage.

7. The method according to claim 6, characterized in that, The inoculation method in step (2) is spray inoculation, and the inoculation pressure is 0.5 to 1 MPa.

8. The application of the method according to any one of claims 3 to 7 in the selection and / or identification of rice blast-resistant plants.

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