Method for identifying resistance of japonica rice sheath blight disease

Abstract

The invention provides a japonica rice sheath blight resistance identification method, and belongs to the technical field of plant disease resistance identification. The invention provides a method for identifying the sheath blight resistance of japonica rice. The method comprises the following steps: planting and culturing japonica rice seeds to be detected in an intelligent greenhouse, performing sheath blight induction from a tillering end stage to a young ear differentiation stage, observing the attack condition of the sheath blight, measuring the average scab length of the japonica rice to be detected, calculating the disease index, and evaluating the sheath blight disease grade of the japonica rice. According to the method, the japonica rice rapidly enters the tillering last stage, the plant requirement of sheath blight identification is met, and the identification period is remarkably shortened; the method can complete simultaneous identification of multiple varieties at the same time, is not limited by other conditions in the field, and reduces the influence of environmental factors, thereby improving the accuracy and repeatability of sheath blight resistance screening. The banded sclerotial blight identification can be carried out for 3-4 times in one year, the dependence on professional equipment and large-area test fields is reduced, the method is more economical and universal, the identification efficiency is improved, and the identification cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease resistance identification, and particularly relates to a method for identifying resistance to sheath blight of japonica rice. Background Art

[0002] Rice is one of the most important food crops in my country. Its production is affected by many diseases, among which sheath blight is a fungal disease that can generally reduce rice production by 10% to 30% and significantly reduce rice quality. Identifying rice germplasm resources resistant to sheath blight and using them in breeding plays a very important role in ensuring my country's food security. Accurate, rapid, and high-throughput evaluation of rice sheath blight resistance is a key link in carrying out rice breeding for sheath blight resistance.

[0003] When identifying the resistance of japonica rice to sheath blight, the traditional strategy is mainly to conduct natural field identification and artificial inoculation in areas with a high incidence of the disease. The natural field identification method is uncontrollable because the spontaneity of the disease is limited by the geographical environment and climatic conditions, and the pathogens are highly variable and the dominant physiological races are frequently replaced. At the same time, areas with a high incidence of the disease are generally far away from japonica rice production and breeding bases, which makes disease resistance identification often time-consuming and laborious, and therefore disease-resistant breeding is difficult to carry out effectively in actual operations. The artificial inoculation method can be carried out in the breeding base, the inoculation site is not restricted by geographical conditions, and the inoculated pathogens are stable and consistent, but these methods are time-consuming and labor-intensive. At the same time, they are affected by field climate and environmental factors and field planting seasons, making it difficult to carry out large-scale disease resistance identification. Summary of the invention

[0004] The present invention provides a method for identifying resistance to sheath blight of japonica rice, which is high efficiency, low cost, rapid and accurate, and has important practical application value for accelerating disease-resistant breeding of japonica rice.

[0005] The invention provides a method for identifying resistance to sheath blight of japonica rice, comprising the following steps: planting and culturing japonica rice seeds to be tested in an intelligent greenhouse, inducing sheath blight from the end of tillering to panicle differentiation, observing the incidence of sheath blight, measuring the average lesion length of the japonica rice to be tested, calculating the disease index, and evaluating the sheath blight disease grade of the japonica rice;

[0006] The temperature in the intelligent greenhouse is set at 25-30° C., the humidity is 80%-90%, the light intensity is 12000-20000 Lux, and the light cycle does not exceed 10 hours of illumination.

[0007] In a preferred embodiment of the present invention, the planting comprises sowing the japonica rice seeds in a seedling hole tray filled with nutrient soil, sowing 10 holes for each variety, sowing 3 seeds in each hole, and repeating each variety 3 times.

[0008] In a preferred embodiment of the present invention, when the plants grow to the 3-leaf 1-heart stage, thinning is performed to retain one seedling in each hole.

[0009] In a preferred embodiment of the present invention, the sheath blight induction comprises using brown sclerotia produced by culturing Rhizoctonia solani as a sheath blight inoculation fungus source, and inducing at a relative humidity of more than 80%.

[0010] In a preferred embodiment of the present invention, the Rhizoctonia solani strain is isolated from japonica rice plants infected with sheath blight in the field, inoculated on a PDA culture medium, and the brown sclerotia produced by the culture are used as the sheath blight inoculation fungus source.

[0011] In a preferred embodiment of the present invention, the method comprises culturing on the PDA medium for 10 to 15 days.

[0012] In a preferred embodiment of the present invention, when the sheath blight inoculation source is used for inoculation, the japonica rice leaf sheath is slightly opened, and the inoculation source is introduced into the opened leaf sheath to restore the leaf sheath.

[0013] In a preferred embodiment of the present invention, each leaf sheath is inoculated with 1 to 3 sources of sheath blight inoculation bacteria.

[0014] In a preferred embodiment of the present invention, the The units of lesion length and leaf sheath length are the same.

[0015] In a preferred embodiment of the present invention, when the disease index is 0, the disease level of sheath blight is immune; when 0<the disease index≤20%, the disease level of sheath blight is resistant; when 20%<disease index≤40%, the disease level of sheath blight is moderately resistant; when 40%<disease index≤60%, the disease level of sheath blight is moderately susceptible; when 60%<disease index≤80%, the disease level of sheath blight is susceptible; when 80%<disease index≤100%, the disease level of sheath blight is highly susceptible.

[0016] Beneficial effects: The present invention provides a method for identifying resistance to sheath blight of japonica rice, wherein the japonica rice seeds to be tested are planted and cultivated in an intelligent greenhouse, sheath blight is induced from the end of tillering to the panicle differentiation stage, the incidence of sheath blight is observed, the average lesion length of the japonica rice to be tested is measured, the disease index is calculated, and the sheath blight disease level of the japonica rice is evaluated. The present invention utilizes the strong photosensitivity of japonica rice, and can enter the end of tillering after 45 days of sowing in the intelligent greenhouse, which meets the plant requirements for sheath blight identification and significantly shortens the identification cycle. The identification method of the present invention is carried out in an intelligent greenhouse, and can simultaneously complete the identification of multiple varieties, and the plant height and growth period of different varieties are consistent, which is not affected by other field conditions, and reduces the influence of environmental factors, thereby improving the accuracy and repeatability of sheath blight resistance screening. The identification method of the present invention does not need to be planted in the field and is not affected by the season. Sheath blight identification can be carried out 3 to 4 times a year, reducing the dependence on professional equipment and large-scale test fields, being more economical and generally applicable, improving identification efficiency, and reducing identification costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A large number of brown sclerotia obtained for the cultivation of sheath blight strains;

[0018] Figure 2 This is a picture of the plant factory processing site for seedling trays;

[0019] Figure 3 This shows the incidence of some japonica rice varieties after inoculation with sheath blight. DETAILED DESCRIPTION

[0020] The invention provides a method for identifying resistance to sheath blight of japonica rice, comprising the following steps: planting and culturing japonica rice seeds to be tested in an intelligent greenhouse, inducing sheath blight from the end of tillering to panicle differentiation, observing the incidence of sheath blight, measuring the average lesion length of the japonica rice to be tested, calculating the disease index, and evaluating the sheath blight disease grade of the japonica rice;

[0021] The temperature in the intelligent greenhouse is set at 25-30° C., the humidity is 80%-90%, the light intensity is 12000-20000 Lux, and the light cycle does not exceed 10 hours of illumination.

[0022] The identification described in the present invention is carried out in an intelligent greenhouse, in which japonica rice can be sown, sheath blight induced and identified. When sowing japonica rice described in the present invention, the japonica rice variety to be identified is sown in a seedling tray, sterilized nutrient soil is placed in the holes of the tray, 10 holes are sown for each variety, 3 seeds are planted in each hole, and the next variety is planted every other row. The sowing is repeated 3 times according to the above method. After germination, wait until the plants grow to the 3-leaf 1-heart stage, and thin out the seedlings, retaining 1 seedling in each hole. In one embodiment of the present invention, the nutrient soil is Miracle-Gro-E nutrient soil.

[0023] Planting japonica rice in the intelligent greenhouse of the present invention, and setting internal control temperature (25-30°C), humidity (80%-90%), light (12000-20000 Lux), and a photoperiod of 10 hours of light / 14 hours of darkness can accelerate the growth of japonica rice and promote the growth of sheath blight. After 20 days of treatment under the conditions, the identified materials quickly entered the late tillering stage and the panicle differentiation stage.

[0024] The present invention does not specifically limit the method for inducing sheath blight, including using a relative humidity of more than 80% for induction, or using a sheath blight inoculation source for the induction. In one embodiment of the present invention, the induction is performed using a sheath blight inoculation source, including isolating a Rhizoctonia solani strain from japonica rice plants infected with sheath blight in the field, inoculating it on a PDA medium and culturing it for 2 to 5 days to obtain japonica rice sheath blight fungi; a large number of brown sclerotia produced after 10 to 15 days of cultivation are used as a sheath blight inoculation source. When the present invention uses the inoculation source for inoculation, the japonica rice leaf sheath is slightly pried open, and the sclerotia are sent into the pried leaf sheath using a skipping needle or tweezers. No moisturizing is required, and the leaf sheath can be restored directly after inoculation. One leaf sheath can be inoculated with 1 to 3 sclerotia.

[0025] The present invention can carry out sheath blight resistance identification 7 days after the inoculation is completed, specifically including observing the incidence of sheath blight, manually measuring the average lesion length of each identified material, calculating the disease index, and classifying the sheath blight disease levels of the identified materials.

[0026] In a preferred embodiment of the present invention, the The units of lesion length and leaf sheath length are the same.

[0027] In a preferred embodiment of the present invention, when the disease index is 0, the disease level of sheath blight is immune; when 0<the disease index≤20%, the disease level of sheath blight is resistant; when 20%<disease index≤40%, the disease level of sheath blight is moderately resistant; when 40%<disease index≤60%, the disease level of sheath blight is moderately susceptible; when 60%<disease index≤80%, the disease level of sheath blight is susceptible; when 80%<disease index≤100%, the disease level of sheath blight is highly susceptible.

[0028] Table 1 Classification of resistance types to sheath blight in japonica rice

[0029] Resistance level Disease measurement ratio index Immunity (I) No symptoms Disease resistance (R) Disease index ≤ 20% Medium resistance (MR) 20%<disease index≤40% Moderate Sensitivity (MS) 40%<disease index≤60% Sickness(S) 60%<disease index≤80% High Sensitivity (HS) 80%<disease index≤100%

[0030] In order to further illustrate the present invention, a method for identifying resistance to sheath blight of japonica rice provided by the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0031] The PDA culture medium of the present invention, namely, potato dextrose agar culture medium (Potato Dextrose Agar), is a commonly used microbial culture medium, and is particularly suitable for the cultivation of fungi such as yeast and mold. When preparing, firstly, 200 grams of peeled potatoes are washed and boiled with water, boiled for 20-30 minutes, filtered with gauze, and the filtrate is supplemented with water to 1000 ml; the filtrate is put back into a pot, 20 grams of glucose and 15-20 grams of pre-crushed agar are added, heated with a low fire and stirred continuously until the agar is completely dissolved, and then water is supplemented to 1000 ml; according to needs, the culture medium is divided into test tubes or triangular flasks, the divided amount of the test tube is about 1 / 5 of the height of the test tube, and an inclined surface is formed after sterilization; the divided amount of the triangular flask does not exceed half of its volume; after the division is completed, sterilization is carried out, generally by high pressure sterilization at 115° C. for about 20 minutes.

[0032] The strain was isolated from the plants infected with rice sheath blight in the field, inoculated on PDA medium and cultured for 2 to 5 days to obtain rice sheath blight bacteria. A large number of brown sclerotia produced after 10 to 15 days of culture were used as the inoculation bacteria source for later use. Figure 1 ).

[0033] Example 1

[0034] On June 10, 2024, seven japonica rice materials were selected for sheath blight resistance identification: the sheath blight resistant variety YSBR1 (Zuo Shimin, Wang Zibin, Chen Xijun, et al. Resistance evaluation of the new resistance source YSBR1 for rice sheath blight. Acta Agronomica Sinica, 2009, 35(04): 608-614), the sheath blight susceptible control variety American japonica rice Lemont (Ji Xuemei. Quantitative trait gene mapping of two rice varieties for sheath blight resistance [D]. Yangzhou University, 2001.), Nanjing 5718 (Su Shen Rice 20210047), the sheath blight resistant, water-saving and drought-resistant rice Hu Han Xiang Ruan 61 (Hu Shen Rice 2024003), Nanjing 5055 (Su Shen Rice 201114), Jinjing 818 (Guo Shen Rice 20180061) and Yinxiang 38 (Hu Shen Rice 2017018).

[0035] The above 7 japonica rice seeds were placed in a water-soaked mesh bag and soaked for 24 hours. Then, they were placed in a germination box in the factory at 40°C for 48 hours until the seeds turned white. Selected seeds with long sprouts and full grains were sown in a sterilized matrix soil (Miracle-Gro-E type nutrient soil, the main components of which are peat soil, perlite, slow-release fertilizer, rice husk ash, coconut husk and river sand) in a 50-hole (hole size of 3.5 cm × 3.5 cm × 4.5 cm) seedling tray (size of 53 cm × 27 cm × 4.5 cm). Each variety was sown in one row, with 10 holes per row and 3 seeds per hole. The sowing specification was 3.5 cm × 3.5 cm; different varieties were sown in alternate rows, and two replicate groups were set for sowing. The sown seedling tray was placed in a stainless steel non-porous tray (size of 60 cm × 30 cm × 7 cm) with a water retention depth of 4 cm. When the seeds have 3 leaves and 1 heart (12 to 15 days after sowing), remove 2 weak seedlings from each hole and set the row spacing to 4 cm × 6 cm.

[0036] The seedlings in the seedling trays obtained from the above steps were placed in a plant factory for 20 days ( Figure 2 ). The plant factory control equipment includes: temperature and humidity control system, artificial lighting system, CO2 concentration regulator, automatic sprayer, etc. Set the temperature of the smart greenhouse to 30℃, relative humidity to 85%, light intensity to 20000Lux, photoperiod to 10 hours light / 14 hours dark, and CO2 concentration to 400ppm. Environmental control system parameters: Design temperature range: 15~40℃, control accuracy ±1℃. Design relative humidity range: 40~90%, control accuracy ±5%. Fresh air volume: ≥300m 3 / h (ventilation frequency ≥ 20 times / h). Air supply volume: ≥ 2800m 3 / h. Indoor wind speed: ≤0.5m / s. Humidification capacity: ≥4kg / h. Dehumidification capacity: ≥90L / d. Cabin noise: ≤55dB(A). Auxiliary electric heating: PTC ceramic heating, total power ≤12kW. Lighting system: equipped with LED lighting tri-proof lamps (Leishi Lighting L305 linear lamps), illuminance ≤20000Lux. To meet the lighting demand of 12000Lux, it can be achieved by adjusting the number and layout of LED lamps. Photoperiod control: Through the intelligent control system, the photoperiod can be set to 10 hours of light / 14 hours of darkness.

[0037] Select plants at the end of tillering stage for inoculation. Prepare sterilized needles or tweezers before inoculation to ensure that the surface of the tools is free of bacteria contamination to avoid affecting the inoculation effect and test accuracy. The disinfection method can be high-temperature sterilization (such as high-pressure steam sterilization at 121°C for 20 minutes) or wiping with 75% alcohol and then drying. Prepare labels, markers, etc. to mark the inoculated japonica rice plants and inoculation sites for subsequent observation and record.

[0038] Carefully select japonica rice plants that meet the requirements in the prepared sheath blight inoculation area. Observe the overall growth of the plants to ensure that they are representative and not significantly affected by other diseases and pests. For the selected japonica rice plants, gently push open their leaf sheaths and select the appropriate leaf sheath site for inoculation. Generally, the leaf sheaths in the middle and lower parts of the plants that are relatively strong, not yet fully aged, and without obvious wounds or lesions are preferred, and inoculation on the newly grown leaf sheaths at the top or severely damaged leaf sheaths is avoided to ensure the stability and observability of the inoculation effect. Use a sterilized skipping needle or tweezers to carefully pick up 1 to 3 sclerotia from the storage container. Handle with care during the operation to prevent the sclerotia from breaking or falling. Slowly put the clamped sclerotia into the opened leaf sheath, and try to place the sclerotia on the inner side of the leaf sheath near the base, where the humidity is relatively high and is conducive to sclerotia germination and hyphae growth. When placing the sclerotia, be careful to avoid excessive damage to the leaf sheath tissue to avoid affecting the normal physiological function of the japonica rice plant and the observation of subsequent disease conditions. After confirming that the sclerotia are securely placed, there is no need for additional moisturizing treatment. Just gently restore the leaf sheath to return it to its natural growth state and minimize human interference with the plant's growth environment.

[0039] After the inoculation is completed, the inoculated japonica rice plants should be marked immediately with labels and markers. The marking content should include detailed information such as the inoculation date, the number of inoculated sclerotia, the japonica rice variety, and the inoculation site, so as to accurately track and record the disease progression of each japonica rice plant.

[0040] After inoculation, the japonica rice plants were cultivated according to normal field management methods to maintain appropriate water, nutrient supply and light conditions, but large-scale agricultural operations (such as fertilization, spraying, etc.) should be avoided during the observation period to avoid affecting the accurate judgment of the incidence of sheath blight. Sheath blight identification work was carried out from the 7th day after inoculation.

[0041] Identification of sheath blight: Prepare a ruler with an accuracy of 0.1 cm, ensure that the measuring tool is calibrated and the measuring surface is clean and free of stains and damage, and can accurately measure the length of the lesion. Prepare a record form to record in detail the measurement data of each japonica rice variety, each japonica rice plant, and each lesion on the inoculated leaf sheath. The record form should include items such as the japonica rice variety name, plant number, leaf sheath number, and lesion length.

[0042] According to the marks made during the previous inoculation, accurately find the japonica rice plants 7 days after inoculation. Inspect and measure the japonica rice plants one by one in the pre-set order to ensure that no inoculated samples are missed. For each japonica rice plant, reconfirm the location of the inoculated leaf sheath. Carefully observe the inoculated leaf sheath and its surrounding tissues. Since the sheath blight lesions may be irregular in shape and may appear in multiple locations on the leaf sheath, it is necessary to fully inspect the surface of the leaf sheath, including the front and back sides and the location where the leaf sheath connects to the stem. Align the measurement starting point of the ruler to one end of the lesion. When measuring, ensure that the measuring tool is parallel to the growth direction of the lesion to accurately obtain the longest length of the lesion. For irregularly shaped lesions, such as curved, branched or ring-shaped lesions, measure along the outline of the lesion and record its longest continuous length as the lesion length data. If multiple lesions appear on an inoculated leaf sheath, measure the length of each lesion separately and record them in the corresponding table. For adjacent lesions that tend to merge, they are measured as a whole lesion, but the fusion of the lesions needs to be noted in the record. During the measurement process, after each lesion or each japonica rice plant is measured, the data is immediately recorded in the prepared table to ensure the accuracy and completeness of the data. When recording, the correspondence between the japonica rice variety name, plant number, leaf sheath number and other information and the measurement data should be checked again to prevent recording errors. The final photo result is as follows: Figure 3 shown.

[0043] Referring to the classification standards in Table 1, the resistance grades of seven varieties to sheath blight were identified (Table 2). The disease index of the resistant variety YSBR1 was 19.20%, and its resistance was resistant (R); the disease index of the resistant variety Huhanxiangruan 61 was 35.10%, and its resistance was moderately resistant (MR); the disease index of Nanjing 5055 was 46.70%, and its resistance was moderately susceptible (MS); the disease index of Jinjing 818 was 48.58%, and its resistance was moderately susceptible (MS); the disease index of Yinxiang 38 was 48.57%, and its resistance was moderately susceptible (MS); the disease index of the susceptible control variety Lemont was 80.71%, and its resistance was highly susceptible (HS); the disease index of Nanjing 5718 was 81.32%, and its resistance was highly susceptible (HS).

[0044] Table 2 Identification results of resistance to sheath blight in japonica rice

[0045]

[0046] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for identifying resistance to sheath blight in japonica rice, characterized in that: The following steps are involved: Planting and culturing japonica rice seeds to be tested in an intelligent greenhouse, inducing sheath blight from the end of tillering to panicle differentiation, observing the incidence of sheath blight, measuring the average lesion length of the japonica rice to be tested, calculating the disease index, and evaluating the sheath blight disease grade of the japonica rice; The temperature in the intelligent greenhouse is set at 25-30° C., the humidity is 80%-90%, the light intensity is 12000-20000 Lux, and the light cycle does not exceed 10 hours of illumination.

2. The identification method according to claim 1, characterized in that: The planting comprises sowing the japonica rice seeds in a seedling hole tray filled with nutrient soil, sowing 10 holes for each variety, sowing 3 seeds in each hole, and repeating each variety 3 times.

3. The identification method according to claim 2, characterized in that: This includes thinning out the seedlings when the plants grow to the 3-leaf 1-heart stage, retaining one seedling in each hole.

4. The identification method according to claim 1, characterized in that: The sheath blight induction comprises using brown sclerotia produced by culturing Rhizoctonia solani as a sheath blight inoculation fungus source, and inducing at a relative humidity of more than 80%.

5. The identification method according to claim 4, characterized in that: The method comprises separating Rhizoctonia solani strains from japonica rice plants infected with sheath blight in the field, inoculating the strains on a PDA culture medium, and using the brown sclerotia produced by the culture as the sheath blight inoculation fungus source.

6. The identification method according to claim 5, characterized in that: The method comprises culturing on the PDA medium for 10 to 15 days.

7. The identification method according to claim 5 or 6, characterized in that: When the sheath blight inoculation fungus source is used for inoculation, the japonica rice leaf sheath is slightly opened, and the inoculation fungus source is sent into the opened leaf sheath to restore the leaf sheath.

8. The identification method according to claim 7, characterized in that: Each leaf sheath is inoculated with 1 to 3 sources of sheath blight inoculation bacteria.

9. The identification method according to claim 1, characterized in that: The units of lesion length and leaf sheath length are the same.

10. The identification method according to claim 9, characterized in that: When the disease index is 0, the disease level of sheath blight is immune; when 0<the disease index≤20%, the disease level of sheath blight is resistant; When 20%<disease index≤40%, the sheath blight disease level is medium resistance; When 40%<disease index≤60%, the disease level of sheath blight is moderately susceptible; when 60%<disease index≤80%, the disease level of sheath blight is susceptible; when 80%<disease index≤100%, the disease level of sheath blight is highly susceptible.

Images