Rapid identification method for rice blast in plant breeding factory

Abstract

The invention relates to the technical field of plant disease technologies, in particular to a method for quickly identifying rice blast in a plant breeding factory, which accelerates the growth speed of rice by utilizing the breeding factory with controllable light, temperature, humidity and carbon dioxide concentration, accelerates the process of identifying the rice blast compared with a field, and improves the identification efficiency in a controllable environment. The induction rate of rice blast inoculation is improved by adopting a self-created method, the growth period of rice can be shortened and the identification process can be accelerated by controlling light, temperature and humidity, the environment suitable for the growth of rice blast germs can be regulated and controlled, and the disease resistance of rice blast can be identified more accurately; the whole process does not have a bacteria-keeping environment, and the influence of other pathogenic bacteria is eliminated; and the formula of the inoculation liquid can ensure infection of the bacterial liquid to the greatest extent, so that the purpose of verification is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease technology, and in particular to a method for quickly identifying rice blast in a plant breeding factory. Background Art

[0002] Rice blast pathogen has very high requirements for humidity conditions. It must maintain high humidity for a long time, especially under relatively low temperature conditions, to induce the occurrence of rice blast. This requirement of continuous high humidity is often not met in the general natural environment, resulting in the failure of rice blast pathogen to germinate.

[0003] In large-scale rice blast induction experiments, such as large-scale variety resistance identification, due to the large scale and difficult to control conditions, insufficient humidity often affects the disease. The existing rice blast induction-related experimental work does not have a controllable environment and a method to increase the rice blast induction rate. This often causes irreparable losses for resistance identification work with strong seasonality and limited number of test variety seeds (such as hybrid rice seeds). Summary of the invention

[0004] The purpose of the present invention is to provide a method for rapid identification of rice blast in a plant breeding factory, which can utilize a breeding factory with controllable light, temperature, humidity and carbon dioxide concentration to accelerate the growth rate of rice, thereby accelerating the process of rice blast identification compared to the field. At the same time, in a controllable environment, an original method is used to improve the induction rate of rice blast inoculation.

[0005] To achieve the above object, the present invention provides a method for rapid identification of rice blast in a plant breeding factory, comprising the following steps:

[0006] Prepare dry filter paper with conidia attached;

[0007] The prepared dried filter paper is used to prepare an inoculation solution of rice blast fungus at the seedling stage and an inoculation solution of rice blast fungus at the booting stage;

[0008] Cultivating rice as identification material, and dividing the cultivated rice into two groups, one group is sprayed at seedling stage, and the other group is transplanted culture group;

[0009] Under specified conditions, the prepared rice blast fungus inoculation solution at the seedling stage is uniformly sprayed on the leaves or ear necks of healthy rice plants in the seedling spraying group until water droplets appear on the leaves or ear necks, and then observation, recording and statistics are performed to calculate the rice blast fungus seedling inoculation induction rate;

[0010] When the growth period of the rice materials in the transplant culture group reaches the booting stage, the rice blast pathogen inoculation solution at the booting stage can be injected into the unopened ears under specified conditions, and then observation, recording and statistics are carried out to calculate the incidence rate of rice blast pathogen at the booting stage and the loss rate of rice blast pathogen at the booting stage.

[0011] Wherein, the specific method of preparing the dry filter paper with conidia attached thereto is:

[0012] Selecting rice blast strains that have strong pathogenicity to rice and can produce abundant conidia;

[0013] The selected strains were cultured in oatmeal medium;

[0014] After the selected strain matures, the oatmeal is washed with sterile water to collect the spore fluid containing conidia;

[0015] The conidia in the spore liquid are separated and attached to the filter paper through the filter paper, and the filter paper attached with the conidia is quickly air-dried to prepare the dry filter paper attached with the conidia.

[0016] The specific preparation methods of the rice blast fungus inoculation solution at the seedling stage and the rice blast fungus inoculation solution at the booting stage are as follows:

[0017] Cutting the dry filter paper with conidia attached thereto into pieces;

[0018] The cut dried filter paper with conidia attached thereto is divided into two groups, and the two groups are respectively put into the seedling stage basal solution and the booting stage basal solution one by one, and the two groups are fully stirred to obtain the seedling stage basal spore solution and the booting stage basal spore solution;

[0019] The conidia concentration of the seedling stage basic spore solution and the booting stage basic spore solution is continuously monitored until the conidia concentration reaches the standard of 104-106 / ml, so as to prepare the corresponding seedling stage rice blast fungus inoculation solution and the booting stage rice blast fungus inoculation solution.

[0020] The specific preparation methods of the seedling spraying group and the transplanting culture group are as follows:

[0021] The japonica rice varieties to be identified were sown in seedling trays, and the soil was sprayed with water to moisten the soil and then darkened for 3 days;

[0022] After germination, when the plants grow to the 3-leaf 1-heart stage, they can be divided into two parts, one for the seedling spraying group and the other for transplanting;

[0023] The transplanting group selected robust seedlings and transplanted them into a sterilized culture box with a water-retaining layer to simulate field cultivation measures, which served as the transplanting culture group.

[0024] Wherein, the specific preparation method of the seedling stage base solution is:

[0025] The invention is prepared by mixing 0.1% polyacrylamide and 0.1% APG by weight, and adding sterile water to make up to 100% and then mixing.

[0026] Wherein, the specific preparation method of the booting stage basic liquid is:

[0027] The invention is prepared by mixing 0.1% polyacrylamide, 0.1% APG and 0.1% hydroxyethyl cellulose according to weight percentage, and adding sterile water to make up to 100% and then mixing.

[0028] The specific methods for observing, recording and counting the induction rate of rice blast fungus at the seedling stage are as follows:

[0029] The observation, investigation and recording began on the second day after the seedling spraying group completed the spraying and inoculation of the rice blast fungus inoculation solution at the seedling stage;

[0030] The inoculation induction rate of rice blast fungus at the seedling stage was calculated on the 10th day.

[0031] The specific methods for observing, recording and counting the incidence of rice blast at the heading stage and the loss rate of rice blast are as follows:

[0032] Starting observation, investigation and recording on the 7th day after the transplanting culture group completes the injection inoculation of the rice blast pathogen in the booting stage;

[0033] On the 14th day, the incidence of rice blast at the heading stage of the rice blast pathogen is counted;

[0034] The rice blast ear blast loss rate was counted after 20 days.

[0035] The invention discloses a method for rapid identification of rice blast in a plant breeding factory. In a rice breeding factory where light, temperature and humidity can be controlled, rice blast inoculation can be carried out quickly. The two time periods of rice blast inoculation are divided into spraying at the seedling stage and injection of inoculation solution at the booting stage. A water retaining agent and a surfactant are added to the inoculation solution at the seedling stage, which can effectively maintain the activity of the bacterial solution, improve the hydrophilicity and spreadability of the surface of the rice plant, effectively increase the efficiency of rice receiving the bacterial solution, and prolong the retention time of the inoculation solution on the rice receptor, thereby providing favorable conditions for the induction of rice blast fungi and significantly improving the induction rate of artificial inoculation of rice blast. Hydroxyethyl cellulose (thickener) is added to the inoculation solution at the booting stage, and the bacterial solution is accurately injected into the unopened ear by a continuous syringe. The bacterial solution will adhere to the husk to prevent the bacterial solution from flowing out from the leaf pillow, thereby improving the inoculation efficiency and the induction rate of artificial inoculation of rice blast. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0037] Figure 1 The present invention is a schematic diagram of inoculating an oat culture medium with the rice blast pathogen.

[0038] Figure 2 It is a schematic diagram of the seedling spraying preparation of the present invention.

[0039] Figure 3 It is a schematic diagram of preparing to inoculate ear blight after transplanting of the present invention.

[0040] Figure 4 It is a schematic diagram of a continuous syringe for inoculating ear blast of the present invention and an example of its use.

[0041] Figure 5 It is a schematic diagram of leaf blight and ear blight at the seedling stage of some materials of the present invention.

[0042] Figure 6 The present invention is a flow chart of a method for rapid identification of rice blast in a plant breeding factory.

[0043] Figure 7 The present invention is a flow chart of a specific method for preparing dry filter paper with conidia attached thereto.

[0044] Figure 8 It is a flow chart of the specific preparation method of the rice blast fungus inoculation solution at the seedling stage and the rice blast fungus inoculation solution at the booting stage. DETAILED DESCRIPTION

[0045] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0046] In the description of the present invention, it should be understood that “plurality” means two or more than two, unless otherwise clearly and specifically defined.

[0047] See also Figures 1 to 8 The present invention provides a method for rapid identification of rice blast in a plant breeding factory, comprising the following steps:

[0048] S1: Preparation of dry filter paper with conidia attached;

[0049] Furthermore, the specific method of preparing the dry filter paper with conidia attached thereto is:

[0050] S101: Select rice blast strains that have strong pathogenicity to rice and can produce abundant conidia;

[0051] S102: The selected strains were cultured in oatmeal medium;

[0052] S103: After the selected strain matures, the oatmeal is washed with sterile water to collect the spore liquid containing conidia;

[0053] S104: Separating the conidia in the spore liquid through the filter paper and attaching them to the filter paper, and quickly air-drying the filter paper with the conidia attached to prepare the dry filter paper with the conidia attached to it.

[0054] Specifically, first, a rice blast strain having strong pathogenicity to rice and capable of producing abundant conidia is selected, the selected strain is cultured in an oatmeal medium, after the strain matures, the oatmeal is washed with sterile water, and the spore liquid containing conidia is collected;

[0055] The conidia in the spore liquid are separated and attached to the filter paper through the filter paper, and the filter paper attached with the conidia is quickly air-dried for subsequent use;

[0056] Among them, the rice blast pathogen preparation: the rice blast pathogen was taken from diseased plants infected with rice blast in the field, and then cultured in the laboratory using oat culture medium;

[0057] Oatmeal medium is a commonly used culture medium for cultivating various microorganisms. Its preparation method is as follows: prepare materials: oat kernels, sucrose, agar powder, and water.

[0058] Production steps:

[0059] Take oat kernels in a beaker, add water to it, and then sterilize it under high pressure steam;

[0060] After sterilization, let the autoclave cool naturally, take it out after cooling, filter it with gauze, add water to the filtrate, and then add sucrose and agar powder, continue heating, stir constantly during heating to avoid sticking to the pot, boil for 5-8 minutes to fully dissolve the sucrose and agar powder;

[0061] The fully dissolved solution is fixed to volume in a beaker, and then divided into Erlenmeyer flasks or test tubes. After the division, plug the cotton plug and wrap it with kraft paper to prevent moisture from entering and wetting the cotton plug during sterilization. After sealing, sterilize it under high pressure steam, and then let the sterilizer cool naturally. After cooling, the oatmeal culture medium is obtained. Finally, the prepared oatmeal culture medium ( Figure 1 ) Refrigerate and store for later use.

[0062] Note:

[0063] During the preparation of oatmeal culture medium, attention should be paid to aseptic operation to avoid contamination;

[0064] The pH value of oatmeal medium needs to be adjusted according to different microorganisms;

[0065] The storage time of oatmeal culture medium should not be too long, generally 1-2 weeks;

[0066] The conidia in the spore solution were separated and attached to the filter paper by passing through the filter paper;

[0067] The filter paper with conidia attached was quickly air-dried for subsequent use.

[0068] S2: using the prepared dried filter paper to prepare an inoculum solution for rice blast fungus at the seedling stage and an inoculum solution for rice blast fungus at the booting stage;

[0069] S3: Cultivate rice as identification material, and divide the cultivated rice into two groups, one is the seedling spraying group, and the other is the transplanting culture group;

[0070] Furthermore, the specific preparation method of the rice blast fungus inoculation solution at the seedling stage and the rice blast fungus inoculation solution at the booting stage is:

[0071] S301: cutting the dry filter paper with conidia attached thereto into pieces;

[0072] S302: dividing the cut dry filter paper with conidia attached into two groups, respectively putting them into the seedling stage basal solution and the booting stage basal solution one by one, and fully stirring them to obtain the seedling stage basal spore solution and the booting stage basal spore solution;

[0073] S303: Continuously monitoring the conidia concentration of the seedling stage basic spore solution and the booting stage basic spore solution until the conidia concentration reaches a standard of 104-106 conidia / ml, thereby preparing the corresponding seedling stage rice blast fungus inoculation solution and the booting stage rice blast fungus inoculation solution;

[0074] Furthermore, the specific preparation method of the seedling stage base solution is:

[0075] 0.1% polyacrylamide and 0.1% APG by weight are mixed and made up to 100% with sterile water;

[0076] Furthermore, the specific preparation method of the booting stage basic solution is:

[0077] 0.1% polyacrylamide, 0.1% APG and 0.1% hydroxyethyl cellulose are mixed and prepared by adding sterile water to 100%;

[0078] The method comprises the following steps: mixing 0.1-4% of a water retaining agent and 0.03-1% of a surfactant according to weight percentage, and supplementing the total amount with sterile water to 100% to form a base liquid, cutting the air-dried filter paper into small pieces, putting the pieces into the base liquid one by one, and stirring the pieces sufficiently to ensure that the conidia on the filter paper are completely detached and evenly dispersed in the liquid, and continuously monitoring the conidia concentration in the spore liquid until the conidia concentration reaches the standard of 104-106 / ml. The liquid at this time is an inoculation liquid for rice blast fungus, which can be used for inoculation and is for standby use;

[0079] Specifically, the preparation of the base liquid:

[0080] First, accurately weigh the water retainer, and make its content in the final mixed solution between 0.1% and 4% by weight. For example, if you want to prepare 100 grams of base liquid, the amount of water retainer should be 0.1 grams to 4 grams. The water retainer can be a common material with good water absorption and water retention, such as sodium polyacrylate. Place the weighed water retainer in a clean and dry container for later use.

[0081] Next, weigh the surfactant, the weight percentage of which should be between 0.03% and 1%. Taking 100 grams of base liquid as an example, the weight of the surfactant should be between 0.03 grams and 1 gram. The surfactant can be a non-ionic surfactant such as Tween-20, which helps to disperse the spores later. Add the weighed surfactant to the container where the water retaining agent is placed.

[0082] Then slowly add sterile water into the container, stirring while adding, until the total amount of the solution reaches 100 grams (i.e., replenished to 100% of the total amount). The stirring process should be continuous and uniform to ensure that the water retaining agent and surfactant are completely dissolved and evenly dispersed in the sterile water to form a base solution. The stirring time can be determined according to the actual situation, generally not less than 5 minutes, to ensure the uniformity of the solution;

[0083] Specifically, spore processing and inoculum preparation:

[0084] Air-dry the filter paper with rice blast fungus conidia in advance. The filter paper can be placed in a well-ventilated, clean and dry environment to avoid contamination by dust and other impurities until the filter paper is completely dry.

[0085] Use sterile scissors to cut the air-dried filter paper into small pieces of uniform size. The size of the small pieces can be determined according to the convenience of actual operation, for example, cut into square pieces with a side length of 1-2 cm;

[0086] Put the cut filter paper pieces into the prepared base liquid one by one. After each piece of filter paper is put in, use a glass rod or magnetic stirrer to stir thoroughly. When stirring, the strength and speed should be controlled to avoid splashing of the liquid. At the same time, make sure that the filter paper is fully rolled in the liquid so that the conidia attached to the filter paper can be completely detached and evenly dispersed in the base liquid. The stirring time can be adjusted according to the actual situation. Generally, stir for 2-3 minutes after each filter paper is put in, until all the filter paper pieces are processed and the spores are observed to be evenly distributed in the liquid;

[0087] Use a hemacytometer or other specialized microbial counting instrument to continuously monitor the conidia concentration in the spore solution;

[0088] During the monitoring process, samples can be taken for testing at regular intervals (e.g., 10-15 minutes). If the spore concentration is found to be lower than 10^4 / ml, the amount of sterile water added for subsequent dilution can be appropriately reduced; if the spore concentration is higher than 10^6 / ml, an appropriate amount of sterile water can be added for dilution;

[0089] Through continuous adjustment and monitoring, the conidia concentration in the spore liquid reaches the standard range of 10^4-10^6 / ml. The liquid obtained at this time is the rice blast fungus inoculation liquid, which can be properly stored and used for subsequent inoculation experiments or related research. When storing, it should be sealed and placed in an environment with a suitable temperature (such as about 4°C) to maintain the activity of the spores and the stability of the inoculation liquid.

[0090] S4: under specified conditions, uniformly spraying the prepared rice blast fungus inoculation solution at the seedling stage on the leaves or ear necks of healthy rice plants in the seedling spraying group until water droplets appear on the leaves or ear necks, and then observing, recording and counting to calculate the rice blast fungus seedling inoculation induction rate;

[0091] Furthermore, the specific methods for observing, recording and counting the induction rate of rice blast fungus at the seedling stage are as follows:

[0092] Starting observation, investigation and recording on the second day after the seedling spraying group completes the spraying and inoculation of the rice blast fungus inoculation solution at the seedling stage;

[0093] On the tenth day, the inoculation induction rate of rice blast fungus at the seedling stage was calculated.

[0094] S5: When the growth period of the rice materials in the transplant culture group reaches the booting stage, the rice blast pathogen inoculation solution at the booting stage can be injected into the unopened ears under specified conditions, and then observation, recording and statistics are carried out to calculate the incidence rate of rice blast pathogen at the booting stage and the loss rate of rice blast pathogen at the booting stage.

[0095] Furthermore, the specific methods for observing, recording and counting the incidence of rice blast at the heading stage and the rice blast loss rate are as follows:

[0096] Starting observation, investigation and recording on the 7th day after the transplanting culture group completes the injection inoculation of the rice blast pathogen in the booting stage;

[0097] On the 14th day, the incidence of rice blast at the heading stage of the rice blast pathogen is counted;

[0098] The rice blast ear blast loss rate was counted after 20 days.

[0099] Specifically, sowing and darkening treatment: prepare seedling plug trays (50-hole plug trays, 53 cm × 27 cm, upper diameter 4.8 cm, depth 4.7 cm, lower diameter 2.3 cm, single hole volume 55 ml), ensure that the plug trays are clean and free of impurities to avoid adverse effects on seed germination;

[0100] Eleven indica rice seeds to be identified were selected, namely Hanyou 737, Hanyou 8210, Yuanfengyou 901, Hanyou 173, Huanghuazhan 1, Huhan 1505, Hanyou 73, Hanyou 623, Jiafuzhan 1, as well as the rice blast susceptible variety Yuanfengzao and the rice blast resistant variety Huhan 1516, and they were evenly sown in each hole of the seedling tray, 3-5 seeds were sown in each hole to ensure sufficient number of seedlings and facilitate subsequent screening of strong seedlings, and two trays were sown for each variety;

[0101] When sowing, pay attention to the placement depth of the seeds, which should generally be controlled at around 1-2 cm to ensure that the seeds can fully contact the moisture and nutrients in the soil while obtaining sufficient oxygen supply to facilitate seed germination.

[0102] After sowing, use a watering can or spray equipment to evenly spray an appropriate amount of water on the surface of the soil in the plug tray to fully moisten the soil, but avoid water accumulation to prevent the seeds from rotting due to lack of oxygen. The degree of moistening should be such that the soil surface is moist and no water seeps out when the soil is lightly pressed by hand.

[0103] The seedling trays moistened with water were placed in a dark environment with relatively stable temperature and humidity for 3 days;

[0104] The ambient temperature of the darkening treatment should be controlled at 28-30℃, and the relative humidity should be maintained at 80%-90%. Darkening treatment can improve the uniformity of seedling emergence, increase the germination rate, promote the accumulation of nutrients, shorten the seedling raising time, and reduce the damage to the initial buds;

[0105] During this period, check the soil moisture regularly. If the soil surface is found to be dry, add a small amount of water in time to ensure that the seeds can carry out physiological activities in the early stage of germination in a suitable humidity environment.

[0106] Among them, seedling management and treatment: after 3 days of darkening treatment, the seedling tray was placed in a plastic culture box (55.7cm×36.7cm×8.2cm), and the water layer in the box was kept at 2cm;

[0107] Transfer the seedling tray and the plastic incubator to a breeding factory environment with controlled light, temperature and humidity, and wait for the seeds to germinate. Generally, the seeds will germinate within 2-3 days after the transfer.

[0108] When the plants grow to the 3-leaf 1-heart stage, the seedlings are selected and processed. First, the seedlings are carefully removed from the plug tray, and the root system is avoided as much as possible.

[0109] Then, two trays of seedlings were given different treatments, and one tray of seedlings remained in the nursery area ( Figure 2 ), prepare to spray rice blast fungi at the seedling stage to detect leaf blast at the seedling stage, and select healthy and strong seedlings with well-developed root systems and no signs of diseases and insect pests for transplanting.

[0110] Transplanting operation: prepare the incubator for transplanting in advance, and the incubator must be strictly disinfected before use. Chemical disinfectants (such as sodium hypochlorite solution) can be used for immersion or spray disinfection, and then rinse thoroughly with clean water to ensure that there is no residual disinfectant to prevent damage to the seedlings;

[0111] A 5-cm-thick layer of sterilized substrate soil (peat soil, garden soil, perlite and sawdust) and a 3-cm-thick water layer were laid at the bottom of the incubator to simulate field transplanting preparations.

[0112] Transplant the selected healthy seedlings into the incubator. When transplanting, pay attention to placing the roots of the seedlings in the soil in a stretched manner, then gently cover the soil and compact it to ensure that the seedlings are firmly upright and the roots are in full contact with the soil.

[0113] After transplanting, spray water again to make the soil and roots closely combined and replenish the water lost during the transplanting process ( Figure 3 ).

[0114] Among them, fertilization and environmental control: both the seedlings in the seedling stage and the plants after transplanting are fertilized according to the conventional rice fertilization treatment plan;

[0115] During the seedling raising period, an appropriate amount of base fertilizer (such as nitrogen, phosphorus and potassium compound fertilizer) can be evenly mixed into the seedling raising soil before sowing. The amount of fertilizer applied is determined according to the soil fertility and the fertilizer requirements of the rice variety. Generally, 1-2 kg of compound fertilizer is added to every cubic meter of seedling raising soil.

[0116] During the growth of the plant, topdressing is carried out according to different growth stages. For example, after the 3-leaf 1-heart stage, a dilute nitrogen fertilizer solution (such as 0.1%-0.2% urea solution) can be sprayed every 7-10 days to promote the growth of the plant's stems and leaves; after the seedling slowing period after transplanting, the application of phosphorus and potassium fertilizers is gradually increased to promote the root development and reproductive growth of the plant;

[0117] When applying fertilizer, pay attention to spraying or spreading the fertilizer evenly to avoid seedling burn caused by excessive local fertilizer concentration.

[0118] The entire operation process is carried out in a breeding factory with controlled light, temperature and humidity. The light intensity can be controlled at 2000-3000 lux during the seedling stage, and gradually increases to 3000-5000 lux as the plants grow. The temperature is maintained at 25-30℃ during the day and 18-22℃ at night.

[0119] Relative humidity is maintained at 70%-80% in the seedling stage and can be appropriately reduced to 60%-70% in the later growth stage. By precisely controlling these environmental factors, the best conditions for the growth and development of rice are provided, and it is also convenient to study and analyze the interaction between rice and rice blast under different environmental conditions;

[0120] In the breeding factory, environmental parameters should be monitored and recorded regularly, and environmental control equipment should be adjusted in time to ensure the stability and consistency of environmental conditions.

[0121] Among them, spray inoculation at seedling stage:

[0122] Temperature and humidity control: Determine the optimal environmental parameters: First, it is necessary to determine the most suitable growth temperature and humidity range for conidia based on the characteristics of the selected rice blast strain and the sensitivity of the rice variety. Generally speaking, the germination and infection activity of rice blast conidia are higher in an environment with a temperature of 25-28°C and a relative humidity of 85%-95%.

[0123] Preparation and setting of environmental conditioning equipment: Control the breeding factory environment. A few hours before inoculation, turn on the temperature and humidity adjustment function, set the temperature precisely at 26°C, and adjust the humidification device through humidity sensor feedback to stabilize the relative humidity at around 90%. At the same time, ensure that the air in the incubator is well ventilated, and set an appropriate ventilation rate, ventilating 1-2 times an hour.

[0124] Inoculation operation: Preparation and inspection of inoculation solution: Before inoculation, confirm again whether the concentration of the prepared inoculation solution reaches the standard range of 10^4-10^6 / ml, and the inoculation solution should be fully mixed without precipitation or impurities. The inoculation solution is loaded into a clean and sterile sprayer. The nozzle of the sprayer should be able to produce uniform and fine droplets. If a pressure sprayer can be used, the spray pressure can be adjusted to 0.2-0.3MPa to ensure that the inoculation solution is evenly dispersed.

[0125] Plant preparation and protection: 11 trays of rice seedlings are used as inoculation objects. Before inoculation, plastic film or other isolation materials can be used to separate the inoculation area from the surrounding environment to prevent the inoculation solution from splashing onto other unrelated plants and causing contamination and cross infection.

[0126] Inoculation process: Hold the sprayer and place the nozzle about 15-20 cm away from the leaves or neck of the rice plant, and spray at a uniform speed and strength. Starting from the top of the plant, spray the front and back sides of the leaves and the neck of the ear in turn to ensure that the inoculation liquid fully covers the target area. During the spraying process, constantly observe the wetness of the surface of the leaves or neck of the ear until evenly distributed small water droplets appear on the surface and are about to begin to flow. For larger planting areas or a large number of plants, inoculations should be carried out in sequence according to the predetermined order to avoid omissions or repeated inoculations, and good inoculation records should be kept, including inoculation time, inoculated plant number, inoculation liquid concentration and other information.

[0127] Environmental maintenance after inoculation: After inoculation, quickly transfer the inoculated rice plants to a culture area or isolated greenhouse with the same temperature and humidity conditions set in advance, turn off the ventilation equipment or reduce the ventilation volume, and keep the environment relatively static for 24-48 hours to facilitate the attachment, germination and infection of pathogen spores. During this period, closely monitor the changes in environmental temperature and humidity, and make timely adjustments if there are fluctuations to ensure that environmental conditions are always within the range suitable for pathogen infection.

[0128] Observation, recording and statistics: Observation, investigation and recording began on the second day after inoculation. On the tenth day, the incidence of rice leaf blast at the seedling stage was calculated based on the disease situation. The calculation formula was incidence rate (%) = (total number of diseased plants / total number of observed plants) × 100%, as shown in Table 1. Rice leaf blast disease grading standards.

[0129] Table 1:

[0130]

[0131] Record the calculation results and note the observation date, rice variety, planting environment and other relevant information for subsequent analysis and comparison.

[0132] Among them, injection vaccination at the booting stage:

[0133] Monitoring and determination of growth period: closely follow the growth process of rice after transplanting, and comprehensively judge whether the rice material has entered the booting stage by observing the morphological characteristics of rice, the number of growth days and previous experience data. In the booting stage, the internodes at the base of the stalk of rice plants begin to elongate, and the panicles begin to differentiate and gradually develop and swell. The precise determination can be made by dissecting the plants and observing the panicle size and development stage.

[0134] Environmental control: After it is determined that the inoculation stage of the booting period is about to begin, the temperature control system in the breeding factory is set to about 30°C in advance;

[0135] At the same time, the humidity regulating device is started, and the air humidity is controlled to be stable at around 80% through the humidity regulator, and the humidity fluctuation range is controlled between 78% and 82%, creating suitable temperature and humidity conditions for inoculation, which is conducive to the survival, germination and infection of rice blast fungi.

[0136] Preparation and storage of inoculum: Carefully prepare the inoculum so that its conidia concentration meets the experimental design requirements (10^4-10^6 conidia / ml);

[0137] For inoculation at the booting stage, hydroxyethyl cellulose is added to the inoculation solution as a thickener. The amount added is determined according to the experimental pre-test, generally 0.5%-2% of the volume of the inoculation solution. The inoculation solution after adding the thickener is fully stirred to ensure that the hydroxyethyl cellulose is completely dissolved and evenly dispersed in the inoculation solution to achieve the best thickening effect;

[0138] Then carefully transfer the inoculum into a continuous syringe. The syringe should be strictly sterilized in advance to prevent bacteria from contaminating the inoculum and affecting the inoculation effect.

[0139] Inoculation site and method: After the operator puts on sterile protective equipment, he holds a continuous syringe filled with inoculation solution and places the syringe close to the rice ear primordium (bulge), and must select the rice ear primordium that is not broken;

[0140] Slowly inject the inoculum solution into the ear from top to bottom with the needle tilted, which is conducive to the bacterial solution covering the entire surface of the husk and allowing the bacterial solution to adhere evenly to the husk ( Figure 4 );

[0141] Due to the addition of hydroxyethyl cellulose, the viscosity of the bacterial solution increases, which can better adhere to the surface of the husk and effectively prevent the bacterial solution from flowing out of the leaf pillow, thereby increasing the residence time and concentration of the inoculum at the inoculation site, greatly enhancing the contact opportunity between the pathogen and the rice panicle tissue, and thus improving the inoculation efficiency;

[0142] During the injection process, attention should be paid to controlling the injection volume. According to the size and development stage of the rice panicle, the injection volume per panicle should be controlled at 1 ml to ensure that the inoculum can fully cover the surface of the husk without flowing and wasting due to excessive amount or causing mechanical damage to the panicle.

[0143] Disease observation and record: 7 days after inoculation, closely observe the disease of rice plants. Starting from the 10th day after inoculation, check the rice panicles and leaves regularly every day to see if they have typical symptoms of rice blast, such as the appearance of lesions, color changes, shape and size of lesions, etc., and record the onset time, location of the disease, symptoms, etc. in detail ( Figure 5 );

[0144] The formula for calculating the rice panicle blast incidence and panicle blast loss rate is: Incidence = (Total number of diseased plants / Total number of investigated plants) × 100%. Panicle blast loss rate = (Total yield loss due to disease / Total theoretical yield without disease) × 100%, as shown in Table 2.

[0145] Table 2:

[0146]

[0147] The comprehensive index of rice blast resistance was calculated based on the leaf blast incidence rate in the early seedling stage, the ear blast incidence rate and the ear blast loss rate. The formula was comprehensive index = seedling leaf blast disease level × 25% + ear blast incidence disease level × 25% + ear blast loss rate disease level × 50%.

[0148] Finally, the comprehensive index of rice blast resistance of 11 rice materials was identified as shown in Table 3 . Results of rapid identification of rice blast resistance in breeding factory.

[0149] Table 3:

[0150]

[0151]

[0152] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A method for rapid identification of rice blast in a plant breeding factory, characterized in that: The following steps are involved: Prepare dry filter paper with conidia attached; The prepared dried filter paper is used to prepare an inoculation solution of rice blast fungus at the seedling stage and an inoculation solution of rice blast fungus at the booting stage; Cultivating rice as identification material, and dividing the cultivated rice into two groups, one group is sprayed at seedling stage, and the other group is transplanted culture group; Under specified conditions, the prepared rice blast fungus inoculation solution at the seedling stage is uniformly sprayed on the leaves or ear necks of healthy rice plants in the seedling spraying group until water droplets appear on the leaves or ear necks, and then observation, recording and statistics are performed to calculate the rice blast fungus seedling inoculation induction rate; When the growth period of the rice materials in the transplant culture group reaches the booting stage, the rice blast pathogen inoculation solution at the booting stage can be injected into the unopened ears under specified conditions, and then observation, recording and statistics are carried out to calculate the incidence rate of rice blast pathogen at the booting stage and the loss rate of rice blast pathogen at the booting stage.

2. The method for rapid identification of rice blast in plant breeding factories according to claim 1, characterized in that: The specific method of preparing the dry filter paper with conidia attached thereto is: Selecting a rice blast strain that is highly pathogenic to rice and can produce conidia; The selected strains were cultured in oatmeal medium; After the selected strain matures, the oatmeal is washed with sterile water to collect the spore fluid containing conidia; The conidia in the spore liquid are separated and attached to the filter paper through the filter paper, and the filter paper attached with the conidia is air-dried to prepare the dry filter paper attached with the conidia.

3. The method for rapid identification of rice blast in plant breeding factories according to claim 1, characterized in that: The specific preparation methods of the rice blast fungus inoculation solution at the seedling stage and the rice blast fungus inoculation solution at the booting stage are as follows: Cutting the dry filter paper with conidia attached thereto into pieces; The cut dried filter paper with conidia attached thereto is divided into two groups, and the two groups are respectively put into the seedling stage basal solution and the booting stage basal solution one by one, and the two groups are fully stirred to obtain the seedling stage basal spore solution and the booting stage basal spore solution; The conidia concentration of the seedling stage basic spore solution and the booting stage basic spore solution is continuously monitored until the conidia concentration reaches the standard of 104-106 / ml, so as to prepare the corresponding seedling stage rice blast fungus inoculation solution and the booting stage rice blast fungus inoculation solution.

4. The method for rapid identification of rice blast in plant breeding factories according to claim 1, characterized in that: The specific preparation methods of the seedling spraying group and the transplanting culture group are as follows: The japonica rice varieties to be identified were sown in seedling trays, and the soil was sprayed with water to moisten the soil and then darkened for 3 days; After germination, when the plants grow to the 3-leaf 1-heart stage, they can be divided into two parts, one for the seedling spraying group and the other for transplanting; The transplanting group selected robust seedlings and transplanted them into a sterilized culture box with a water-retaining layer to simulate field cultivation measures, which served as the transplanting culture group.

5. The method for rapid identification of rice blast in plant breeding factories according to claim 3, characterized in that: The specific preparation method of the seedling stage base solution is: The invention is prepared by mixing 0.1% polyacrylamide and 0.1% APG by weight, and adding sterile water to make up to 100% and then mixing.

6. The method for rapid identification of rice blast in plant breeding factories according to claim 4, characterized in that: The specific preparation method of the booting stage basic liquid is as follows: The invention is prepared by mixing 0.1% polyacrylamide, 0.1% APG and 0.1% hydroxyethyl cellulose according to weight percentage, and adding sterile water to make up to 100% and then mixing.

7. The method for rapid identification of rice blast in plant breeding factories according to claim 1, characterized in that: The specific methods for observing, recording and counting the induction rate of rice blast fungus inoculation at the seedling stage are as follows: Starting observation, investigation and recording on the second day after the seedling spraying group completes the spraying and inoculation of the rice blast fungus inoculation solution at the seedling stage; On the tenth day, the inoculation induction rate of rice blast fungus at the seedling stage was calculated.

8. The method for rapid identification of rice blast in plant breeding factories according to claim 1, characterized in that: The specific methods for observing, recording and counting the incidence of rice blast at the heading stage and the loss rate of rice blast are as follows: Starting observation, investigation and recording on the 7th day after the transplanting culture group completes the injection inoculation of the rice blast pathogen in the booting stage; On the 14th day, the incidence of rice blast at the heading stage of the rice blast pathogen is counted; The rice blast ear blast loss rate was counted after 20 days.

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