Sesame stem blight resistance identification and evaluation method

Abstract

The invention belongs to the technical field of sesame disease control, and particularly relates to a sesame stem blight resistance identification and evaluation method. The method is used for screening and identifying sesame stem blight resistant germplasm or evaluating the sesame stem blight resistance of specific sesame germplasm, and comprises the following steps: preparing sesame seedlings, performing root-irrigation inoculation treatment, identifying and evaluating the resistance and the like. On the basis of a root irrigation infection method, the sesame seedlings are inoculated, the concentration of inoculated strains is easy to adjust, indoor disease resistance identification and evaluation in the sesame seedling stage are facilitated, a solid foundation is laid for sesame stem blight resistance mechanism research and disease prevention and control, and reference is provided for research on stem blight of other crops. In the experiment process, in order to prevent plant roots from being mechanically damaged and influence disease degree investigation, the inventor adopts pure vermiculite with loose texture as a culture medium, and designs a specifically improved Hoagland nutrient solution as an irrigation material, so that a good foundation is laid for judging the disease resistance of sesame.

Description

Technical Field

[0001] The invention belongs to the technical field of sesame disease prevention and control, and particularly relates to a method for identifying and evaluating sesame stem blight resistance. Background Art

[0002] During sesame cultivation, sesame stem blight is a common and one of the most serious fungal diseases. Macrophomina phaseolina , MP), which not only infects sesame, but also a variety of legumes, carrots, cotton and other crops, causing great harm. For sesame, sesame stem blight mainly occurs in the seedling and flowering stages, with rapid onset and difficult prevention and control; in severe cases, it can reach more than 30%, seriously affecting the yield and quality of sesame seeds.

[0003] Accurate assessment and evaluation of resistance identification is one of the top priorities for scientific research work such as disease prevention and control and screening of disease-resistant germplasm. Therefore, researchers have carried out relevant resistance identification and evaluation work by using methods such as seed inoculation and toothpick inoculation, combined with the pathogenic characteristics of Phaseolus vulgaris to sesame germplasm. However, through research and analysis of relevant screening, identification and evaluation methods, it can be seen that the existing publicly available methods have the disadvantages of poor stability of identification results and too young seedlings of inoculated samples to reflect the resistance level of germplasm to stem blight pathogens, which in turn makes it impossible to widely use them in the precise identification and genetic analysis of sesame germplasm resistance to stem blight. Therefore, there is an urgent need to establish an efficient and stable method for precise identification and evaluation of sesame stem blight resistance in the indoor seedling stage to explore the disease resistance characteristics of sesame, and ultimately provide theoretical and technical support for the effective prevention and control of sesame stem blight. Summary of the invention

[0004] In the present application, the inventor aims to provide a method for accurately identifying and evaluating the resistance of sesame germplasm to stem blight pathogens based on root inoculation, thereby laying a certain technical and material foundation for the screening of sesame germplasm with high resistance to stem blight and the prevention and control of stem blight.

[0005] The technical solution adopted by the present invention is described in detail as follows.

[0006] A method for identifying and evaluating sesame stem blight resistance is provided, and the method is used for screening and identifying sesame stem blight resistance germplasm, and specifically comprises the following steps: 1. Preparing sesame samples for testing Select an appropriate amount of healthy and mature seeds of the sesame variety to be tested, sterilize them, and culture them until the seeds turn white (reference culture conditions: 25℃~28℃, 100~120rpm shaking culture); The sterilization treatment is specifically performed as follows: first treat with 70% alcohol for 30 seconds, then treat with 3% sodium hypochlorite for 10 to 15 minutes, and finally rinse with sterile water for 3 to 5 times; Then, the white sesame seeds are sown in a container containing sterile vermiculite; the container is, for example, a paper cup with a capacity of 250 mL, and 10 seeds can be sown in each paper cup; After sowing, the sesame seeds were regularly irrigated with modified Hoagland nutrient solution according to their growth conditions, and cultured in an artificial climate incubator until they reached the stage of three pairs of true leaves (approximately 30-35 days). The culture conditions were as follows: 28°C, 80% relative humidity, and 15h / 9h light-dark alternation. The modified Hoagland nutrient solution formula is: 945 mg / L calcium nitrate tetrahydrate, 506 mg / L potassium nitrate, 80 mg / L ammonium nitrate, 136 mg / L potassium dihydrogen phosphate, 493 mg / L magnesium sulfate, 2.5 mL iron salt solution and 5 mL trace element solution, pH=6.0; Iron salt solution: 2.78 g ferrous sulfate heptahydrate and 3.73 g disodium ethylenediaminetetraacetate (EDTA-Na), 500 ml distilled water, pH = 5.5; Trace element liquid: potassium iodide 0.83mg / l, boric acid 6.2mg / L, manganese sulfate 22.3mg / L, zinc sulfate 8.6mg / L, sodium molybdate 0.25mg / L, copper sulfate 0.025mg / L, cobalt chloride 0.025mg / L; (II) Root inoculation treatment A sterile water suspension of Phaseolus vulgaris strain (1×10 3 -2×10 4 cfu / mL) as the infection solution, select the sesame plants that are healthy and growing consistently in step (I) as the experimental samples, and inject the infection solution (20-50mL) into the base of the sesame stem with a syringe (when injecting the infection solution, it is mainly used for root infection, so pay attention to the liquid level during injection). At the same time, ensure that the infection solution is evenly sprinkled around the base of each sesame seedling stem; After the infection solution was injected, the plants were covered with sterile vermiculite with a thickness of about 1 cm and continued to be cultured in an artificial climate incubator (28°C, relative humidity of 80%, 15h / 9h light and dark alternation). During this period, the plants were watered with modified Hoagland nutrient solution once every 3 days to ensure the nutrients needed for plant growth. Under the same operation mode, sesame seedlings irrigated with clean water were used as negative controls without inoculation. The preparation method of the sterile aqueous bacterial suspension of the Phaseolus vulgaris strain can be referred to as follows: First, the preserved Phaseolus vulgaris strain was inoculated into 300 mL of PDB liquid culture medium and cultured at 28°C and 150-180 rpm for 3-5 days; After the incubation, filter the filtrate with 4 layers of sterile gauze, add an appropriate amount of sterile water to mix, and then transfer it to a food processor for further crushing (to ensure uniform mixing). Finally, dilute it to 1×10 3 -2×10 4 cfu / mL is enough; The PDB liquid culture medium contains 200 g of potato and 20 g of glucose in every 1000 mL of distilled water.

[0007] 3. Identification and evaluation of sesame varieties’ resistance to spot blight Observe and record the occurrence of stem blight of sesame seedlings after inoculation in step (ii) every day, investigate and record the number of diseased plants and the disease grade every 7 days (generally, the investigation can be continued until 28 days after inoculation), calculate the stem blight disease index, and determine the disease resistance of sesame varieties or the pathogenicity of specific strains to sesame based on it; The calculation formula of stem blight index is: Disease index (DI) = Σ (disease grade × corresponding number of diseased plants) / total number of plants; The sesame stem blight classification standard is set as follows: Level 0: Good growth without symptoms, normal above-ground stems and leaves, and normal rhizomes; Level 1: The disease occurs at the base of the stem or less than 1 / 4 of the stem, and the leaves wilt slightly; Level 2: 1 / 4-1 / 2 of the stems are diseased, less than 1 / 2 of the leaves are obviously wilted, and the plants are slightly dwarfed; Level 3: More than 1 / 2 of the stems or branches are diseased, more than 1 / 2 of the leaves are obviously wilted, and the plant is significantly dwarfed; Level 4: The entire stem is infected, the plant is severely dwarfed and close to death; According to the disease index, the resistance of different sesame varieties to stem blight can be divided into five levels, as follows: Level 1: High disease resistance, 0≤DI≤15; Second level: Disease resistance, 15 <DI≤30; Third level: medium resistance, 30 <DI≤50; Level 4: Sickness, 50 <DI≤70; Fifth level: High sensitivity, 70 <DI≤100。

[0008] In this application, the inventors inoculated sesame seedlings (3 pairs of true leaves to bud stage) based on the root irrigation infection method. The concentration of the inoculated strain is easy to adjust, which is conducive to the indoor disease resistance identification and evaluation of sesame seedlings, laying a solid foundation for the study of the mechanism of sesame resistance to stem blight and disease prevention and control, and also providing reference and reference for the study of stem blight of other crops. During the experiment, in order to avoid mechanical damage to the roots of the plants and affect the investigation of the degree of disease, the inventors used loose pure vermiculite as the culture matrix, and designed a specific modified Hoagland nutrient solution as the irrigation material, which ensured the normal nutrient supply of sesame plants while ensuring the healthy growth of the roots of the plants, thus laying a good foundation for the determination of sesame disease resistance.

[0009] In general, the technical method of this application is simple and easy to operate, has strong repeatability, and the identification results are stable and reliable. It can more accurately reflect the interaction between Phaseolus vulgaris and sesame varieties. It can lay a good theoretical and technical foundation for the study of the infection mechanism of stem blight fungus, the disease resistance mechanism of sesame and the screening of control agents, and has good scientific research and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The phenotypic characteristics of Yuzhi No. 11 at different stages after root irrigation with infected bacterial solution; a is the 14th day after inoculation; b is the 21st day after inoculation; c is the 28th day after inoculation; d is the 42nd day after inoculation; the plants from left to right in each figure are: water control, 5×10 3 cfu / ml treatment, 1×10 4 cfu / ml treatment and 2×10 4 cfu / ml treatment; Figure 2 The resistance phenotypes of different sesame germplasms on the 28th day after root irrigation with infected bacterial solution; a is 18YM63; b is 18YM61; c is 18YM64; d is 18YM51; e is 18YM45; f is 18YM44; each pot of 1×10 4 The results were repeated three times under each treatment. DETAILED DESCRIPTION

[0011] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental background conditions in the following embodiments are briefly introduced as follows.

[0012] Biomaterials: Sesame variety Yuzhi No. 11, a common and commonly used germplasm material in existing sesame planting, can be obtained through public channels; The germplasm materials such as "18YM43~66" involved in the embodiments are all from the germplasm resource bank owned by the applicant Henan Academy of Agricultural Sciences (the relevant germplasm materials are randomly selected, so the relevant numbers are all self-numbered for record keeping and have no special technical meaning); The highly pathogenic Phaseolus vulgaris strain 0909-4C used in the embodiment was collected and isolated in the early stage by the Fungal Laboratory of the Institute of Plant Protection, Henan Academy of Agricultural Sciences of the applicant. The embodiments only use this as an example for related experiments. When the sesame stem blight resistance identification and evaluation method provided by this application is actually applied, it is not limited to this specific strain, and the main technical concept of this application does not involve the application of this strain.

[0013] Example 1 Taking the sesame variety Yuzhi No. 11 and a highly pathogenic Phaseolus vulgaris strain 0909-4C isolated and collected by the inventors in the early stage as examples, the screening and identification method for stem blight of sesame varieties provided in this application is specifically introduced as follows.

[0014] 1. Preparing sesame samples for testing Select an appropriate amount of healthy and mature seeds of Yuzhi 11, sterilize them, and culture them at 28℃ and 120rpm until the seeds turn white; During the sterilization process, the specific operation is as follows: first treat with 70% alcohol for 30 seconds, then treat with 3% sodium hypochlorite for 10 minutes, and finally rinse with sterile water for 5 times; Then, the white sesame seeds were sown in sterile vermiculite pots, with 10 seeds sown in each pot; After sowing, the plants were regularly irrigated with modified Hoagland nutrient solution and cultured in an artificial climate incubator until they reached the stage of three pairs of true leaves (approximately 30-35 days). The culture conditions were: 28°C, 80% relative humidity, and 15h / 9h light-dark alternation. The modified Hoagland nutrient solution formula is: 945 mg / L calcium nitrate tetrahydrate, 506 mg / L potassium nitrate, 80 mg / L ammonium nitrate, 136 mg / L potassium dihydrogen phosphate, 493 mg / L magnesium sulfate, 2.5 mL iron salt solution and 5 mL trace element solution, pH=6.0; Iron salt solution: 2.78g ferrous sulfate heptahydrate and 3.73g disodium ethylenediaminetetraacetate (EDTA-Na), 500ml distilled water, pH=5.5; Trace element solution: potassium iodide 0.83 mg / l, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L.

[0015] (II) Root inoculation treatment Use the sterile water suspension of Phaseolus vulgaris strain as the infection solution, select the sesame plants that are strong and uniform in growth in step (i) as the experimental samples, and inject the infection solution (50mL) into the base of the sesame stem with a syringe (when injecting the infection solution, ensure that the injection liquid level is not higher than 1 cm from the planting base of the stem), and at the same time, ensure that the solution is evenly sprinkled around the base of the stem of each sesame seedling; After the infection solution was injected, the plants were covered with sterile vermiculite with a thickness of about 1 cm and continued to be cultured in an artificial climate incubator (28°C, relative humidity of 80%, 15h / 9h light and dark alternation). During this period, the plants were watered with modified Hoagland nutrient solution once every 3 days to ensure the nutrients needed for plant growth. Three replicates were set for each treatment. Under the same operation mode, sesame seedlings irrigated with clean water were used as negative controls without inoculation. The sterile aqueous bacterial suspension of the Phaseolus vulgaris strain is prepared as follows: First, the bacterial block of Phaseolus phaseoli (MP) 0909-4C stored at -20°C was inoculated into 300 mL of PDB liquid culture medium and cultured at 28°C and 160 rpm for 3 days; After the culture, filter with 4 layers of sterile gauze, add appropriate amount of sterile water to the filtrate and mix well, then transfer to a food processor for further crushing (to ensure uniform mixing), and finally dilute with sterile water to (as different experimental groups): 5×10 3 cfu / mL, 1×10 4 cfu / ml and 2×10 4 cfu / ml is enough; The PDB liquid culture medium contains 200 g of potato and 20 g of glucose in every 1000 mL of distilled water.

[0016] (III) Identification and evaluation of sesame varieties’ resistance to spot blight, Observe and record the occurrence of stem blight of sesame seedlings after inoculation in step (ii) every day, investigate and record the number of diseased plants and the disease grade every 7 days, calculate the stem blight disease index, and determine the disease resistance of sesame varieties or the pathogenicity of specific strains to sesame based on the index; The calculation formula of stem blight index is: Disease index (DI) = Σ (disease level × corresponding number of diseased plants) / total number of plants.

[0017] The growth and disease conditions of sesame plants in each pot on days 7, 14, 21, 28, 35 and 42 after inoculation and the calculation results of the disease index are shown in Table 1 below.

[0018] Table 1. Changes in disease index of sesame plants treated with different concentrations of MP strains From the results in the table above, we can see that in 5×10 3 cfu / ml, 1×10 4 cfu / ml and 2×10 4 Under the condition of inoculation concentration of cfu / ml, MP strain 0909-4C can infect Yuzhi No. 11 and cause the plant to become sick (part of the actual disease situation comparison is shown in the figure below) Figure 1 as shown).

[0019] Combined with the previous field disease resistance identification experience and the actual growth of sesame, the sesame stem blight classification standards can be further set as follows: Level 0: Good growth without symptoms, normal above-ground stems and leaves, and normal rhizomes; Level 1: The disease occurs at the base of the stem or less than 1 / 4 of the stem, and the leaves wilt slightly; Level 2: 1 / 4-1 / 2 of the stems are diseased, less than 1 / 2 of the leaves are obviously wilted, and the plants are slightly dwarfed; Level 3: More than 1 / 2 of the stems or branches are diseased, more than 1 / 2 of the leaves are obviously wilted, and the plant is significantly dwarfed; Level 4: The entire stem is infected and the plant is severely stunted and close to death.

[0020] Combined with the above disease index (DI), the resistance of different sesame varieties to stem blight can be further divided into five levels, as follows: Level 1: High disease resistance, 0≤DI≤15; Second level: Disease resistance, 15 <DI≤30; Third level: medium resistance, 30 <DI≤50; Level 4: Sickness, 50 <DI≤70; Fifth level: High sensitivity, 70 <DI≤100; In addition, from the above disease index results, it can be seen that under the condition of root irrigation inoculation, the disease index of sesame stem blight is positively correlated with the inoculation concentration. The higher the inoculation concentration, the higher the disease index. From the 7th day to the 28th day after inoculation, the disease index of the test material showed a gradual upward trend and was relatively stable after 28 days. This result can also provide a basis for the inoculation concentration and investigation time in the future.

[0021] Example 2 This example takes the MP strain (0909-4C) as an example and 20 randomly selected sesame germplasms as examples to identify and distinguish the resistance of different sesame germplasms to stem blight. The relevant experimental process is briefly introduced as follows.

[0022] 1. Preparing sesame samples for testing Referring to the operation of Example 1, different sesame germplasm materials were cultured to the three pairs of true leaves stage for use.

[0023] (II) Root inoculation treatment Refer to Example 1 and operate at 1×10 4 cfu / mL of infected bacterial solution was used for root irrigation.

[0024] (III) Identification and evaluation of sesame varieties’ resistance to spot blight, The plant growth and disease status of each germplasm were investigated on the 7th, 14th, 21st and 28th day after inoculation. The statistical results are shown in Table 2. Figure 2 shown.

[0025] Table 2. Changes in disease index of 20 sesame germplasms after treatment with MP strains .

[0026] From the above table, we can see that in 1×10 4 Under the inoculum concentration of cfu / ml, the DI range of the above 20 germplasms was 18.33-100, and the disease resistance levels were different.

[0027] Further combined with the aforementioned "Standards for Classification of Sesame Stem Blight Disease" and "Five Levels of Sesame Varieties' Stem Blight Resistance", it can be seen that: on the 28th day after treatment with MP strain 0909-4C, the disease indexes of 18YM55, 18YM63, 18YM64, and 18YM65 were all 100.00, indicating a high susceptibility level; the disease index of 18YM51 was 18.33, indicating a resistant level; the disease indexes of 18YM44 and 18YM45 were 61.67 and 60.83, indicating a susceptible level.

[0028] This result shows that the above-mentioned resistance identification method can be used to effectively identify and differentiate the resistance of different germplasm resources to sesame stem blight, thereby laying a good technical foundation for the screening of resistant varieties and the breeding of new varieties.

Claims

1. A method for identifying and evaluating resistance to sesame stem blight, characterized in that: The method is used for screening and identifying sesame stem blight resistance germplasm or for evaluating the resistance of a specific sesame germplasm to sesame stem blight, and specifically comprises the following steps:

1. Preparing sesame samples for testing Select an appropriate amount of healthy and mature seeds of the sesame variety to be tested, sterilize them, and culture them until the seeds turn white; Then, sow the white sesame seeds in a container filled with sterile vermiculite; After sowing, sesame seeds were regularly irrigated with modified Hoagland nutrient solution according to their growth conditions until they reached the stage of three pairs of true leaves. (II) Root inoculation treatment Using a sterile aqueous suspension of Phaseolus vulgaris strain as the infection solution, selecting sesame plants that are robust and have uniform growth in step (i) as experimental samples, and injecting the infection solution into the base of the sesame stem with a syringe; After the infection solution is injected, cover with sterile vermiculite about 1 cm thick and continue culturing; Under the same operation mode, sesame seedlings irrigated with clean water were used as negative controls without inoculation.

3. Identification and evaluation of sesame varieties’ resistance to spot blight Observe and record the occurrence of stem blight of sesame seedlings after inoculation in step (ii) every day, record and count the number of diseased plants and the disease grade, calculate the stem blight disease index, and determine the disease resistance of sesame varieties or the pathogenicity of specific strains to sesame based on the index; The calculation formula of stem blight index is: Disease index DI = Σ (disease grade × corresponding number of diseased plants) / total number of plants; The sesame stem blight classification standard is set as follows: Level 0: Good growth without symptoms, normal above-ground stems and leaves, and normal rhizomes; Level 1: The disease occurs at the base of the stem or less than 1 / 4 of the stem, and the leaves wilt slightly; Level 2: 1 / 4-1 / 2 of the stems are diseased, less than 1 / 2 of the leaves are obviously wilted, and the plants are slightly dwarfed; Level 3: More than 1 / 2 of the stems or branches are diseased, more than 1 / 2 of the leaves are obviously wilted, and the plant is significantly dwarfed; Level 4: The entire stem is infected, the plant is severely dwarfed and close to death; Based on the disease index, the resistance of different sesame varieties to stem blight is divided into five levels, as follows: Level 1: High disease resistance, 0≤DI≤15; Second level: Disease resistance, 15 <DI≤30; Third level: medium resistance, 30 <DI≤50; Level 4: Sickness, 50 <DI≤70; Fifth level: High sensitivity, 70 <DI≤100。 2. The method for identifying and evaluating sesame stem blight resistance according to claim 1, characterized in that: In step (i), when culturing sesame seeds, the culturing conditions are: shaking culturing at 25°C-28°C and 100-120 rpm.

3. The method for identifying and evaluating sesame stem blight resistance according to claim 1, characterized in that: In step (i), the modified Hoagland nutrient solution formula is: 945 mg / L calcium nitrate tetrahydrate, 506 mg / L potassium nitrate, 80 mg / L ammonium nitrate, 136 mg / L potassium dihydrogen phosphate, 493 mg / L magnesium sulfate, 2.5 mL iron salt solution and 5 mL trace element solution, pH=6.0; Iron salt solution: 2.78g ferrous sulfate heptahydrate and 3.73g disodium ethylenediaminetetraacetate, 500ml distilled water, pH=5.5; Trace element solution: potassium iodide 0.83 mg / l, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L.

4. The method for identifying and evaluating sesame stem blight resistance according to claim 1, characterized in that: In step (ii), the content of Phaseolus vulgaris strain in the infected bacterial solution is 1×10 3 -2×10 4 cfu / mL; root irrigation volume is 20-50mL.

5. The method for identifying and evaluating sesame stem blight resistance according to claim 1, characterized in that: In step (ii), the culture conditions are: 28°C, 80% relative humidity, and 15h / 9h light / dark alternation.

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