Method for identifying seedling stage resistance of asparagus stem blight

Abstract

The invention relates to a seedling stage resistance identification method for asparagus stem blight, belongs to the technical field of stem blight identification, can rapidly and accurately identify the resistance of asparagus seedlings to stem blight, and is suitable for disease resistance screening in the breeding process of asparagus germplasm resources and new varieties. The disease resistance identification experiment is carried out by adopting an improved indoor artificial spore spraying method in the seedling stage, so that time and labor are saved, the disease resistance of the asparagus in the seedling stage can be well and truly reflected, and the influence of the external environment is small. The method also provides help for experiments under the condition of more materials.

Description

Technical Field

[0001] The invention relates to the technical field of breeding, and in particular to a method for identifying resistance to asparagus stem blight at the seedling stage. Background Art

[0002] Asparagus is tender, delicious and nutritious. It is known as the "king of vegetables" and is also called lazy vegetables because it is easy to grow and manage. However, the incidence of stem blight of asparagus is still very high. The main sites of asparagus stem blight are the stems and leaves, and gradually spread to the stems. In the early stages of the disease, small milky white spots will appear, and then expand to spindles or oval brown spots. As the lesions spread rapidly, the central part turns brown and sunken, forming many small black spots (conidiophores), and the surrounding area turns reddish brown. Infected plants show inhibition at the base of the stem and dry stems and leaves, leading to the rapid death of the entire plant.

[0003] Establishing an efficient and reliable method for identifying resistance to asparagus stem blight is an important basis for breeding and using disease-resistant varieties. At present, there are many methods for identifying resistance to asparagus stem blight in the seedling stage, which are divided into natural identification and artificial inoculation identification. However, identification in the adult stage is greatly affected by the field environment, takes a long time to identify, has poor phenotypic stability, or has complex methods, large workload, and long identification time, which is not suitable for large-scale resistance identification. Summary of the invention

[0004] The present invention aims to solve the above technical problems and provides a method for identifying resistance to asparagus stem blight at the seedling stage.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that it comprises the following steps:

[0007] S1. Asparagus planting:

[0008] Soak the asparagus seeds in 50-54°C hot water for 10-15 minutes, remove and rinse with cold water, and then soak in 40-45% carbendazim WP 1000 times solution for 30-45 hours, take out and germinate at 28°C for 60-70 hours under moisturizing conditions to obtain the germinated asparagus seeds, and then sow;

[0009] S2. Asparagus seedlings:

[0010] Asparagus seedlings with asparagus stem blight were collected for strain isolation;

[0011] S3. Preparation of spore suspension

[0012] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 10-15 days to grow stroma and conidiophores, and the conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, and placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia, and after filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension;

[0013] S4. Cultivation of asparagus seedlings:

[0014] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed. When the asparagus seedlings are 10-12 cm tall, the asparagus seedlings are transplanted into a greenhouse, cultured in light for 4-5 days, and then switched to dark culture for 1-2 days;

[0015] S5. Spraying:

[0016] Formula 1x10 6 spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture;

[0017] S6. Calculation of medical condition:

[0018] The incidence of asparagus stem blight of the asparagus seedlings is investigated, the asparagus stem blight resistance of the asparagus seedlings at the seedling stage is predicted, and the asparagus seedlings are graded according to the severity of the disease and the disease index.

[0019] Preferably, the sowing in the steps S1 and S4 is carried out in a seedling tray, with 1-2 seeds sown in each hole, the germination rate is measured 20-25 days after sowing, and the seedling height is measured 40-55 days after sowing.

[0020] Preferably, the asparagus seedlings in step S2 have a plant height of 18-25 cm and a diameter of 6-10 cm.

[0021] Preferably, the temperature in the greenhouse is 20-25 degrees Celsius.

[0022] Preferably, the disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest level representative value).

[0023] Preferably, the classification is as follows:

[0024] Level 1 high resistance: disease index is 0;

[0025] Level 2 disease resistance: mild disease, 0<disease index ≤ 10;

[0026] Level 3: 10<disease index≤20;

[0027] Level 4 moderate infection: 20<sickness index≤40;

[0028] Level 5: 40<disease index≤60;

[0029] Level 6: Highly susceptible: greater than 60.

[0030] After adopting the above structure, the present invention has the following advantages:

[0031] The invention can quickly and accurately identify the resistance of asparagus seedlings to stem blight, and is suitable for disease resistance screening in the process of asparagus germplasm resources and new variety breeding. This experiment adopts an improved method of indoor artificial spraying of spores in the seedling stage to perform disease resistance identification experiments, which saves time and labor, and can better and truly reflect the disease resistance of asparagus seedlings, and is less affected by the external environment. This method also provides help for experiments with a large number of materials.

[0032] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] none DETAILED DESCRIPTION

[0034] Specific embodiments of the present invention will now be mentioned in detail. Although the present invention is described in conjunction with these specific embodiments, it should be appreciated that it is not intended to limit the present invention to these specific embodiments. On the contrary, these embodiments are intended to cover substitutions, changes or equivalent embodiments that may be included in the spirit and scope of the invention defined by the claims. In the following description, a large number of specific details are set forth in order to provide a comprehensive understanding of the present invention. The present invention can be implemented without some or all of these specific details. In other cases, in order not to make the present invention unnecessarily obscure, well-known process operations are not described in detail.

[0035] When used in conjunction with "including," "methods comprising," or similar language in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] The present invention is further described in detail below in conjunction with the full text.

[0037] In conjunction with the full text, a method for identifying resistance to asparagus stem blight at the seedling stage is characterized in that it comprises the following steps:

[0038] S1. Asparagus planting:

[0039] Soak the asparagus seeds in 50-54°C hot water for 10-15 minutes, remove and rinse with cold water, and then soak in 40-45% carbendazim WP 1000 times solution for 30-45 hours, take out and germinate at 28°C for 60-70 hours under moisturizing conditions to obtain the germinated asparagus seeds, and then sow;

[0040] S2. Asparagus seedlings:

[0041] Asparagus seedlings with asparagus stem blight were collected for strain isolation;

[0042] S3. Preparation of spore suspension

[0043] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 10-15 days to grow stroma and conidiophores, and the conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, and placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia, and after filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension;

[0044] S4. Cultivation of asparagus seedlings:

[0045] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed. When the asparagus seedlings are 10-12 cm tall, the asparagus seedlings are transplanted into a greenhouse, cultured in light for 4-5 days, and then switched to dark culture for 1-2 days;

[0046] S5. Spraying:

[0047] Prepare a spore suspension with a concentration of 1x106 spores / mL, and use a high-pressure or air-blown sprayer to spray the spore suspension onto the asparagus seedlings in step S4, spraying the asparagus seedlings before and after they are transplanted into the greenhouse, and spraying once on the second day of light culture and the first day of dark culture;

[0048] S6. Calculation of medical condition:

[0049] The incidence of asparagus stem blight of the asparagus seedlings is investigated, the asparagus stem blight resistance of the asparagus seedlings at the seedling stage is predicted, and the asparagus seedlings are graded according to the severity of the disease and the disease index.

[0050] In the spraying process in step S5 of the present invention, the operator should maintain a consistent walking speed to avoid missed spraying or repeated spraying. The swing of the spray head should be uniform to ensure that the agent can evenly cover all parts of the asparagus seedlings.

[0051] For asparagus fields with dense canopies, use high-pressure or air-blown sprayers so that the droplets can penetrate the canopy and achieve better spraying effects.

[0052] In high temperature and windy weather conditions, spraying operations should be avoided to prevent the pesticide from being blown away by the wind or affecting the spray effect.

[0053] Record the disease: First, it is necessary to record the occurrence of asparagus stem blight in the field in detail. According to the symptoms of asparagus stem blight, small milky white spots will appear in the early stage, and then expand to spindle or oval brown spots. As the lesions expand rapidly, the central part becomes brown and sunken, forming many small black spots (conidiophores), and the surrounding area becomes reddish brown.

[0054] In order to prevent the distilled water on the culture dish cover from flowing onto the culture dish during the culture process, causing the colonies to form sheets and making it impossible to count or separate, and to reduce the possibility of bacterial contamination, the conidia in the culture dish are scraped off with a glass slide and wrapped with gauze in step S1 of the present invention.

[0055] The sowing in the steps S1 and S4 is performed in a seedling tray, with 1-2 seeds sown in each hole, the germination rate is measured 20-25 days after sowing, and the seedling height is measured 40-55 days after sowing.

[0056] The asparagus seedlings in step S2 have a plant height of 18-25 cm and a diameter of 6-10 cm.

[0057] The temperature in the greenhouse is 20-25 degrees Celsius.

[0058] Statistical analysis of the incidence of various qualities and calculation of the disease index. This step is to evaluate the differences in asparagus's resistance to stem blight. The disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value). According to the disease index, the disease resistance of asparagus seedlings is evaluated. The standard stipulates specific disease resistance identification and resistance evaluation standards to ensure that the results are accurate and reliable.

[0059] The classification is as follows:

[0060] Level 1 high resistance: disease index is 0;

[0061] Level 2 disease resistance: mild disease, 0<disease index ≤ 10;

[0062] Level 3: 10<disease index≤20;

[0063] Level 4 moderate infection: 20<sickness index≤40;

[0064] Level 5: 40<disease index≤60;

[0065] Level 6: Highly susceptible: greater than 60.

[0066] Embodiment 1:

[0067] A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that it comprises the following steps:

[0068] S1. Asparagus planting:

[0069] The asparagus seeds were soaked in 54℃ hot water for 15min, washed out and rinsed with cold water, and then soaked in 45% carbendazim WP 1000 times solution for 45h. After being taken out, they were germinated at 28℃ for 70h under moisturizing conditions to obtain the germinated asparagus seeds, and then sowed in the seedling tray, sowing 2 seeds per hole. The germination rate was measured 25d after sowing, and the seedling height was measured 55d after sowing.

[0070] S2. Asparagus seedlings:

[0071] Asparagus seedlings with asparagus stem blight were collected, with a plant height of 25 cm and a diameter of 10 cm, for strain isolation;

[0072] S3. Preparation of spore suspension

[0073] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 15 days to allow it to grow stroma and conidiophores. The conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia. After filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension.

[0074] S4. Cultivation of asparagus seedlings:

[0075] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed, sowed in a seedling tray, sowed 2 seeds per hole, germination rate 25 days after sowing, and seedling height is measured 55 days after sowing. When the asparagus seedlings are 12 cm tall, the asparagus seedlings are transplanted into a greenhouse, the temperature in the greenhouse is 25 degrees Celsius, light culture is carried out for 5 days, and then switched to dark culture for 2 days;

[0076] S5. Spraying:

[0077] Formula 1x10 6 spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture;

[0078] S6. Calculation of medical condition:

[0079] The incidence of asparagus stem blight of the asparagus seedlings was investigated, the resistance of the asparagus seedlings to asparagus stem blight at the seedling stage was predicted, and the asparagus seedlings were graded according to the severity of the disease and the disease index, wherein the disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves investigated × highest representative value).

[0080] Embodiment 2:

[0081] A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that it comprises the following steps:

[0082] S1. Asparagus planting:

[0083] Soak the asparagus seeds in 50℃ hot water for 10min, take them out and rinse them with cold water, then soak them in 40% carbendazim WP 1000 times solution for 30h, take them out and germinate them at 28℃ for 60h under moisturizing conditions to obtain the germinated asparagus seeds, then sow them in the seedling tray, sow 1-2 seeds per hole, the germination rate will be detected 20-25d after sowing, and the seedling height will be measured 40-55d after sowing;

[0084] S2. Asparagus seedlings:

[0085] Asparagus seedlings with asparagus stem blight were collected, with a plant height of 18 cm and a diameter of 6 cm, for strain isolation;

[0086] S3. Preparation of spore suspension

[0087] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 10 days to allow it to grow stroma and conidiophores. The conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia. After filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension.

[0088] S4. Cultivation of asparagus seedlings:

[0089] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed, sowed in a seedling tray, sowed one seed per hole, germination rate 20 days after sowing, and seedling height is measured 40 days after sowing. When the asparagus seedlings are 10 cm tall, the asparagus seedlings are transplanted into a greenhouse, the temperature in the greenhouse is 20 degrees Celsius, light culture is carried out for 4 days, and then switched to dark culture for 1 day;

[0090] S5. Spraying:

[0091] Formula 1x10 6spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture;

[0092] S6. Calculation of medical condition:

[0093] The incidence of asparagus stem blight of the asparagus seedlings was investigated, the resistance of the asparagus seedlings to asparagus stem blight at the seedling stage was predicted, and the asparagus seedlings were graded according to the severity of the disease and the disease index, wherein the disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves investigated × highest representative value).

[0094] Embodiment three:

[0095] A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that it comprises the following steps:

[0096] S1. Asparagus planting:

[0097] The asparagus seeds were soaked in 52℃ hot water for 12min, taken out and rinsed with cold water, and then soaked in 42% carbendazim WP 1000 times solution for 35h. After being taken out, they were germinated at 28℃ for 63h under moisturizing conditions to obtain the germinated asparagus seeds, and then sowed in the seedling tray, sowing one seed per hole. The germination rate was measured 22d after sowing, and the seedling height was measured 44d after sowing.

[0098] S2. Asparagus seedlings:

[0099] Asparagus seedlings with asparagus stem blight were collected, with a plant height of 22 cm and a diameter of 8 cm, for strain isolation;

[0100] S3. Preparation of spore suspension

[0101] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 12 days to grow stroma and conidiophores. The conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia. After filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension.

[0102] S4. Cultivation of asparagus seedlings:

[0103] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed, sowed in a seedling tray, sowed one seed per hole, germination rate 22 days after sowing, and seedling height is measured 50 days after sowing. When the asparagus seedlings are 11 cm tall, the asparagus seedlings are transplanted into a greenhouse, the temperature in the greenhouse is 22 degrees Celsius, light culture is carried out for 4 days, and then switched to dark culture for 1 day;

[0104] S5. Spraying:

[0105] Formula 1x10 6 spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture;

[0106] S6. Calculation of medical condition:

[0107] The incidence of asparagus stem blight of the asparagus seedlings was investigated, the resistance of the asparagus seedlings to asparagus stem blight at the seedling stage was predicted, and the asparagus seedlings were graded according to the severity of the disease and the disease index, wherein the disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves investigated × highest representative value).

[0108] Embodiment 4:

[0109] A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that it comprises the following steps:

[0110] S1. Asparagus planting:

[0111] The asparagus seeds were soaked in 51℃ hot water for 14min, taken out and rinsed with cold water, and then soaked in 43% carbendazim WP 1000 times solution for 44h. After being taken out, they were germinated at 28℃ for 68h under moisturizing conditions to obtain the germinated asparagus seeds, and then sowed in the seedling tray, sowing one seed per hole. The germination rate was measured 23d after sowing, and the seedling height was measured 51d after sowing.

[0112] S2. Asparagus seedlings:

[0113] Asparagus seedlings with asparagus stem blight were collected, with a plant height of 23 cm and a diameter of 8 cm, for strain isolation;

[0114] S3. Preparation of spore suspension

[0115] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 11 days to grow stroma and conidiophores, and the conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia. After filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension;

[0116] S4. Cultivation of asparagus seedlings:

[0117] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed, sowed in a seedling tray, sowed one seed per hole, germination rate 20 days after sowing, and seedling height is measured 45 days after sowing. When the asparagus seedlings are 12 cm tall, the asparagus seedlings are transplanted into a greenhouse, the temperature in the greenhouse is 22 degrees Celsius, light culture is performed for 5 days, and then switched to dark culture for 2 days;

[0118] S5. Spraying:

[0119] Formula 1x10 6 spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture;

[0120] S6. Calculation of medical condition:

[0121] The incidence of asparagus stem blight of the asparagus seedlings was investigated, the resistance of the asparagus seedlings to asparagus stem blight at the seedling stage was predicted, and the asparagus seedlings were graded according to the severity of the disease and the disease index, wherein the disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves investigated × highest representative value).

[0122] Embodiment five:

[0123] A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that it comprises the following steps:

[0124] S1. Asparagus planting:

[0125] The asparagus seeds were soaked in 54℃ hot water for 14min, washed out and rinsed with cold water, and then soaked in 43% carbendazim WP 1000 times solution for 41h. After being taken out, they were germinated at 28℃ for 66h under moisturizing conditions to obtain the germinated asparagus seeds, and then sowed in the seedling tray, sowing one seed per hole. The germination rate was measured on the 25th day after sowing, and the seedling height was measured on the 51st day.

[0126] S2. Asparagus seedlings:

[0127] Asparagus seedlings with asparagus stem blight were collected, with a plant height of 19 cm and a diameter of 9 cm, for strain isolation;

[0128] S3. Preparation of spore suspension

[0129] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 13 days to grow stroma and conidiophores, and the conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia. After filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension;

[0130] S4. Cultivation of asparagus seedlings:

[0131] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed, sowed in a seedling tray, sowed one seed per hole, germination rate 25 days after sowing, and seedling height is measured 52 days after sowing. When the asparagus seedlings are 11 cm tall, the asparagus seedlings are transplanted into a greenhouse, the temperature in the greenhouse is 25 degrees Celsius, light culture is carried out for 5 days, and then switched to dark culture for 1 day;

[0132] S5. Spraying:

[0133] Formula 1x10 6 spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture;

[0134] S6. Calculation of medical condition:

[0135] The incidence of asparagus stem blight of the asparagus seedlings was investigated, the resistance of the asparagus seedlings to asparagus stem blight at the seedling stage was predicted, and the asparagus seedlings were graded according to the severity of the disease and the disease index, wherein the disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves investigated × highest representative value).

[0136] Embodiment six:

[0137] A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that it comprises the following steps:

[0138] S1. Asparagus planting:

[0139] The asparagus seeds were soaked in 52℃ hot water for 11min, taken out and rinsed with cold water, and then soaked in 43% carbendazim WP 1000 times solution for 38h. After being taken out, they were germinated at 28℃ for 61h under moisturizing conditions to obtain the germinated asparagus seeds, and then sowed in the seedling tray, sowing one seed per hole. The germination rate was measured 21d after sowing, and the seedling height was measured 44d after sowing.

[0140] S2. Asparagus seedlings:

[0141] Asparagus seedlings with asparagus stem blight were collected, with a plant height of 19 cm and a diameter of 7 cm, for strain isolation;

[0142] S3. Preparation of spore suspension

[0143] The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 12 days to grow stroma and conidiophores. The conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia. After filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension.

[0144] S4. Cultivation of asparagus seedlings:

[0145] The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed, sowed in a seedling tray, sowed one seed per hole, germination rate 20 days after sowing, seedling height measured 40 days, when the asparagus seedlings are 11 cm tall, transplanted into a greenhouse, the temperature in the greenhouse is 25 degrees Celsius, light culture for 5 days, and then switched to dark culture for 1 day;

[0146] S5. Spraying:

[0147] Formula 1x10 6 spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture;

[0148] S6. Calculation of medical condition:

[0149] The incidence of asparagus stem blight of the asparagus seedlings was investigated, the resistance of the asparagus seedlings to asparagus stem blight at the seedling stage was predicted, and the asparagus seedlings were graded according to the severity of the disease and the disease index, wherein the disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves investigated × highest representative value).

[0150] Embodiment 1, embodiment 2, embodiment 3, embodiment 4, embodiment 5 and embodiment 6 are selected in sequence

[0151] Asparagus varieties including New Dynasty F1 (USA), Jinglu Lu No. 1 (Asparagus Center of Beijing Academy of Agricultural and Forestry Sciences), TC (Weifang Academy of Agricultural Sciences, Shandong), SD09-6 (Weifang Academy of Agricultural Sciences, Shandong), 16523-B (Japan), and Dali Tianmendong were used as materials. The resistance of asparagus to stem blight at the seedling stage was identified according to the methods provided in Examples 1, 2, 3, 4, 5, and 6. The results are detailed in the data in Table 1.

[0152] Table 1

[0153] variety Disease index Resistance level Embodiment 1 New Dynasty F1 22.6 Level 4 Embodiment 2 Jinglu No.1 13.8 Level 3 Embodiment 3 TC 8.6 Level 2 Embodiment 4 SD09-6 11.6 Level 3 Embodiment 5 16523-B 23.8 Level 4 Embodiment 6 Dali Asparagus 0 Level 1

[0154] From the data in Table 1, it can be seen that among the tested varieties, Dali Asparagus cochinchinensis has the strongest resistance to stem blight at the seedling stage, while 16523-B has the worst disease resistance.

[0155] The invention can quickly and accurately identify the resistance of asparagus seedlings to stem blight, and is suitable for disease resistance screening in the process of asparagus germplasm resources and new variety breeding. This experiment adopts an improved method of indoor artificial spraying of spores in the seedling stage to perform disease resistance identification experiments, which saves time and labor, and can better and truly reflect the disease resistance of asparagus seedlings, and is less affected by the external environment. This method also provides help for experiments with a large number of materials.

[0156] The above examples prove that the method for identifying asparagus seedling resistance provided by the present invention can identify the seedling resistance of different varieties of asparagus seedlings to stem blight according to corresponding standards, thereby unifying the identification standards and achieving the purpose of objectively evaluating the seedling resistance of different varieties of asparagus seeds to stem blight.

[0157] The present invention and its embodiments are described above, and such description is not restrictive. What is shown in the full text is only one of the embodiments of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A method for identifying resistance to asparagus stem blight at the seedling stage, characterized in that: The following steps are involved: S1. Asparagus planting: Soak the asparagus seeds in 50-54°C hot water for 10-15 minutes, remove and rinse with cold water, and then soak in 40-45% carbendazim WP 1000 times solution for 30-45 hours, take out and germinate at 28°C for 60-70 hours under moisturizing conditions to obtain the germinated asparagus seeds, and then sow; S2. Asparagus seedlings: Asparagus seedlings with asparagus stem blight were collected for strain isolation; S3. Preparation of spore suspension The asparagus stem blight strain was cultured on a potato dextrose agar medium plate for 10-15 days to grow stroma and conidiophores, and the conidiophores in the culture dish were scraped off with a glass slide, wrapped with gauze, and placed in a glass mortar with a diameter of 8 cm, 30 mL of sterile water was added, and the conidia were gently crushed with a pestle to release the conidia to obtain conidia, and after filtering through three layers of gauze, the spore concentration was determined with a hemocytometer, and the concentration was adjusted by adding sterile water to obtain a spore suspension; S4. Cultivation of asparagus seedlings: The asparagus seeds in step S1 are induced, the conidia in step S3 are added to the asparagus seeds, and then sowed. When the asparagus seedlings are 10-12 cm tall, the asparagus seedlings are transplanted into a greenhouse, cultured in light for 4-5 days, and then switched to dark culture for 1-2 days; S5. Spraying: Formula 1x10 6 spores / mL concentration of spore suspension, using a high pressure or air-assisted spraying device to spray the spore suspension onto the asparagus seedlings in step S4, spraying before and after the asparagus seedlings are transplanted into the greenhouse, spraying once on the second day of light culture and the first day of dark culture; S6. Calculation of medical condition: The incidence of asparagus stem blight of the asparagus seedlings is investigated, the asparagus stem blight resistance of the asparagus seedlings at the seedling stage is predicted, and the asparagus seedlings are graded according to the severity of the disease and the disease index.

2. The method for identifying resistance to asparagus stem blight at the seedling stage according to claim 1, characterized in that: The sowing in the steps S1 and S4 is performed in a seedling tray, with 1-2 seeds sown in each hole, the germination rate is measured 20-25 days after sowing, and the seedling height is measured 40-55 days after sowing.

3. The method for identifying resistance to asparagus stem blight at the seedling stage according to claim 1, characterized in that: The asparagus seedlings in step S2 have a plant height of 18-25 cm and a diameter of 6-10 cm.

4. The method for identifying resistance to asparagus stem blight at the seedling stage according to claim 1, characterized in that: The temperature in the greenhouse is 20-25 degrees Celsius.

5. The method for identifying resistance to asparagus stem blight at the seedling stage according to claim 1, characterized in that: The disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves investigated × highest level representative value).

6. The method for identifying resistance to asparagus stem blight at the seedling stage according to claim 1, characterized in that: The classification is as follows: Level 1 high resistance: disease index is 0; Level 2 disease resistance: mild disease, 0<disease index ≤ 10; Level 3: 10<disease index≤20; Level 4 moderate infection: 20<sickness index ≤ 40; Level 5: 40<disease index≤60; Level 6: Highly susceptible: greater than 60.