Method for identifying peanut crown rot resistance at seedling stage

Abstract

The invention discloses a method for identifying peanut crown rot resistance at seedling stage, comprising: (1) culturing Aspergillus niger pathogen AN1 on a PDA plate and a wheat grain culture medium to prepare an inoculum; (2) culturing seeds that have turned white after indoor germination in an Aspergillus niger spore suspension overnight; (3) inserting the infected seeds into sterile soil for culturing; when the cotyledons just open and the first true leaves are exposed, burying the wheat grain inoculum uniformly around the gap between the embryo and the cotyledons of the peanut stem base; (4) culturing for about 1 week after inoculation, grading according to the degree of disease occurrence, calculating the disease index, and evaluating resistance. The invention provides a method for identifying peanut crown rot resistance at seedling stage that is simple to operate, accurate, efficient, and stable, and can provide important technical support for the future screening of disease-resistant peanut germplasm resources and disease-resistant breeding research, thereby laying an important research foundation for the effective prevention and control of peanut crown rot at seedling stage.

Description

Technical Field

[0001] The invention relates to a method for identifying peanut crown rot resistance, in particular to a method for identifying peanut crown rot resistance at the seedling stage. Background Art

[0002] Peanut (Arachis hypogaea L.) is an important economic and oil crop in my country. It can be used as an oil crop, processed as protein and eaten, and can also be exported to earn foreign exchange. With the acceleration of the peanut industrialization process, the huge oilseed processing industry has an increasing demand for peanuts, and therefore, there are higher requirements for peanut yield and disease resistance. Peanut diseases not only affect the quality of peanuts, but have also been a limiting factor in increasing yields. In recent years, with the adjustment of agricultural structure, the continuous increase in crop planting, and the return of straw to the field, peanut crown rot has become more and more serious and has gradually become the main disease in peanut production. Peanut crown rot is a soil-borne disease that mainly occurs in the seedling stage. It is difficult to prevent and control it using pesticide seed dressing and biological control. Therefore, breeding and application of disease-resistant varieties are the most economical and effective prevention and control methods.

[0003] Peanut crown rot is a disease that has become more serious in recent years. There is currently no method to identify peanut crown rot resistance at the seedling stage, which seriously restricts the breeding of peanut crown rot-resistant varieties. Therefore, it is urgently necessary to study and establish a method to identify peanut crown rot resistance at the seedling stage. Summary of the invention

[0004] The invention aims to provide a method for identifying the resistance of peanut to crown rot at the seedling stage.

[0005] In order to achieve the purpose of the present invention, the present invention provides a method for identifying the resistance of peanut to crown rot at the seedling stage, comprising the following steps:

[0006] (1) Preparation of peanut crown rot inoculum: The isolated and purified Aspergillus niger was transferred to a PDA plate and cultured in the dark at 28-30°C for 2-3 days, and then transferred to a wheat grain culture medium and cultured in the dark at 28-30°C for 4-5 days. The resulting infected wheat grains were the peanut crown rot inoculum. Alternatively, after culturing for 4-5 days until the mycelium covered the wheat grains, the infected wheat grains (inoculum) were aseptically transferred to a sterilized non-woven bag or kraft paper bag, sealed, and stored at 4°C for later use;

[0007] (2) Seed germination: Soak the seeds of the peanut variety to be tested in warm water at about 30-40°C for 6-7 hours, take them out, wrap them with wet gauze, and place them in an incubator at 27-30°C in the dark for 1-2 days. During this period, spray water frequently to keep the gauze moist, and germinate until the seeds turn white.

[0008] (3) Seed infection: Place the germinated white seeds in a 6.0×10 7 ~12.0×10 7 100 / ml Aspergillus niger spore suspension, cultured overnight;

[0009] The preparation method of the Aspergillus niger spore suspension comprises: transferring the separated and purified Aspergillus niger to PDA, culturing in the dark at 28-30° C., waiting for the mycelium to grow all over the plate (about 6-7 days), repeatedly washing and blowing the colony with sterile water by a pipette, filtering with three layers of sterile gauze, counting the number of spores with a hemocytometer under a microscope, and adjusting the concentration of the spore suspension to 6.0×10 7 ~12.0×10 7 Pieces / m1.

[0010] (4) Pathogen inoculation: The infected white seeds were sown in sterile soil and cultured in the dark. When the cotyledons just opened and the first true leaf was exposed, the inoculum from step (1) was evenly buried in the gap between the embryo and cotyledons at the base of the peanut stem. Each plant was inoculated with 20 infected wheat grains (with a bacterial load of 3.0×10 5 ~10.0×10 6 CFU / grain), water every day for 3 consecutive days to keep the soil moist; after inoculation, the incubator conditions were controlled at: 35-38°C, 13h of light and 28-30°C, 11h of darkness, humidity was kept above 95%, and the light intensity was 80000-100000lx;

[0011] (5) After 5 to 10 days of cultivation, observe the disease status of the peanut seedlings and determine the disease grade of each seedling according to the following standards;

[0012] The incidence level is divided into 0-5 levels, and the specific classification standards are as follows:

[0013] Level 0: There are no lesions on the cotyledons and stem bases, and no disease symptoms;

[0014] Level 1: Only the cotyledons have slight lesions;

[0015] Level 2: There are lesions at the base of the stem, but the lesions do not exceed 1 / 3 of the base of the stem (i.e., the area of ​​the lesions does not exceed 1 / 3 of the area of ​​the base of the stem);

[0016] Level 3: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 3 of the base of the stem, but does not exceed 1 / 2 of the base of the stem;

[0017] Level 4: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 2 of the base of the stem;

[0018] Level 5: The entire seed kernel and cotyledons rot or the entire seedling dies;

[0019] Then calculate the disease index of the peanut variety to be tested according to the disease level of each peanut seedling, disease index (DI) = [Σ (number of diseased plants at each level × corresponding disease level) / (total number of plants × highest disease level)] × 100%

[0020] (6) Classifying the resistance level according to the disease index and identifying the disease resistance of the peanut variety to be tested; the classification standard of the resistance level is:

[0021] Immunity (I): Disease index (DI) ≤ 5%;

[0022] High resistance (HR): 5%<disease index (DI) ≤15%;

[0023] Disease resistance (R): 15%<disease index (DI) ≤ 25%;

[0024] Moderate resistance (MR): 25%<disease index (DI) ≤ 45%;

[0025] Moderate cold (MS): 45%<disease index (DI) ≤ 65%;

[0026] Susceptible (S): 65%<disease index (DI) ≤ 75%;

[0027] High sensitivity (HS): Disease index (DI) > 75%.

[0028] Furthermore, in step (1), the isolated and purified Aspergillus niger is transferred to a PDA plate, cultured in the dark at 28-30° C. for 2-3 days, fresh bacterial blocks with a size of 5-10 mm at the edge of the colony are picked, and then transferred to a wheat grain culture medium for culture.

[0029] Furthermore, in step (2), peanut seeds with bright color and full seeds are selected for germination.

[0030] Furthermore, in step (3), the germinated white seeds are placed in a suspension of Aspergillus niger spores and cultured in the dark at 30-35° C. for 24-36 hours.

[0031] The Aspergillus niger used for inoculation in the present invention is Aspergillus niger AN1 separated and purified from a typical diseased plant with basal rot in the seedling stage in the main peanut producing area. Aspergillus niger AN1 has been deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postal code 430072, deposit number CCTCC NO: M2023126, and deposit date February 15, 2023.

[0032] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0033] (I) The method for identifying crown rot resistance in peanut seedlings provided by the present invention is to directly inoculate and cause the disease indoors, which is not affected by other conditions in the field. The identification is accurate and reliable, and is not limited by the peanut growing season, and the identification can be done at any time.

[0034] (ii) Simple operation, fast onset, low cost and high efficiency. The identification methods used in the present invention are all conventional laboratory operation methods, which are low in cost and short in time, and the identification results can be obtained in about ten days.

[0035] (3) This identification method can quickly identify the crown rot resistance of peanut germplasm materials at the seedling stage, screen out breeding materials with resistance, eliminate non-disease-resistant parents or intermediate breeding materials, reduce the breeding workload, and has good application prospects for the cultivation of disease-resistant peanut varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The inoculation symptoms of different peanut plants at the stem base in the preferred embodiment of the present invention are shown in Figure 1. From left to right, they are Z651, Z639, Z600, Z644, Z646, Z636, and Z641. DETAILED DESCRIPTION

[0037] The present invention provides a method for rapid identification of crown rot resistance in peanut seedling stage, comprising the following steps:

[0038] 1. Inoculum preparation: The Aspergillus niger AN1 (preservation number CCTCC NO: M 2023126) pathogen isolated and purified from typical diseased plants with crown rot in the main peanut producing areas was transferred to a PDA plate and cultured in the dark at 28-30°C for 2-3 days. Fresh fungus blocks with a size of 5 mm on the edge of the colony were picked and inoculated on the wheat grain culture medium. The culture was kept at 28-30°C in the dark for 4-5 days until the mycelium covered the wheat grains. The infected wheat grains (inoculum) were transferred to sterile non-woven bags or kraft paper bags under sterile conditions, sealed, and stored at 4°C for later use.

[0039] 2. Seed germination: Select peanut seeds with bright color and full grains for germination for 1 to 2 days (soak in warm water at about 30 to 40 degrees Celsius for 6 to 7 hours, take out and wrap them with moist gauze and place them in an incubator at 27 to 30 degrees Celsius. After 1 to 2 days, select seeds with uniform white length).

[0040] 3. Seed infection: Place the germinated white seeds in (6.0~12.0)×10 7 Incubate in a suspension of Aspergillus niger spores / ml at 30-35°C overnight for 24-36 hours.

[0041] 4. Pathogen inoculation: Insert the fully infected white seeds into sterile soil for cultivation, and then bury the grain inoculum evenly around the gap between the embryo and cotyledons at the base of the peanut stem. Water every day for 3 consecutive days to keep the soil moist.

[0042] 5. Observe and record the disease situation, classify it according to the severity of the disease, calculate the disease index, and conduct resistance evaluation.

[0043] In the above method, in step 3, the time for wheat grain inoculation is when the cotyledons have just opened and the first true leaf is exposed, and the inoculation site is around the gap between the embryo at the base of the plant stem and the cotyledons.

[0044] In the aforementioned method, in step 4, the incubator temperature is controlled at 35-38°C, 13h of light and 28-30°C, 11h of darkness, alternating, and the humidity is maintained above 95%, and the light intensity is 80000-1000001x.

[0045] In the above method, in step 5, the extremely susceptible control variety YH1 in the field all became ill (about 1 week after wheat grain inoculation), and the disease situation at the base of the plant stem was observed and recorded.

[0046] In the above method, step 5 includes the following operations:

[0047] The disease is classified according to the severity of the disease, which is divided into 0-5 levels. The specific classification standards are:

[0048] Level 0: There are no lesions on the cotyledons and stem bases, and no disease symptoms;

[0049] Level 1: Only the cotyledons have slight lesions;

[0050] Level 2: There are lesions at the base of the stem, but the lesions do not exceed 1 / 3 of the base of the stem;

[0051] Level 3: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 3 of the base of the stem, but does not exceed 1 / 2 of the base of the stem root;

[0052] Level 4: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 2 of the base of the stem;

[0053] Level 5: The entire seed kernel and cotyledons rot or the entire seedling dies.

[0054] Disease index (DI) = [Σ(number of diseased plants at each level × disease level) / (total number of plants surveyed × highest disease level)] × 100%

[0055] 6. Classify the resistance level according to the disease index and identify the disease resistance of the peanut; the resistance level classification standard is:

[0056] Immunity (I): Disease index (DI) ≤ 5%;

[0057] High resistance (HR): 5%<disease index (DI) ≤15%;

[0058] Disease resistance (R): 15% < disease index (DI) ≤ 25%:

[0059] Moderate resistance (MR): 25%<disease index (DI) ≤ 45%;

[0060] Moderate cold (MS): 45%<disease index (DI) ≤ 65%;

[0061] Susceptible (S): 65%<disease index (DI) ≤ 75%;

[0062] High sensitivity (HS): Disease index (DI) > 75%.

[0063] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0064] The culture medium used in the following examples is:

[0065] PDA plate: 200 g potato, 20 g agar, 18 g glucose, 1 L distilled water.

[0066] Preparation method: Wash, peel and slice the potatoes, add distilled water, bring to a boil and cook for about 25 minutes, filter through double-layer gauze, add agar and glucose, heat to melt, then add distilled water to 1L, and sterilize at 120℃ for 20 minutes.

[0067] Wheat grain culture medium: 1kg wheat grains, 0.1-0.5kg fermented air-dried peanut straw, 10g calcium carbonate, and 10g gypsum powder.

[0068] Preparation method: Select normal, plump and germ-free wheat grains (average grain size 3.5mm), rinse with clean water 2 to 3 times, soak in 1% lime water for 10 to 15 hours. When the temperature is low, the soaking time can be extended by 3 to 5 hours. Take out the wheat grains and put them in a pot to boil. Continue to cook until the wheat grains are cooked through and do not bloom. Take them out in time and spread them out to dry in the sun to drain the surface moisture. When the moisture content of the wheat grains is about 50% (the wheat grains are not sticky), put them away in time, add fermented and air-dried peanut straw, calcium carbonate and gypsum powder in proportion, stir evenly, divide them into culture bottles, and sterilize them in a high-pressure sterilizer at 120℃ for 2 to 2.5 hours.

[0069] Among them, the preparation method of fermented and air-dried peanut straw is as follows: take 10kg of peanut straw, clean it, cut it in the middle and chop it into 3cm size, and then blow dry the surface moisture; take 20g of fermentation liquid (Nongshengle straw fermentation liquid), add 3.5L of water and stir thoroughly; mix the mixed liquid with 10kg of chopped peanut straw evenly, put it in a plastic basin and compact it, wrap it with plastic wrap to make it airtight, ferment it anaerobically at 28°C for 7 days, and then dry it for storage.

[0070] Nongshengle straw fermentation liquid was purchased from Anhui Nong Leyuan Agricultural Technology Service Co., Ltd. It contains more than 80 kinds of microorganisms from ten genera, including lactic acid bacteria, yeast, Bacillus, photosynthetic bacteria, filamentous fungi, etc., and the effective live bacteria count is ≥20 billion CFU / mL.

[0071] Example 1 Rapid identification method for peanut crown rot resistance at seedling stage

[0072] The peanut varieties Yuanza 9102 (highly resistant), WH14 (medium resistant), Baisha 1016 (susceptible) and YH1 (highly susceptible) with different resistance in the field were used as test materials. The peanut seed materials were provided by the Peanut Germplasm Creation and Application Team of the Crop Research Institute of Anhui Academy of Agricultural Sciences.

[0073] Peanut crown rot pathogen strain and source: Aspergillus niger strain AN1 (preservation number CCTCC NO: M 2023126) was isolated and purified from diseased plants in an area with severe peanut crown rot in Anhui Province.

[0074] Specific steps:

[0075] (1) Preparation of wheat grain inoculum: Transfer the preserved Aspergillus niger pathogen to a PDA plate, culture it in the dark at 30°C for 3 days, pick a 5 mm fresh bacterial block from the edge of the colony, inoculate it on the wheat grain culture medium, and culture it in the dark at 30°C for 5 days until the mycelium covers the wheat grains. Transfer the infected wheat grains (inoculum) to a sterilized non-woven bag or kraft paper bag under aseptic conditions, seal it, and store it at 4°C for later use.

[0076] (2) Peanut seeds with bright colors and full seeds were soaked in warm water at about 30°C for 6 hours. After being fished out, they were wrapped in moist gauze and placed in an incubator at 30°C for incubation. After 2 days, 30 seeds with the same length of whitening seeds were selected from each material.

[0077] (3) The above 30 seeds were placed in a spore suspension of peanut basal rot pathogen AN1 and cultured at 30°C overnight for 24 hours.

[0078] The preparation method of the spore suspension of peanut basal rot pathogen AN1 includes: transferring the separated and purified Aspergillus niger AN1 to PDA, culturing in the dark at 28-30°C, waiting for the hyphae to grow all over the plate (about 6-7 days), repeatedly washing and blowing the colony with sterile water by a pipette, filtering with three layers of sterile gauze, counting the number of spores with a hemocytometer under a microscope, and adjusting the concentration of the spore suspension to about 8.0×10 7 Pieces / mL.

[0079] (4) The 30 seeds fully infected were divided into three replicates (10 seeds in each), and were placed in sterile soil for dark culture. When the cotyledons just opened and the first true leaf was exposed, wheat grains inoculated with pathogens were evenly buried around the gap between the peanut embryo and cotyledons. Each plant was inoculated with 20 infected wheat grains (with a bacterial load of about 6.0×10 6 CFU / grain), water every day for 3 consecutive days to keep the soil moist. The incubator temperature was controlled at: 38°C, 13h of light and 30°C, 11h of darkness, and the humidity was kept above 95%. The light intensity was 1000001x.

[0080] (5) When all the susceptible control strains YH1 become diseased (about 1 week after inoculation), observe the disease situation, grade them according to the severity of the disease, calculate the disease index, and evaluate the resistance. The disease grade is divided into 0-5 levels, and the specific classification criteria are:

[0081] Level 0: There are no lesions on the cotyledons and stem bases, and no disease symptoms;

[0082] Level 1: Only the cotyledons have slight lesions;

[0083] Level 2: There are lesions at the base of the stem, but the lesions do not exceed 1 / 3 of the base of the stem;

[0084] Level 3: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 3 of the base of the stem, but does not exceed 1 / 2 of the base of the stem;

[0085] Level 4: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 2 of the base of the stem;

[0086] Level 5: The entire seed kernel and cotyledons rot or the entire seedling dies.

[0087] Disease index (DI) = [Σ(number of diseased plants at each level × disease level) / (total number of plants surveyed × highest disease level)] × 100%

[0088] The disease index was calculated according to the formula, and the average of three repeated values ​​was taken as the final disease index. The resistance level was divided according to the final disease index to identify the disease resistance of peanut varieties. The results are shown in Table 1. The classification standard of the resistance level is:

[0089] Immunity (I): Disease index (DI) ≤ 5%;

[0090] High resistance (HR): 5%<disease index (DI) ≤15%;

[0091] Disease resistance (R): 15%<disease index (DI) ≤ 25%;

[0092] Moderate resistance (MR): 25%<disease index (DI) ≤ 45%;

[0093] Moderate cold (MS): 45%<disease index (DI) ≤ 65%;

[0094] Susceptible (S): 65%<disease index (DI) ≤ 75%;

[0095] High sensitivity (HS): Disease index (DI) > 75%.

[0096] Table 1 Results of identification of peanut varieties' resistance to crown rot at seedling stage

[0097]

[0098] This method was used to determine the crown rot resistance of different peanut varieties, and the disease index was consistent with the known crown rot resistance of the variety.

[0099] Example 2 Practical application of the rapid identification method for peanut crown rot resistance at the seedling stage

[0100] The method of Example 1 was used to identify the resistance of 16 peanut germplasm materials such as Z600 to crown rot at the seedling stage. All peanut germplasm seeds were provided by the peanut germplasm creation and application team of the Crop Research Institute of Anhui Academy of Agricultural Sciences. The results are shown in Table 2, which shows that there are obvious differences in the resistance of different peanut varieties to crown rot at the seedling stage, and the resistance identification results are consistent with the actual resistance results shown in the field.

[0101] Table 2 Identification results of crown rot resistance in different peanut varieties at seedling stage

[0102]

[0103] Symptoms of inoculation at the base of the stems of different peanut plants Figure 1 .

[0104] Example 3 Effects of different vaccination methods on disease incidence

[0105] Peanut varieties Yuanza 9102 (highly resistant), WH14 (medium resistant), Baisha 1016 (susceptible) and YH1 (highly susceptible) with different field resistance performance were used as test materials, and the spore suspension and wheat grain inoculum in Example 1 were used as inoculum. Three inoculation treatments were set: direct injection of spore suspension into the cotyledon gap at the seedling stage (treatment 1), burying the wheat grain inoculum into the gap between the embryo and cotyledon (treatment 2), and first infecting seeds with spore suspension and then inoculating wheat grains at the seedling stage (treatment 3).

[0106] The results showed that only the inoculation method used in treatment 3 showed obvious differences between resistant and susceptible varieties. In treatment 1, the four varieties were basically disease-free (DI < 5%); in treatment 2, the susceptible varieties developed disease later and were easily affected by other pathogens. All varieties were seriously diseased, and the identification results were inaccurate.

[0107] Example 4 Effect of different inoculation periods on disease incidence

[0108] Peanut varieties Yuanza 9102 (highly resistant), WH14 (medium resistant), Baisha 1016 (susceptible) and YH1 (highly susceptible) with different field resistance performance were used as test materials, and the spore suspension and wheat grain inoculum in Example 1 were used as inoculum. Wheat grain inoculation was divided into three periods: cotyledon stage, first true leaf stage (seedling stage), and seedling stage.

[0109] The results showed that after inoculation at the cotyledon stage and the first true leaf stage (seedling stage), the disease index (DI) between highly resistant and highly susceptible varieties was significantly different; at the seedling stage, due to the increase in seedling age and the increase in disease resistance of peanut seedlings, the disease situation of each variety was relatively mild, and the difference in disease index (DI) between resistant and susceptible varieties was not obvious, and it was difficult to distinguish between resistant and susceptible varieties. The cotyledons of highly susceptible and susceptible varieties rotted seriously at the cotyledon stage, and the disease index was very high, making it difficult to distinguish between highly susceptible and susceptible varieties; at the first true leaf stage (seedling stage), the disease index (ID) of highly resistant, moderately resistant, susceptible and highly susceptible varieties was significantly different, which is the best period for resistance identification (Table 3)

[0110] Table 3 Effect of different vaccination periods on the incidence of disease

[0111]

[0112]

[0113] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for identifying peanut crown rot resistance at the seedling stage, characterized in that: The following steps are involved: (1) Preparation of peanut crown rot inoculum: Separate and purify Aspergillus niger ( Aspergillus niger ), transferred to PDA plate, cultured in the dark at 28-30℃ for 2-3 days, then transferred to wheat grain culture medium, cultured in the dark at 28-30℃ for 4-5 days, the obtained wheat grains with bacteria are the peanut crown rot inoculum; (2) Seed germination: Soak the seeds of the peanut variety to be tested in warm water at 30-40°C for 6-7 hours, take them out, wrap them with wet gauze and place them in an incubator at 27-30°C for 1-2 days in the dark. During this period, spray water to keep the gauze moist and germinate until the seeds turn white. (3) Seed infection: Place the germinated white seeds in a 6.0×10 7 ~12.0×10 7 100 / ml Aspergillus niger spore suspension, cultured overnight; The preparation method of the Aspergillus niger spore suspension comprises: transferring the separated and purified Aspergillus niger to PDA, culturing in the dark at 28-30°C, and repeatedly washing and blowing the colonies with sterile water by a pipette until the mycelium covers the entire plate, and then filtering with three layers of sterile gauze, counting the number of spores with a hemocytometer under a microscope, and adjusting the concentration of the spore suspension to 6.0×10 7 ~12.0×10 7 Pieces / ml; (4) Pathogen inoculation: sow the infected white seeds in sterile soil for dark culture. When the cotyledons just open and the first true leaf is exposed, evenly bury the inoculum of step (1) around the gap between the embryo and cotyledons of the peanut stem. Inoculate 20 infected wheat grains per plant. Water every day for 3 consecutive days to keep the soil moist. After inoculation, the incubator conditions are controlled at: 35-38°C, 13h of light and 28-30°C, 11h of darkness, and the humidity is kept above 95%; the light intensity is 80,000-100,000 lx. (5) After 5-10 days of cultivation, observe the disease status of the peanut seedlings and determine the disease grade of each seedling according to the following standards; The incidence level is divided into 0-5 levels, and the specific classification standards are as follows: Level 0: There are no lesions on the cotyledons and stem bases, and no disease symptoms; Level 1: Only the cotyledons have slight lesions; Level 2: There are lesions at the base of the stem, but the lesions do not exceed 1 / 3 of the base of the stem; Level 3: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 3 of the base of the stem, but does not exceed 1 / 2 of the base of the stem; Level 4: There are lesions at the base of the stem, and the size of the lesions exceeds 1 / 2 of the base of the stem; Level 5: The entire seed kernel and cotyledons rot or the entire seedling dies; Then calculate the disease index of the peanut variety to be tested according to the disease level of each peanut seedling. Disease index = [∑(number of diseased plants at each level × corresponding disease level) / (total number of plants × highest disease level)] × 100% (6) Classifying the resistance level according to the disease index and identifying the disease resistance of the peanut variety to be tested; the classification standard of the resistance level is: Immunity: disease index ≤5%; High resistance: 5%<disease index≤15%; Disease resistance: 15%<disease index≤25%; Medium resistance: 25%<disease index≤45%; Moderate cold: 45%<disease index≤65%; Infectious disease: 65%<disease index≤75%; High sensitivity (HS): Disease index (DI)> 75%, among which, The Aspergillus niger is Aspergillus niger AN1, the deposit number is CCTCC NO: M 2023126.

2. The method according to claim 1, characterized in that Step (1) The isolated and purified Aspergillus niger was transferred to a PDA plate and cultured in the dark at 28-30°C for 2-3 days, fresh bacterial blocks with a size of 5-10 mm at the edge of the colony were picked and then transferred to a wheat grain culture medium for culture.

3. The method according to claim 1, characterized in that Step (2) Select peanut seeds with bright color and full seeds for germination.

4. The method according to claim 1, characterized in that: Step (3) placing the germinated white seeds into a suspension of Aspergillus niger spores and culturing them in the dark at 30-35° C. for 24-36 hours.

5. The method according to claim 1, characterized in that The bacterial load of the infected wheat grains in step (4) is 3.0×10 5 ~10.0×10 6 CFU / particle.

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