An indoor inoculation and identification method for peanut net spot disease based on rooted detached leaves

Abstract

The present invention relates to an indoor inoculation and identification method for peanut net spot disease based on rooted detached leaves. The detached leaves are rooted by detached leaf sampling, plant hormone treatment and greenhouse culture, and then the net spot disease resistance of peanut materials is evaluated by indoor inoculation of conidia suspension of net spot disease pathogens, leaf scanning and lesion area analysis. Based on the rooting of detached leaves and the condition that the normal growth and development of plants is not affected, the present invention realizes the net spot disease inoculation and resistance identification of scarce materials under indoor environmental conditions, thereby improving the identification efficiency of excellent germplasm net spot disease resistance. After the leaves are rooted, the chromosome composition of the materials can be analyzed by root tips to identify the identity of the materials; on the other hand, the rooted leaves have life functions, which improves the accuracy of resistance identification. The method of the present invention is simple and efficient to operate, and can evaluate the net spot disease resistance of a single or a few rare peanut materials, effectively shortening the disease-resistant breeding cycle.

Description

Technical Field

[0001] The invention relates to an indoor inoculation identification method for peanut net blotch based on rooted detached leaves, and belongs to the technical field of crop disease resistance identification. Background Art

[0002] As an important oil and cash crop, peanut (Arachis hypogaea L.) is crucial to ensure the stability of its yield and quality in actual production practice. Peanut growth and development are often invaded by a variety of pests and diseases. Peanut net blotch is a leaf disease caused by fungi, which often causes a 10%-20% reduction in production in disease-prone production areas, and more than 30% in severe cases. Therefore, it is of great significance to identify and create genetic germplasm resources for peanut resistance to net blotch.

[0003] There are three main methods for identifying resistance to peanut net spot disease: the first is identification based on natural disease occurrence in the field, and the disease situation is greatly affected by the natural environment; the second is identification based on artificial inoculation in the field to induce disease occurrence, but it is affected by the season and has a long cycle; the third is identification based on artificial inoculation indoors, which has a short cycle, high efficiency, and easy control of environmental conditions, and good repeatability of identification results. Using natural disease occurrence and artificial inoculation in the field, researchers have screened out some highly resistant or disease-resistant germplasms in different regions, such as P12, Qunyu 101, Luhua No. 11, Huayu 36, Zheng 8903, etc. In the process of studying wild species and their interspecific hybrid offspring, the applicant found that the wild peanut species A.duranensis is resistant to net spot disease, and obtained a series of heterochromatic systems of four red seeds and A.duranensis through interspecific hybridization. Due to the special chromosome composition, the fertility of some hybrid offspring is low, and the number of early generation materials is scarce. The currently commonly used methods for identifying peanut net blotch disease usually use the whole plant as the object of identification and inoculation, which consumes a lot of identification materials. This seriously affects the efficiency of identifying net blotch disease resistance in rare peanut germplasm, especially interspecific hybrid offspring, thereby affecting the utilization of excellent net blotch disease genes in wild germplasm. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides an indoor inoculation and identification method for peanut net blotch based on rooted detached leaves. The detached peanut leaves are rooted through α-naphthaleneacetic acid (NAA) induction and 1 / 2MS culture solution culture to obtain living leaves with life functions, and then the net blotch inoculation and identification are carried out, which solves the problems of net blotch inoculation and resistance identification of peanut germplasm materials and improves the efficiency of mining excellent net blotch resistance genes.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] An indoor inoculation and identification method for peanut net spot disease based on rooted and detached leaves comprises the following steps:

[0007] (1) Peanut in vitro leaf culture and rooting induction: Cut the third-to-last leaf from the top of a healthy and undamaged peanut plant and soak it in rooting solution for 6 h. Insert the soaked leaf into a nutrient pot filled with vermiculite, pour nutrient solution, and cover with plastic film to keep moisture and cultivate roots.

[0008] (2) Cytological identification of interspecific hybrid offspring materials of peanut: fresh root tips were cut for chromosome preparation, and then fluorescence in situ hybridization was performed to identify interspecific hybrids or additional line materials using a chromosome-specific oligonucleotide library as a probe;

[0009] (3) Inoculation of spore suspension: Before inoculation, Tween 20 was added to the conidia suspension of the net spot pathogen to make the final concentration of Tween 20 1% (v / v). The conidia suspension was then sprayed evenly onto the leaves and covered with a film for moisture preservation at 25°C-28°C for 20 days. The inoculation concentration of the conidia suspension was 2×10 6 ~2.5×10 6 Pieces / mL;

[0010] (4) Identification of resistance to net blotch disease in detached peanut leaves.

[0011] The leaf cut in step (1) is a whole peanut pinnate compound leaf comprising 4 leaflets. The incision position when cutting the peanut leaf is the base of the petiole, and an oblique cut is used during cutting.

[0012] The rooting culture conditions in step (1) are: constant temperature culture at 25° C.-28° C. for 15-20 days; the rooting solution is 0.01 mg / mL α-naphthylacetic acid; the vermiculite is sterilized and rehydrated; and the nutrient solution is 1 / 2MS nutrient solution without sucrose and agar.

[0013] Furthermore, the leaves sprayed with the conidia solution in step (3) are healthy leaves with roots.

[0014] Furthermore, the grading standard used in the identification of resistance to net blotch disease in step (4) is:

[0015] The disease resistance of detached peanut leaves to net spot disease was identified by calculating the disease level according to the ratio of lesion area to leaf area. The disease grading standard is as follows: Level 0: no lesions detected; Level 1: 0 < lesion area < 1%; Level 2: 1% ≤ lesion area < 3%; Level 3: 3% ≤ lesion area < 5%; Level 4: 5% ≤ lesion area < 7%; Level 5: 7% ≤ lesion area < 10%; Level 6: 10% ≤ lesion area < 15%; Level 7: 15% ≤ lesion area < 18%; Level 8: 18% ≤ lesion area < 20%; Level 9: lesion area ≥ 20%. The disease resistance index was further determined according to the disease level.

[0016] Furthermore, the resistance evaluation standard of the disease resistance index is:

[0017] The disease index DI of each material was calculated according to the disease stage, disease index = {[Σ(disease plants at each level / number of leaves × disease stage)] / (total plants surveyed / number of leaves × 9)} × 100. The relative disease resistance index RDI was further calculated according to the disease index, relative disease resistance index = 1-disease index of a certain variety / disease index of the most susceptible variety. The disease resistance of the materials was evaluated according to the relative disease resistance index, and the disease resistance was divided into 6 levels: RDI: 1.0 for no lesion reaction I; RDI: 0.80-0.99 for high resistance HR; RDI: 0.60-0.79 for resistance R; RDI: 0.40-0.59 for moderate resistance MR; RDI: 0.20-0.39 for susceptible S; RDI<0.20 for highly susceptible HS.

[0018] Beneficial effects of the present invention:

[0019] The present invention can realize the inoculation and resistance identification of scarce materials under indoor environmental control conditions without affecting the normal growth and development process of the plants based on the in vitro leaf rooting, thereby improving the identification efficiency of excellent germplasm resistance to net blotch. Moreover, after the leaves are rooted, on the one hand, the chromosome composition of the materials can be analyzed by using the root tip to identify the identity of the materials; on the other hand, the leaves have life functions, which improves the accuracy of resistance identification.

[0020] The method of the invention is simple and efficient to operate, and can evaluate the resistance to net blotch disease of a single or a few rare peanut materials, thereby effectively shortening the disease-resistant breeding cycle.

[0021] The method of the invention has important potential application value in breeding for resistance to peanut web blotch and other leaf diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Rooting images of detached leaves after α-NAA induction and nutrient solution culture treatment;

[0023] Figure 2 :The cytological identification results of the artificially synthesized hexaploid Am1210 material and its parents;

[0024] Figure 3 :The growth of detached leaves after inoculation of conidia of net spot disease indoors;

[0025] Note: Figure a: Growth of detached leaves, Figure b: Net spot disease susceptibility of detached leaves;

[0026] Figure 4 : Cytological identification results of additional line materials of A.duranensis-Si Li Hong;

[0027] Figure 5: Susceptibility of A.duranensis-Si Li Hong additional line materials and their parents to net spot disease on detached leaves;

[0028] Note: Figure a: Growth of the detached leaves of the additional line materials and parents; Figure b: Status of the detached leaves of some materials;

[0029] Figure 6 :Scan images of net spot disease on detached leaves of the additional line materials of A.duranensis-Si Li Hong and its parents. DETAILED DESCRIPTION

[0030] The specific implementation of the present invention is further described in detail below in conjunction with the examples. Unless otherwise specified, the percentages in the text are all percentages by weight.

[0031] Some of the reagents used in the present invention are as follows:

[0032] 2×SSC buffer: composed of 0.3 M trisodium citrate dihydrate and 3 M sodium chloride;

[0033] 1×TNT buffer: composed of 0.1 M Tris hydrochloride, 0.15 M sodium chloride and 0.05% (v / v) Tween-20;

[0034] 1×TNB buffer: composed of 0.5 M Tris hydrochloride, 0.15 M sodium chloride and 0.5% blocking reagent;

[0035] DAPI buffer: composed of 0.1 M citric acid and 0.05 M disodium hydrogen phosphate;

[0036] 1 / 2MS nutrient solution: 2.47 g of 1 / 2MS solid culture medium without sucrose and agar is dissolved in 1000 ml of distilled water by heating and sterilizing by high pressure.

[0037] Example 1 Method for identifying net blotch resistance of peanut Silihong, wild species A. duranensis (Zw55) and its synthetic hexaploid peanut Am1210 based on in vitro leaf rooting

[0038] 1. Rooting of Peanut Leaves in Vitro

[0039] On plants with good growth conditions planted in the field (tetraploid Silihong, diploid Zw55, and hexaploid Am1210 materials), the third-to-last leaf from the top that is healthy and undamaged was cut from the base of the petiole. The leaf was a whole peanut pinnate compound leaf with 4 leaflets, and then rinsed with sterile water 2-3 times. The obliquely cut detached leaves were placed in 0.01 mg / mL α-naphthaleneacetic acid (NAA) rooting solution, and the α-NAA solution was immersed in the leaf cut for 6 hours. Then the leaves soaked in α-NAA solution were taken out and inserted into a nutrient pot filled with sterilized and rehydrated vermiculite, and irrigated with 1 / 2MS nutrient solution without sucrose and agar, covered with plastic film to keep moisture, and kept at 25℃-28℃ for 15-20 days. Each material was cultured in vitro three times, and 3-4 leaves were placed in each nutrient pot for each replication. The detached leaves cultured for 15 days have successfully grown healthy root tips and formed functional living leaves ( Figure 1 ), indicating that this method can induce rooting of detached leaves.

[0040] 2. Cytological Identification of Chromosomes in Root Tips of Detached Leaves of Hexaploid Am1210

[0041] 2.1. Preparation and denaturation of chromosome sections from isolated leaf root tip cells

[0042] The detached leaves of Am1210 cultured for 15 days were pulled out, and the root tips of the detached leaves with a length of 1-2 cm were cut and treated with 0.002% 8-hydroxyquinoline solution for 3 hours, and then placed in a 3:1 (v / v) alcohol:acetic acid fixative. The chromosomes of metaphase were obtained by chromosome pressing method, frozen at -80℃ for 12-14 hours, the cover glass was removed, dehydrated in anhydrous ethanol for 12 hours and air-dried, and the air-dried slides containing chromosomes were placed in 70% formamide solution at 75℃ for denaturation for 70 seconds, and then dehydrated in 70%, 95%, and 100% (v / v) alcohol at -20℃ for 5 minutes each and air-dried.

[0043] 2.2. Preparation and denaturation of hybridization solution

[0044] First, prepare the hybridization solution. The composition of the hybridization solution system is: 7.5 μL of analytical grade deionized formamide, 1.5 μL of 20×SSC buffer, 2 μL of 50% dextran sulfate solution, 0.5 μL of 10 mg / mL salmon sperm DNA, 2 μL of each 200 ng / μL chromosome-specific single copy oligonucleotide probe library (chromosome probe libraries 1-10 can be used individually or in combination), then denature the hybridization solution at 105°C for 13 min, and finally place it in anhydrous ethanol at -20°C for 10-15 min.

[0045] 2.3. Chromosome fluorescence in situ hybridization

[0046] Drop the denatured hybridization solution onto the slide obtained in step 2.1, cover with a coverslip, and hybridize at 37°C for 36h; discard the coverslip, rinse with 42°C 2×SSC buffer 4-5 times; then wash twice with 2×SSC buffer at 42°C, each time for 5min; then wash with 1×TNT buffer at room temperature for 5min; drain the slides, add 50μL 1×TNB buffer and 1μL anti-digoxin rhodamine / anti-biotin on each slide, and incubate in a dark box at 37°C for 50min; then wash the slides with 1×TNT buffer at room temperature for 3 times, each time for 5min; finally, blow dry the slides, add 500μL of DAPI buffer with a concentration of 0.5μg / mL on each slide, stain for 3-4min, rinse with DAPI buffer 4-5 times, drop 6-7μL of anti-fluorescence quenching sealing agent, cover with a 20mm×20mm coverslip, and obtain images with a fluorescence microscope.

[0047] Figure 2 The results of cytological identification showed that the chromosome numbers of Silihong, wild species Zw55, and hexaploid Am1210 were 40, 20, and 60, respectively. Figure 2 ac), using the oligonucleotide library specific to chromosomes 1-10 as probes to identify interspecific hybrids or additional line materials by fluorescence in situ hybridization; from the cytological images of the artificially synthesized hexaploid material Am1210 identified by the probe library, it can be seen that all chromosomes of Am1210 can be stained with DAPI signals. Am1210 has 60 chromosomes, with 6 chromosomes from 1 to 10 ( Figure 2 ), indicating that roots grown from detached leaves can be used for cytological experiments such as chromosome preparation and fluorescence in situ hybridization.

[0048] in, Figure 2 a: DAPI staining results of four red cells; Figure 2 b: DAPI staining results of wild species Zw55 (A. duranensis); Figure 2 c: DAPI staining results of Am1210; Figure 2 d: Signal overlay of DAPI and chromosome 1 and 5 probe libraries; Figure 2 e: Signal overlay of DAPI and chromosome 2 and 6 probe libraries; Figure 2 f: Signal overlay of DAPI and chromosome 3 and 4 probe libraries; Figure 2 g: Signal overlay of DAPI and chromosome 7 and 8 probe libraries; Figure 2 h: Signal overlay of DAPI and chromosome 9 and 10 probe libraries; Figure 2dh: The red arrows point to chromosomes 1, 2, 3, 8, and 9, and the green arrows point to chromosomes 5, 6, 4, 7, and 10.

[0049] The cytological identification of the present invention can be extended to other peanut varieties, but usually the chromosome number of common varieties is certain and cytological identification is not required.

[0050] 3. Incubation of peanut detached leaves with net spot disease and identification of resistance

[0051] Select the in vitro leaves of Silihong, A.duranensis and Am1210 with good growth status, set up three replicates, set up one nutrient pot, and plant three in vitro culture leaves. Prepare the conidia suspension of net spot disease before inoculation: scrape the culture medium full of conidia into a grinder, use a sampler (30Hz / 40s) to break the conidia, collect the broken conidia and add sterile water to them to mix, filter with 3 layers of sterile gauze to remove the culture medium and the hyphae of the net spot disease pathogen, obtain the conidia suspension, measure the concentration of the conidia suspension with a hemocytometer, and add an appropriate amount of sterile water to make the concentration of the conidia suspension reach 2×10 6 ~2.5×10 6 / mL, it can be used for the net spot disease inoculation experiment. Before inoculation, Tween 20 should be added to the conidia suspension (to make the final concentration of Tween 20 1% (v / v)), and then the mixed solution is evenly sprayed on the leaves. The leaves are covered with a film for moisturizing and cultured at 25°C-28°C for 20 days. On the 21st day, EPSON scanner (Epson PerfectionV39) software is used to take pictures, and leaf area analysis software (Wanshen LA-Sv.2.3.0.3, China) is used to analyze the lesion area, and the disease level is calculated according to the ratio of lesion area to leaf area. The disease index (Disease Index, DI) of different materials is calculated according to the disease level, and the relative disease resistance index (Relative disease index, RDI) is further calculated according to the disease index. Finally, the net spot disease resistance is evaluated by the relative disease resistance index.

[0052] Disease level classification standard: Level 0: no lesions detected; 0 < lesion area < 1%; Level 2: 1% ≤ lesion area < 3%; Level 3: 3% ≤ lesion area < 5%; Level 4: 5% ≤ lesion area < 7%; Level 5: 7% ≤ lesion area < 10%; Level 6: 10% ≤ lesion area < 15%; Level 7: 15% ≤ lesion area < 18%; Level 8: 18% ≤ lesion area < 20%; Level 9: lesion area ≥ 20%;

[0053] Disease index (DI) = {[Σ(number of diseased plants / leaves at each level × disease level)] / (total number of investigated plants / leaves × 9)} × 100;

[0054] Relative disease resistance index (RDI) = 1-disease index of a certain variety (line) / disease index of the most susceptible variety (line); Disease resistance evaluation classification criteria: RDI: 1.0 is no lesion reaction I; RDI: 0.80-0.99 is high resistance HR; RDI: 0.60-0.79 is resistance R; RDI: 0.40-0.59 is medium resistance MR; RDI: 0.20-0.39 is susceptible S; RDI < 0.20 is highly susceptible HS. It can be seen that the RDI of Silihong is 0.23, which is susceptible to web spot disease S, and the wild species Zw55 and hexaploid Am1210 have almost no lesions, which is no lesion reaction I, indicating that web spot disease resistance can be effectively identified based on detached leaves.

[0055] Example 2 Identification of Net Blotch Resistance of A.duranensis-Si Li Hong Addition Line Material Using Detached Leaves

[0056] 1. Cytological identification of chromosomes from the root tips of leaves of A.duranensis-Si Li Hong addition line

[0057] First, we backcrossed the hexaploid Am1210 with its cultivated parent for two generations and selfed it for 2-3 generations. We obtained different additional line materials through leaf rooting and cytological identification.

[0058] The chromosome composition information of the materials is shown in Table 1: including DA10A du 、MA5A du 、MA1A du 、MA4A du 、MA(1A du +3A du )、MA(1A du +2A du +3A du +9A du )、MA7A du -1. MA7A du -2. MA9A du The detached leaves of Silihong, Zw55, hexaploid Am1210 and different additional line materials were selected as identification materials to be inoculated with the conidia suspension of net spot pathogen. Referring to step 1 of Example 1, the detached leaves of the above materials were collected for indoor culture at 25°C for 15 days, and the grown root tips were cut for cytological identification according to step 2 of Example 1.

[0059] Figure 4 The results of cytological identification are as follows: MA1A du There are 5 chromosomes stained with green signals by the chromosome 1 probe library, indicating that it is 1A du Monosomic addition line of chromosome; MA(1A du +2A du +3Adu +9A du ) each has 5 that can be 1A du , 2A du , 3A du and 9A du The single copy probe library is stained with a signal, indicating that it is 1A du , 2A du , 3A du and 9A du Monosomic addition line of chromosome; MA(1A du +3A du ) Five chromosomes were stained with green signals by the chromosome 1 probe library, and five chromosomes were stained with red signals by the chromosome 3 probe library, indicating that it is 1A du and 3A du Monosomic addition line of chromosome; MA4A du There are 5 chromosomes stained with green signals by the chromosome 4 probe library, indicating that it is 4A du Monosomic addition line of chromosome; MA5A du There are 5 chromosomes stained with green signals by the chromosome 5 probe library, indicating that it is 5A du Monosomic addition line of chromosome; MA7A du There are 5 chromosomes stained with green signals by the chromosome 7 probe library, indicating that it is 7A du Monosomic addition line of chromosome; MA9A du There are 5 chromosomes stained with red signals by the chromosome 9 probe library, indicating that it is 9A du Monosomic addition line of chromosome; DA10A du Six chromosomes are stained with green signals by the chromosome 10 probe library, indicating that it is 10A du Disomic addition lines of chromosomes; from this, it can be seen that they are all heterosomal addition line materials of A. duranensis and A. duranensis.

[0060] in, Figure 4 a:MA1A du Signal overlay of DAPI and chromosome 1 probe library, the green arrow points to chromosome 1; Figure 4 b: MA(1A du +2A du +3A du +9A du ) and the signal overlay of the probe pools for chromosomes 1 and 3. The green arrow points to chromosome 1, and the red arrow points to chromosome 3. Figure 4 c: MA(1A du +2A du +3A du +9Adu ) and the signal overlay of the probe libraries of chromosomes 2 and 9, the green arrow points to chromosome 9, and the red arrow points to chromosome 2; Figure 4 d: MA(1A du +3A du ) and the signal overlay of the probe libraries of chromosomes 1 and 3. The red arrow points to chromosome 3, and the green arrow points to chromosome 1; Figure 4 e:MA4A du The green arrow in the signal overlay of DAPI and chromosome 4 probe library points to chromosome 4; Figure 4 f:MA5A du Signal overlay of DAPI and chromosome 5 probe library, the green arrow points to chromosome 5; Figure 4 g:MA7A du Signal overlay of DAPI and chromosome 7 probe library, the green arrow points to chromosome 7; Figure 4 h:MA9A du The signal overlay of DAPI and chromosome 9 probe library, the red arrow points to chromosome 9; Figure 4 i:DA10A du Signal overlay of DAPI and chromosome 10 probe library. The green arrow points to chromosome 10.

[0061] Table 1 Chromosome composition information of A.duranensis-Si Li Hong addition line materials

[0062]

[0063] 2. Evaluation of resistance to net spot disease in A.duranensis-Si Li Hong additional line

[0064] Refer to step 3 of Example 1 to prepare 2×10 6 The prepared conidia suspension of net spot pathogen was evenly sprayed on the cultured detached leaves, and covered with plastic film to keep moisture to promote the infection of pathogens. The constant temperature of 25℃-28℃ was maintained for 20 days. On the 21st day, the resistance to net spot disease was evaluated. Figure 5 This is the resistance and susceptibility of Silihong, Zw55, hexaploid Am1210 and different additional line materials to net blotch disease on the 21st day after inoculation with conidia suspension of net blotch pathogen, indicating that in vitro rooted leaves inoculated with net blotch pathogen indoors can be used for identification of net blotch disease resistance.

[0065] in, Figure 5 a is the growth of the in vitro rooted leaves of Silihong, Zw55, hexaploid Am1210 and different addition lines in nutrient pots 20 days after being inoculated with net spot disease; Figure 5 b shows the state of the detached leaves of some materials 20 days after inoculation with the net spot pathogen. At this time, the detached leaves are still in a living state.

[0066] 3. Identification of resistance to net spot disease in A.duranensis-Si Li Hong additional line

[0067] The detached leaves after inoculation on the 21st day were taken and scanned with an EPSON scanner (Epson Perfection V39). The leaf area analysis software (Wanshen LA-Sv.2.3.0.3, China) was used to analyze the lesion area of ​​each leaf, and the disease level was calculated based on the ratio of lesion area to leaf area. The resistance rating of different materials to net spot disease was obtained based on the leaf scanning results. Among them, the additional line material MA1A du and MA(1A du +2A du +3A du +9A du ) materials had RDI of 0.95 and 0.92 respectively, and the disease resistance was high resistance to HR, while the other materials were susceptible to net spot disease S( Figure 6 ), the statistical results of resistance identification are shown in Table 1 above.

[0068] The above are only preferred embodiments of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement or improvement made under the spirit and principle of the present invention is within the protection scope of the present invention.

Claims

1. A method for indoor inoculation and identification of peanut net spot based on rooted detached leaves, characterized in that: The following steps are involved: (1) Peanut in vitro leaf culture and rooting induction: Cut the third-to-last leaf from the top of a healthy and undamaged peanut plant and soak it in rooting solution for 6 h. Insert the soaked leaf into a nutrient pot filled with vermiculite, pour nutrient solution, and cover with plastic film to keep moisture and cultivate roots. (2) Cytological identification of interspecific hybrid offspring materials of peanut: fresh root tips were cut for chromosome preparation, and then fluorescence in situ hybridization was performed to identify interspecific hybrids or additional line materials using a chromosome-specific oligonucleotide library as a probe; (3) Inoculation of spore suspension: Before inoculation, add Tween 20 to the conidia suspension of the net spot pathogen to make the final concentration of Tween 20 1% (v / v). Then spray the conidia suspension evenly onto the leaves and cover with a film to keep them moist for 20 days at 25℃-28℃. The inoculation concentration of the conidia suspension is 2×10 6 ~2.5×10 6 Pieces / mL; (4) Identification of resistance to net blotch disease in detached peanut leaves; The leaf cut in step (1) is a whole peanut pinnate compound leaf comprising 4 leaflets. The incision position when cutting the peanut leaf is the base of the petiole, and the cutting is performed obliquely. The rooting culture conditions are: constant temperature culture at 25° C.-28° C. for 15-20 days; and the rooting solution is 0.01 mg / mL of α-naphthylacetic acid.

2. The method for indoor inoculation and identification of peanut web blotch based on rooted detached leaves according to claim 1, characterized in that: The vermiculite in step (1) has been sterilized and rehydrated.

3. The method for indoor inoculation and identification of peanut web blotch based on rooted detached leaves according to claim 1, characterized in that: The nutrient solution in step (1) is 1 / 2 MS nutrient solution without sucrose and agar.

4. The method for indoor inoculation and identification of peanut web blotch based on rooted detached leaves according to claim 1, characterized in that: The leaves sprayed with the conidia solution in step (3) are healthy leaves with roots.

5. The method for indoor inoculation and identification of peanut web blotch based on rooted detached leaves according to claim 1, characterized in that: The grading standard used in the identification of net spot disease resistance in step (4) is: The disease resistance of detached peanut leaves to net spot disease was identified by calculating the disease level according to the ratio of lesion area to leaf area. The disease grading standard is as follows: Level 0: no lesions detected; Level 1: 0 < lesion area < 1%; Level 2: 1% ≤ lesion area < 3%; Level 3: 3% ≤ lesion area < 5%; Level 4: 5% ≤ lesion area < 7%; Level 5: 7% ≤ lesion area < 10%; Level 6: 10% ≤ lesion area < 15%; Level 7: 15% ≤ lesion area < 18%; Level 8: 18% ≤ lesion area < 20%; Level 9: lesion area ≥ 20%. The disease resistance index was further determined according to the disease level.

6. The method for indoor inoculation and identification of peanut web blotch based on rooted detached leaves according to claim 5, characterized in that: The resistance evaluation criteria of the disease resistance index are: The disease index DI of each material was calculated according to the disease level. The disease index = {[Σ(disease plants at each level / number of leaves × disease level)] / (total plants surveyed / number of leaves × 9)} × 100. The relative disease resistance index RDI was further calculated according to the disease index. The relative disease resistance index = 1-disease index of a certain variety / disease index of the most susceptible variety. The disease resistance of the material was evaluated according to the relative disease resistance index, and the disease resistance was divided into 6 levels: RDI: 1.0 is no disease spot reaction I; RDI: 0.80-0.99 is high resistance HR; RDI: 0.60-0.79 is resistance R; RDI: 0.40-0.59 is moderate resistance MR; RDI: 0.20-0.39 is susceptible S; RDI < 0.20 is high sensitivity HS.

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