Method for identifying SMV resistance of genetically improved soybeans with gene editing, interference and like

Abstract

The invention provides a method for identifying soybean mosaic virus (SMV) resistance of genetically improved soybeans obtained by utilizing technologies of gene editing, transgenosis, RNAi interference and the like. The method comprises the following steps: (1) providing a soybean variety to be evaluated; (2) respectively preparing a first solution containing carborundum and a second solution containing soybean mosaic virus, and mixing the first solution and the second solution in proportion to obtain a working solution; (3) spraying the working solution obtained in the step (2) to leaves of a soybean variety to be evaluated by adopting a spray gun inoculation method so as to enable the soybean mosaic virus to intrude into the leaves; and (4) investigating the disease condition after inoculating in the step (3) for a period of time, and identifying the soybean mosaic virus resistance grade of the soybean variety to be evaluated according to the disease performance.

Description

Technical Field

[0001] The present invention relates to the technical field of plant virus detection and plant disease resistance identification, and particularly to a method for identifying soybean mosaic virus (SMV) resistance in soybean varieties. The present invention particularly provides a method for identifying SMV resistance in soybean varieties obtained through modern molecular breeding techniques such as transgenic technology, RNAi, and gene editing. Background Art

[0002] In global soybean production, soybean mosaic virus (SMV) remains a key constraint on soybean yield and quality. SMV infection can cause mottling and wrinkling of leaves, stunted plants, and mottled seeds, leading to significant yield losses in severe cases.

[0003] With the continuous advancement of agricultural science and technology, modern genetic improvement technologies have brought new hope for the development of new soybean varieties resistant to soybean mosaic virus (SMV) through transgenic, RNAi, and gene-edited methods. Research results from Huazhong Agricultural University have shown that transgenic tobacco plants overexpressing the cowpea lipid transport protein LTP1 have significantly enhanced resistance to SMV, demonstrating the feasibility of using transgenic technology to enhance resistance through specific genes in plant virus resistance breeding. Professor Zhi Haijian's research group at Nanjing Agricultural University is also conducting research on using transgenic technology to silence eEF1A and cultivate transgenic SMV-resistant soybean varieties. They have identified the soybean eEF1A protein that interacts with the SMV pathogenicity determinant P3, providing new insights into SMV prevention and control. Furthermore, Professor Chen Qingshan's team and Professor Xin Dawei's team at Northeast Agricultural University have constructed single-cell transcriptome maps of soybean leaves infected with SMV, which are being used to identify genes associated with soybean resistance and provide an important theoretical basis for modern genetic improvement technologies such as gene editing.

[0004] When using transgenic technology, RNAi, gene editing, and other methods to obtain soybean varieties, a large number of transformation events are often involved. Taking transgenic technology as an example, during the process of introducing exogenous genes into the soybean genome, due to the random nature of gene integration, different transformation events may result in the exogenous genes being inserted at different locations in the soybean genome, and the copy number and expression level of the inserted genes also vary. Similarly, in RNAi technology, the interference efficiency and off-target effects of dsRNA vary among different transformation events. Although gene editing technology can precisely modify specific gene loci, in actual operation, the editing efficiency and the presence of off-target mutations will also vary depending on the different transformation processes. Therefore, in order to obtain soybean lines with optimal SMV resistance and genetic stability, it is necessary to screen from a large number of transformation events.

[0005] For example, when using gene editing to cultivate SMV-resistant soybeans, various types of edits may occur, such as single-base substitutions, small fragment insertions or deletions, and not all edits can achieve the expected resistance effect. High-throughput detection technology can quickly and comprehensively analyze these transformation events, detect multiple indicators such as gene expression levels, protein activity, and plant resistance performance, and thus efficiently screen out the best strains. However, in the current research on soybean resistance to SMV, high-throughput detection technology for these transformation events is seriously insufficient. Traditional detection methods and analysis methods are not only time-consuming and labor-intensive, but also difficult to meet the needs of rapid detection of large numbers of samples. This greatly limits the efficiency of screening the best SMV-resistant soybean strains from numerous transformation events, and hinders the progress of new SMV-resistant soybean varieties from research and development to actual production applications.

[0006] While modern genetic improvement technologies have achieved theoretical breakthroughs in breeding new SMV-resistant soybean varieties, practical application still faces numerous challenges. The critical bottleneck hindering the transition from R&D to production is the lack of high-throughput technology to identify resistance in these new varieties. Traditional identification methods, such as manual friction inoculation, are not only cumbersome and inefficient, but also struggle to ensure consistent inoculation results, making them unable to meet the demand for rapid identification of a large number of new varieties. Summary of the Invention

[0007] The present invention provides a method for identifying soybean mosaic virus resistance in soybean varieties, the method comprising the following steps:

[0008] (1) Provide the soybean varieties to be evaluated;

[0009] (2) preparing a first solution containing corundum and a second solution containing soybean mosaic virus respectively, and mixing the first solution and the second solution in proportion to obtain a working solution;

[0010] (3) spraying the working solution obtained in step (2) onto leaves of the soybean variety to be evaluated using a spray gun inoculation method to allow soybean mosaic virus to invade the leaves;

[0011] (4) After a period of time after inoculation in step (3), the disease situation is investigated and the soybean mosaic virus resistance level of the soybean variety to be evaluated is identified based on the disease manifestations.

[0012] In one embodiment, the first solution is prepared using corundum and the surfactant Silwet L-77, and the second solution contains the surfactant Silwet L-77;

[0013] In one embodiment, the first solution in step (2) is prepared as follows: corundum with a particle size of 800-1200 mesh (preferably 1000 mesh) and a surfactant Silwet L-77 are selected, the corundum and the surfactant are respectively mixed with water, and then the two are mixed to form a uniform suspension; wherein the mass volume ratio of the corundum is 1%-5% (preferably 5%), and the volume fraction of Silwet L-77 is 0.05%-0.1% (preferably 0.05%); in a preferred embodiment, the corundum solution and the surfactant solution are mixed under ultrasonic conditions.

[0014] In one embodiment, the second solution in step (2) can be prepared by any of the following methods: culturing soybean mosaic virus in a culture medium; or, using a soybean variety susceptible to soybean mosaic virus (e.g., Williams82, abbreviated as W82) to propagate soybean mosaic virus.

[0015] In one embodiment, soybean mosaic virus is propagated using a soybean variety susceptible to soybean mosaic virus. After symptoms appear, diseased leaves are collected, crushed and ground into a suspension, and diluted with a buffer containing Silwet L-77 (the volume fraction of Silwet L-77 is 0.05%) to prepare a second solution.

[0016] In one embodiment, the first solution and the second solution are mixed in a ratio of 1:3-5 (eg, 1:4).

[0017] In one embodiment, when using a spray gun for inoculation, the spray pressure is 5-20 kg / 10 mm 2 (For example, 10-15kg / 10mm 2 ), the nozzle is 2cm-10cm (for example, 3cm-5cm) away from the inoculated plants, and the inoculation is completed and left to stand for a period of time. Optionally, after the virus invades, the step of removing excess inoculation solution is also included.

[0018] In one embodiment, the soybean variety to be evaluated is a soybean variety from conventionally bred soybean varieties or soybean varieties obtained by biological breeding; for example, soybean varieties obtained by transgenic, RNAi or gene editing technology.

[0019] In one embodiment, in step (4), the disease development is investigated 2-3 times between 15 and 20 days after vaccination.

[0020] In one embodiment, soybean mosaic virus resistance rating is evaluated according to the following criteria:

[0021] (1) The disease level is divided into the following 6 levels according to the disease manifestation of each plant:

[0022] Level 0: The plant leaves have no lesions, discoloration, deformities, etc. and are growing normally;

[0023] Level 1: The leaves have a small number of scattered, slightly different-colored patches, which account for less than 10% of the total leaf area and do not affect the leaf morphology and function;

[0024] Level 3: The leaves are obviously mottled, with the patch area accounting for 10%-30%, and there is slight wrinkling, which does not affect the overall growth of the plant;

[0025] Level 5: The leaves are severely mottled, with patches covering 30%-50% of the area, and are obviously wrinkled. Some leaves are twisted and deformed, and plant growth is affected to a certain extent.

[0026] Level 7: Leaves are mottled over a large area, covering 50%-70% of the area, severely wrinkled and twisted, with necrotic spots, and the plant is obviously dwarfed and has reduced branching;

[0027] Level 9: Leaves are almost completely covered with lesions, accounting for more than 70% of the area, and are severely wrinkled and necrotic. The plant is severely dwarfed, with extremely poor growth or even death.

[0028] (2) Investigate each individual plant of the material and record the disease index and the total number of plants investigated according to the following formula:

[0029]

[0030] Calculate the disease index (DI). Where s is the representative value for each disease level, n is the number of plants at each disease level, N is the total number of plants surveyed, and S is the representative value for the highest disease level. Soybean resistance to mosaic virus disease is evaluated based on the disease index (DI): 0 ≤ DI ≤ 20 is highly resistant (HR); 20 < DI ≤ 35 is resistant (R); 35 < DI ≤ 50 is moderately resistant (MR); 50 < DI ≤ 70 is susceptible (S); and 70 < DI ≤ 100 is highly susceptible (HS).

[0031] Based on the above invention content, the present invention proposes the following technical solutions:

[0032] A method for identifying soybean mosaic virus resistance in soybean varieties, comprising the following steps:

[0033] (1) Provide the soybean varieties to be evaluated;

[0034] (2) preparing a first solution containing corundum and a second solution containing soybean mosaic virus, respectively, and mixing the first solution and the second solution in proportion to obtain a working solution; wherein the first solution is prepared using corundum and a surfactant, Silwet L-77, and the second solution contains the surfactant, Silwet L-77;

[0035] (3) Using a spray gun inoculation method, the working solution obtained in step (2) is sprayed onto the leaves of the soybean variety to be evaluated so that the soybean mosaic virus invades the leaves; during the spray gun inoculation, the spray pressure is 5-20 kg / 10 mm 2 ;

[0036] (4) After a period of time after inoculation in step (3), the disease situation is investigated and the soybean mosaic virus resistance level of the soybean variety to be evaluated is identified based on the disease manifestations.

[0037] Furthermore, the mesh size of the corundum is 800-1200 mesh, preferably 1000 mesh; the mass volume ratio of the corundum in the first solution is 1%-5%, preferably 5%.

[0038] Furthermore, the volume fraction of Silwet L-77 in the first solution is 0.05%-0.1%, preferably 0.05%.

[0039] Furthermore, the volume fraction of Silwet L-77 in the second solution is 0.05%-0.1%, preferably 0.05%.

[0040] Furthermore, the second solution is prepared by any of the following methods: culturing soybean mosaic virus in a culture medium; or, using a soybean variety susceptible to soybean mosaic virus to propagate soybean mosaic virus.

[0041] Furthermore, soybean mosaic virus is propagated using a soybean variety susceptible to soybean mosaic virus. After symptoms appear, diseased leaves are collected, crushed and ground into a suspension, and diluted with a buffer containing Silwet L-77 to prepare a second solution.

[0042] Furthermore, the first solution and the second solution are mixed in a ratio of 1:3-5 (eg, 1:4) to obtain a working solution.

[0043] Furthermore, in step (3), the spray gun inoculation pressure is 10-15kg / 10mm 2 ; The distance between the nozzle and the inoculated plants should be 2cm-10cm, and the inoculation should be left to stand for a while.

[0044] Furthermore, the soybean variety to be evaluated comes from a conventionally bred soybean variety or a soybean variety obtained through biological breeding; for example, a soybean variety obtained through genetic modification, RNAi or gene editing technology.

[0045] Furthermore, in step (4), the disease situation is investigated 2-3 times between 15 and 20 days after vaccination.

[0046] Below in conjunction with embodiment, embodiment of the present invention is described in detail, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, rather than to limit the scope of the invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art. Implementation Method

[0047] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0048] Example 1. Experimental materials

[0049] A variety of soybean experimental materials obtained through modern breeding techniques were selected, including SMV-P3-RNAi-transgenic soybean B5B9013-4 (using RNA interference technology to target the SMV P3 gene), SMV-NIB-RNAi-transgenic soybean B5C9123-5, SMV-CP-RNAi-transgenic soybean D8F7021-3, Rsv1-edited disease-resistant soybean E9G8132-4, GmWRKY54 gene-overexpressing soybean F6H9243-5, EMS-induced disease-resistant soybean G7I8031-2, and space-induced disease-resistant soybean H8J9142-3. W82, which is highly susceptible to all SMV races, was used as a control material. Material information was entered in detail using a standardized form using the system of the present invention.

[0050] Example 2, experimental process

[0051] Launch the Python program in the system, call the pandas toolkit, and use the DataFrame.sample() method to set up three biological replicates and randomize the experimental and control materials. Based on actual field conditions, local control is performed and a planting plan is generated to guide planting. Prepare the working solution according to the inoculant formula, inoculate using a high-pressure electric sprayer, and rinse with clean water after inoculation. Within 15-20 days of inoculation, regularly survey and record the disease status of individual plants according to the disease classification standard.

[0052] In this embodiment, soybean mosaic virus (SMV) inoculation is performed using the following method.

[0053] (1) Reagent formula:

[0054] Solution A: Select corundum of a certain mesh size, and control the mass-to-volume ratio at 0.5%-5% (the optimal is 5%). Add a surfactant, and its volume fraction in the suspension is 0.05%-0.2% (a concentration of 0.05% can effectively reduce surface tension, enhance wetting and penetration capabilities, and balance cost and effect). When preparing, first pour the corundum into a beaker and add a small amount of deionized water to stir into a thick paste, then add the surfactant to the remaining deionized water to dissolve it, and then mix the two and ultrasonically treat for 10-15 minutes (ultrasonic power 100-300 watts), and finally transfer to a magnetic stirrer and stir for 15-20 minutes (300-500 rpm) to form a uniform suspension.

[0055] Solution B: Soybean mosaic virus (SMV) inoculation was prepared by propagating the susceptible cultivar W82 in a greenhouse. Fresh diseased leaves were collected from the tops after symptoms appeared, chopped, crushed, and freeze-ground to form a homogenous suspension. The homogenate was diluted 10-fold with 0.05% by volume Silwet L-77 and 0.1 mol / L phosphate buffer (pH 7.0) to prepare the SMV inoculum.

[0056] Working solution: Mix solution A and solution B in a ratio of 1:4. Slowly add solution A to solution B while stirring with a magnetic stirrer at 300-500 rpm until completely mixed to ensure even distribution of the virus and corundum to improve the inoculation effect.

[0057] (2) Vaccination method:

[0058] Use the spray gun inoculation method, use the spray gun spray pressure to spray the emery and virus juice on the leaves, the emery hits the leaf surface to cause micro-injury, allowing the virus to invade. When operating, use a high-pressure electric sprayer and set the spray pressure to 10-15kg / 10mm 2 The nozzle should be 3-5 cm away from the inoculated plants. After inoculation, let it rest for 30-45 minutes. After the virus has penetrated, spray the surface with clean water to remove excess inoculation solution to avoid interference with subsequent experimental observations.

[0059] (3) Investigation and evaluation

[0060] The disease situation was investigated 15-20 days after inoculation, and the investigation was conducted three times before and after. At this time, the symptoms caused by the virus are obvious, and multiple investigations can help to more accurately understand the disease situation. The disease level of each plant is divided into 6 levels: 0, 1, 3, 5, 7, and 9:

[0061] –Level 0: The leaves of the plant have no spots, discoloration, deformities or other symptoms and are growing normally.

[0062] -Level 1: The leaves have a small number of scattered spots of slightly different colors, which account for less than 10% of the total leaf area and do not affect the leaf morphology and function.

[0063] –Level 3: The leaves are obviously mottled, with the patch area accounting for 10%-30%, and are slightly wrinkled, but do not affect the overall growth of the plant.

[0064] -Level 5: The leaves are severely mottled, with the patch area accounting for 30%-50%, and are obviously wrinkled. Some leaves are twisted and deformed, and the growth of the plant is affected to a certain extent.

[0065] -Level 7: Leaves are mottled over a large area, accounting for 50%-70% of the area, and are severely wrinkled and twisted, with necrotic spots. The plant is obviously dwarfed and has reduced branches.

[0066] -Level 9: The leaves are almost completely covered with lesions, accounting for more than 70% of the area, and are severely wrinkled and necrotic. The plants are severely dwarfed, with extremely poor growth or even death.

[0067] Investigate each individual plant of the material, record the disease index and the total number of plants investigated, according to the following formula:

[0068]

[0069] Calculate the disease index (DI). Where s is the representative value for each disease level, n is the number of plants at each disease level, N is the total number of plants surveyed, and S is the representative value for the highest disease level. Soybean resistance to mosaic virus disease is evaluated based on the disease index (DI): 0 ≤ DI ≤ 20 is highly resistant (HR); 20 < DI ≤ 35 is resistant (R); 35 < DI ≤ 50 is moderately resistant (MR); 50 < DI ≤ 70 is susceptible (S); and 70 < DI ≤ 100 is highly susceptible (HS).

[0070] Example 3: Optimization of Soybean Mosaic Virus (SMV) Inoculation Efficiency

[0071] In order to improve the efficiency and consistency of soybean mosaic virus (SMV) inoculation, and thus improve the efficiency of SMV resistance identification, this example optimizes parameters such as the inoculation method, corundum, spray gun conditions, and surfactants.

[0072] 3.1 Analysis of corundum particles and inoculation effect

[0073] The sizes of corundum particles were selected as 500, 800, 1000, 1200, 1500, and 2000 mesh, and solution A was prepared and tested. Untreated soybean W82 was set as the control group, and W82 with different corundum particle suspensions sprayed at high pressure was set as the experimental group. Each experimental group had 100 plants, and 3 groups of experiments were repeated. The high-pressure spray corundum method was used to investigate the disease incidence within 15-20 days of inoculation. The 6 levels of disease severity were statistically analyzed according to the disease performance of each plant, and the ICC value was obtained by calculating the variance between groups and the variance within groups. The specific formula is:

[0074]

[0075] Where MSB is the between-group mean square, MSW is the within-group mean square, and k is the number of observations in each group. The results are shown in the following table.

[0076]

[0077] As shown in the table above, the ICC values of soybean plants tested within the 1000-mesh corundum experimental group were closest to 1, and the consistency of soybean mosaic virus disease grade across the experimental groups was the highest. Therefore, this corundum granule is most suitable for inoculation for soybean mosaic virus resistance assessment. Furthermore, the ICC values of the 800-1200 mesh corundum experimental groups were all greater than 0.8, making them also effective parameters for inoculation corundum granules used in soybean mosaic virus resistance assessment.

[0078] 3.2 Analysis of injection pressure and inoculation effect

[0079] Untreated soybean W82 was set as the control group, and W82 sprayed with different pressures was set as the experimental group. Each experimental group had 100 plants, and 3 groups of experiments were repeated. The spray gun inoculation method was used. The corundum and virus juice were sprayed on the leaves using the spray gun spray pressure. The corundum hit the leaf surface to cause micro-injuries, allowing the virus to invade. In the specific operation, a high-pressure electric sprayer was used, and the spray pressure was set to 1-5kg / 10mm. 2 , 5-10kg / 10mm 2 , 10-15kg / 10mm 2 , 15-20kg / 10mm 2 , 20-25kg / 10mm 2 A total of five adjustable pressure gradient settings were used, with the nozzle positioned 3-5 cm from the inoculated plants. After inoculation, the sprayer was left to rest for 30-45 minutes. After the virus had penetrated, the leaves were sprayed with clean water to remove any excess inoculation solution. Disease activity was monitored 15-20 days later, and the number of diseased plants in each treatment group was counted and the incidence rate calculated. The results are shown in the table below.

[0080]

[0081] From the table above, we can see that when the injection pressure is lower than 10kg / 10mm 2 Therefore, the inoculation method of the present invention needs to ensure that the injection pressure of the diamond inoculation liquid reaches 10kg / 10mm. 2 above.

[0082] Then, the injection pressure is 10-15kg / 10mm 2 , 15-20kg / 10mm 2 , 20-25kg / 10mm 2The disease status of soybeans was monitored 15-20 days after treatment. Six disease levels were assigned to each plant based on disease severity. The ICC values were calculated by calculating the between-group and within-group variances. The results are shown in the table below.

[0083]

[0084] When the injection pressure is 10-15kg / 10mm 2 When spraying at higher pressures, the disease grade consistency within the group was the highest, with an ICC value of 0.9923456. However, the two groups with higher spray pressures experienced leaf damage, which prevented consistency between treated soybeans and prevented consistent disease grade distribution across all plants within the experimental group, hindering the use of statistical analysis for both disease and resistance ratings.

[0085] 3.3 Analysis of surfactants and leaf chemical damage

[0086] Surfactants are substances with fixed hydrophilic and lipophilic groups that can be oriented on the surface of a solution and significantly reduce surface tension. They are crucial for stabilizing the properties of corundum suspensions and helping the SMV pathogen adhere to plant surfaces and penetrate into tissues. Therefore, we tested different surfactants (sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), polyoxyethylene sorbitan fatty acid esters (Tween-20), sorbitan fatty acid esters (Span), dodecyl dimethyl betaine, and Silwet L-77) to test their effectiveness in stabilizing the SMV inoculation working solution (a mixture of Solution A and Solution B at a ratio of 1:4) and preventing leaf damage. The results are shown in the table below. While all of the surfactants, except SDS, improved solution stability, only Tween-20 and Silwet L-77 did not cause significant damage to plant leaves.

[0087]

[0088] The inoculation working solution of 0.1% Tween-20 and 0.05% Silwet L-77 was tested respectively. 2The inoculation experiment was carried out under the spraying pressure. Untreated soybean W82 was used as the control group, and W82 sprayed with different pressures was used as the experimental group. There were 100 plants in each experimental group, and 3 groups of experiments were repeated. The disease incidence was investigated by statistically investigating the period of 15-20 days after inoculation, and the 6 levels of disease severity were statistically analyzed according to the disease manifestations of each plant. The ICC value was obtained by calculating the variance between groups and the variance within groups. The results showed that the consistency of plant disease severity in the experimental group using Silwet L-77 for inoculation was significantly better than that in the experimental group using Tween-20. The ICC value of the former reached 0.94, while that of the latter was less than 0.7. Therefore, the present invention uses an inoculation working solution configured with a concentration of 0.05% Silwet L-77.

[0089]

[0090] 3.4 Inoculation efficiency comparison experiment:

[0091] Two experimental fields, each 100 square meters in area, were selected and labeled Field A and Field B. The same number of soybean plants (500 plants) were planted. Field A was inoculated using the spray gun method of the present invention, while Field B was inoculated using the traditional manual friction method. The inoculation operations were performed by the same number of professionals under the same environmental conditions.

[0092] Field A was prepared with working solution according to the method of the present invention and inoculated using a high-pressure electric sprayer with a spray pressure set at 10-15 kg / 10 mm 2 The nozzle was kept 3-5 cm away from the inoculated plants. After inoculation, the plants were left to rest for 30-45 minutes before being rinsed with clean water. Field B used the traditional manual friction inoculation method. A cotton ball dipped in virus sap was gently rubbed 3-5 times on each soybean leaf to ensure that the leaf surface was slightly damaged and the virus sap adhered.

[0093] The experimental results showed that the spray gun inoculation method in field A took 2 hours to complete the inoculation of 500 plants, and only one person was required to complete the operation. The manual friction inoculation method in field B took 8 hours to complete the inoculation of 500 plants. Due to the more cumbersome operation, it required 3-4 people to complete the inoculation task of the same number of plants. The spray gun inoculation method is four times more efficient than the manual friction inoculation method and also has a significant advantage in labor costs. The specific data comparison is shown in the table below:

[0094]

[0095] Example 4: SMV resistance test on the material of Example 1

[0096] In this example, the optimized method of the above example was used to inoculate the material of Example 1 with SMV and evaluate its resistance. The specific optimized inoculation method is as follows:

[0097] (1) Reagent formula:

[0098] Solution A: Use 1000-mesh corundum at a mass-to-volume ratio of 5%. Add the surfactant Silwet L-77, with a volume fraction of 0.05% in the suspension. To prepare, pour the corundum into a beaker and stir with a small amount of deionized water to form a thick paste. Dissolve the surfactant in the remaining deionized water. The mixture is then ultrasonically treated for 10-15 minutes (ultrasonic power 100-300 watts). Finally, transfer to a magnetic stirrer and stir for 15-20 minutes (300-500 rpm) to form a uniform suspension.

[0099] Solution B: Soybean mosaic virus (SMV) inoculation was prepared by propagating the susceptible cultivar W82 in a greenhouse. Fresh diseased leaves were collected from the tops after symptoms appeared, chopped, crushed, and freeze-ground to form a homogenous suspension. The homogenate was diluted 10-fold with 0.05% by volume Silwet L-77 and 0.1 mol / L phosphate buffer (pH 7.0) to prepare the SMV inoculum.

[0100] Working solution: Mix solution A and solution B in a ratio of 1:4. Slowly add solution A to solution B while stirring with a magnetic stirrer at 300-500 rpm until completely mixed to ensure even distribution of the virus and corundum to improve the inoculation effect.

[0101] (2) Vaccination method:

[0102] Use the spray gun inoculation method, use the spray gun spray pressure to spray the emery and virus juice on the leaves, the emery hits the leaf surface to cause micro-injury, allowing the virus to invade. When operating, use a high-pressure electric sprayer and set the spray pressure to 10-15kg / 10mm 2 The nozzle should be 3-5 cm away from the inoculated plants. After inoculation, let it rest for 30-45 minutes. After the virus has penetrated, spray the surface with clean water to remove excess inoculation solution to avoid interference with subsequent experimental observations.

[0103] (3) Investigation and evaluation

[0104] The disease was investigated 15-20 days after inoculation, with three investigations conducted before and after. At this time, the symptoms caused by the virus are obvious, and multiple investigations can provide a more accurate understanding of the disease. The material from Example 1 was evaluated for SMV resistance according to the levels in Example 2. The results are shown in the following table.

[0105]

[0106] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for identifying soybean mosaic virus resistance in soybean varieties, the method comprising the following steps: (1) Provide the soybean varieties to be evaluated; (2) preparing a first solution containing corundum and a second solution containing soybean mosaic virus respectively, and mixing the first solution and the second solution in proportion to obtain a working solution; wherein, The first solution was prepared using corundum and the surfactant Silwet L-77, and the second solution contained the surfactant Silwet L-77; (3) Using a spray gun inoculation method, the working solution obtained in step (2) is sprayed onto the leaves of the soybean variety to be evaluated so that the soybean mosaic virus invades the leaves; during the spray gun inoculation, the spray pressure is 5-20 kg / 10 mm 2 ; (4) After a period of time after inoculation in step (3), the disease situation is investigated and the soybean mosaic virus resistance level of the soybean variety to be evaluated is identified based on the disease manifestations.

2. The method according to claim 1, characterized in that The mesh size of the corundum is 800-1200 mesh, preferably 1000 mesh; the mass volume ratio of the corundum in the first solution is 1%-5%, preferably 5%.

3. The method according to claim 1, characterized in that The volume fraction of Silwet L-77 in the first solution is 0.05%-0.1%, preferably 0.05%.

4. The method according to claim 1, wherein The volume fraction of Silwet L-77 in the second solution is 0.05%-0.1%, preferably 0.05%.

5. The method according to claim 1, wherein The second solution is prepared by any of the following methods: culturing soybean mosaic virus in a culture medium; or using a soybean variety susceptible to soybean mosaic virus to propagate soybean mosaic virus.

6. The method according to claim 5, characterized in that Soybean mosaic virus is propagated using a soybean variety susceptible to soybean mosaic virus. After symptoms appear, diseased leaves are collected, crushed and ground into a suspension, and diluted with a buffer containing Silwet L-77 to prepare a second solution.

7. The method according to claim 1, characterized in that The first solution and the second solution are mixed in a ratio of 1:3-5 (eg, 1:4) to obtain a working solution.

8. The method according to claim 1, characterized in that When the spray gun is inoculated in step (3), the injection pressure is 10-15kg / 10mm 2 ; The distance between the nozzle and the inoculated plants should be 2cm-10cm, and the inoculation should be left to stand for a while.

9. The method according to claim 1, characterized in that The soybean variety to be evaluated is a soybean variety selected by conventional breeding or a soybean variety obtained by biological breeding; for example, a soybean variety obtained by genetic modification, RNAi or gene editing technology.

10. The method according to claim 1, characterized in that In step (4), the disease situation is investigated 2-3 times between 15 and 20 days after vaccination.

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