Method for removing pepper mild mottle viruses in pepper seeds

Abstract

The invention provides a method for removing capsicum light mottle viruses in capsicum seeds. The method comprises the following steps: (1) carrying out protease hydrolysis treatment on the capsicum seeds by adopting a protease aqueous solution capable of hydrolyzing virus protein shells; and (2) the pepper seeds treated in the step (1) are subjected to light irradiation in a solution containing a photosensitizer to excite the photosensitizer, so that the pepper seeds without the pepper mild mottle viruses are obtained, and the photosensitizer is a visible light excited photosensitizer. Compared with an existing PMMoV virus resisting method, the virus removing method has the advantages that PMMoV viruses of the pepper seeds can be effectively removed, the germination potential and the germination rate of the pepper seeds are not reduced, and the method is more efficient, economical, environmentally friendly and convenient to popularize; the method can promote the prevention and control of pepper virus diseases and the breeding of virus-free pepper seedlings, and guarantee the long-distance transportation of seeds.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to a method for removing pepper mild mottle virus from pepper seeds. Background Art

[0002] Viral diseases are an important type of disease that restricts the production of hot (sweet) peppers in my country. Among them, peppermild mottle virus (PMMoV) has become the main virus type in many areas of my country.

[0003] PMMoV virus belongs to the Tobamovirus genus of the Virgaviridae family and is a non-enveloped positive single-stranded RNA virus. It mainly harms the leaves and fruits of peppers. After being infected by PMMoV virus, the symptoms of pepper leaves are not obvious or show mild chlorosis and shrinkage. In severe cases, mottled or yellow-green mosaic symptoms will appear; plant growth is obviously slow, and the earlier the disease occurs, the more serious the dwarfing will be; the symptoms of fruit disease are relatively obvious, and the diseased fruit is often flat, smaller, and deformed, showing light brown to dark brown necrotic spots, stripes or light yellow mottled, and sometimes a sunken necrotic area will appear, and the fruit loses its commercial quality and edible value.

[0004] PMMoV virus is widely distributed in leaves, petals, anthers, pollen grains, fruits, seeds, roots and other tissues. As a seed-borne virus, it is very easy to spread rapidly with the transportation of pepper seeds and infected seedlings. The virus can also be spread over long distances through human feces, water sources, pepper products, etc., so it has a very high potential for transmission. Therefore, strengthening the quarantine of pepper seeds and pepper products, and removing viruses from seeds to control the source of infection are the most effective measures to control and prevent the risk of pepper seed virus transmission.

[0005] At present, the most commonly used method to deal with pepper virus disease is the trisodium phosphate (TSP) method. However, treating seeds with TSP can only inactivate the virus, but cannot eliminate the virus's infectivity. In addition, although long-term TSP treatment can remove PMMoV in some seeds, it will also reduce the germination rate of pepper seeds and significantly increase the number of abnormal seedlings.

[0006] Although the stem tip virus removal technology can effectively remove viruses from pepper seeds without affecting the quality of the seeds, it is difficult to promote due to its complex procedures, high requirements for operators and environment, and high economic and time costs.

[0007] In addition, there are seed treatment technologies such as dry heat treatment, warm water soaking, dry heat / warm water-agent combined treatment, irradiation, and other chemical agent treatments. However, due to the different characteristics of the seeds, the treatment effects vary and it is difficult to meet the requirements. Therefore, how to simply and efficiently remove the PMMoV virus from pepper seeds while ensuring that the seed quality is not affected is a key issue that needs to be urgently addressed in the current pepper seed virus removal technology. The development of a scientific and reasonable method for removing viruses from pepper seeds is also one of the effective guarantees for the safety of pepper seeds for production, and it is also one of the important driving forces for promoting the sustained and vigorous development of the pepper industry.

[0008] At present, the methods for PMMoV detection at home and abroad include biological methods, electron microscopy technology, enzyme-linked immunosorbent assay and molecular biological assay, with enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (RT-PCR) being the most commonly used. However, if only ELISA or RT-PCR is used to detect seeds after virus removal, the activity of the virus carried by the seeds cannot be confirmed, and the impact of virus removal on seed quality cannot be evaluated.

[0009] Based on the current status of detecting PMMoV virus in pepper seeds, it has become an urgent need to provide a method for removing pepper mild mottle virus from pepper seeds that is simple to operate, fast, efficient and more effective. Summary of the invention

[0010] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for removing pepper mild mottle virus from pepper seeds. The virus removal method provided by the present invention combines protease hydrolysis treatment, photosensitizer and light irradiation treatment, which can effectively remove PMMoV virus from pepper seeds without reducing the germination potential and germination rate of pepper seeds, thereby solving the problems of incomplete virus removal and reduced pepper seed vitality in the prior art.

[0011] The objective of the present invention is achieved through the following technical solutions:

[0012] In a first aspect, the present invention provides a method for removing pepper mild mottle virus from pepper seeds, comprising:

[0013] (1) using a protease aqueous solution capable of hydrolyzing the viral protein shell to hydrolyze the pepper seeds;

[0014] (2) Irradiating the pepper seeds treated in step (1) in a solution containing a photosensitizer to excite the photosensitizer, thereby obtaining pepper seeds free of pepper mild mottle virus, wherein the photosensitizer is a photosensitizer excited by visible light.

[0015] According to some embodiments of the present invention, the protease is subtilisin, such as one or more of savinnase everis 900L, subtilisin A and proteinase K. Among them, savinnase everis 900L is an alkaline protease with high quality and broad spectrum endo-cutting function derived from Bacillus subtilis, which can deeply hydrolyze the protein coat of the virus.

[0016] According to some embodiments of the present invention, the mass concentration of protease in the protease aqueous solution is 0.5-1.5%, preferably 1%.

[0017] According to some embodiments of the present invention, the volume of the protease aqueous solution is 3 to 10 times the volume of the pepper seeds.

[0018] According to some embodiments of the present invention, the protease hydrolysis treatment comprises soaking the pepper seeds in an aqueous solution of protease for 10 to 20 minutes; preferably, the aqueous solution of protease is stirred during the soaking process, for example, stirred 1 to 2 times.

[0019] According to some embodiments of the present invention, the protease hydrolysis treatment further comprises washing the pepper seeds with clean water after the soaking is completed, preferably washing 3 to 5 times, and finally soaking the pepper seeds with clean water, preferably soaking for 10 to 20 minutes.

[0020] According to some embodiments of the invention, the protease hydrolysis treatment is performed at room temperature.

[0021] According to some embodiments of the present invention, before the pepper seeds are subjected to protease hydrolysis treatment, the pepper seeds are pre-soaked in clean water for 3 to 10 hours.

[0022] According to some embodiments of the present invention, the visible light excited photosensitizer is selected from one or more of methylene blue (MB), dihydrochlorin E6, tetraphenylporphyrin and 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine zinc, preferably methylene blue.

[0023] According to some embodiments of the present invention, the wavelength range of the visible light is 400-750 nm.

[0024] According to some embodiments of the present invention, light irradiation is performed using light of the maximum absorption wavelength of the photosensitizer excited by visible light.

[0025] According to some embodiments of the present invention, the solution containing the photosensitizer is an aqueous solution of methylene blue.

[0026] According to some embodiments of the present invention, the concentration of the photosensitizer in the photosensitizer-containing solution is 150-300 μg / mL, preferably 250-300 μg / mL.

[0027] According to some embodiments of the present invention, the volume of the photosensitizer-containing solution is 3 to 10 times the volume of the pepper seeds.

[0028] According to some embodiments of the present invention, H2O2 is added to the photosensitizer-containing solution before light irradiation; preferably, before adding H2O2, the pepper seeds are soaked in the photosensitizer-containing solution for 16 to 18 hours, and then part of the photosensitizer-containing solution is removed; preferably, the concentration of H2O2 in the photosensitizer-containing solution is 1 to 10 mM; preferably, H2O2 is added in the form of an aqueous solution.

[0029] According to some embodiments of the present invention, the light irradiation is performed for 2 to 6 hours, preferably 4 to 6 hours.

[0030] According to some embodiments of the present invention, after the light irradiation, the pepper seeds are washed with clean water, preferably washed 3 to 5 times, and finally the pepper seeds are soaked in clean water, preferably for 15 minutes.

[0031] According to some embodiments of the present invention, during the light irradiation process, the photosensitizer-containing solution containing pepper seeds is stirred, for example, stirred 2 to 3 times.

[0032] Figure 1 The photochemical reaction principle diagram of the light irradiation treatment of the present invention is shown. Figure 1 It can be seen that the use of light of a specific wavelength to make the solution containing the photosensitizer selectively absorb light energy will promote the occurrence of photochemical reactions. For example, methylene blue (MB) will enter an excited state after absorbing light in the wavelength range of 650-670nm, and then transfer energy to the surrounding oxygen to generate highly active singlet oxygen. These active molecules can undergo oxidation reactions with nearby biological macromolecules (nucleic acids, proteins, lipids, etc.), damaging their structure and function; at the same time, a certain amount of H2O2 is added to the solution, and with the participation of catalase in the plant body, it decomposes to produce O2, increasing the surrounding oxygen concentration, promoting the production of singlet oxygen to a certain extent, and thereby enhancing the photochemical reaction.

[0033] The inventors of the present invention have found, based on the principle of photodynamic therapy and after a large number of experiments and studies, that in view of the easy and widespread spread of the PMMoV virus, a protease and a photosensitizer are used for combined treatment. After the protease destroys the PMMoV virus protein shell, the photosensitizer can enter the virus more quickly, and then destroy the nucleic acid and protein under light irradiation, making the virus unable to replicate and lose its infectivity. Furthermore, the addition of a certain concentration of H2O2 can generate oxygen through the catalysis of catalase in pepper seeds, promote the generation of singlet oxygen, and further enhance the effect of virus removal. Moreover, since the reaction products are only oxygen and water, compared with other chemical reagent treatment methods, the virus removal method of the present invention is more environmentally friendly.

[0034] According to some preferred embodiments of the present invention, the method comprises the steps of:

[0035] (1) soaking the pepper seeds in a 0.5-1.5% protease aqueous solution for 10-20 minutes for protease hydrolysis, wherein the volume of the protease aqueous solution is 3-10 times the volume of the pepper seeds;

[0036] (2) Soaking the pepper seeds treated in step (1) in a water-soluble photosensitizer aqueous solution excited by red light at a concentration of 150-300 μg / mL for 16-18 hours, wherein the volume of the photosensitizer aqueous solution is 3-10 times the volume of the pepper seeds; removing part of the photosensitizer aqueous solution so that the volume of the photosensitizer aqueous solution is 1-4 times the volume of the pepper seeds, adding a H2O2 aqueous solution so that the concentration of H2O2 in the photosensitizer aqueous solution is 1-10 mM, and irradiating the seeds with light having the maximum absorption wavelength of the photosensitizer for 2-6 hours, thereby obtaining pepper seeds free of pepper mild mottle virus.

[0037] According to some preferred embodiments of the present invention, the method comprises the steps of:

[0038] (1) Hydrolase treatment:

[0039] At room temperature, the pepper seeds are pre-soaked in clean water for 5 hours, the clean water is removed, and then the pepper seeds are soaked in a savinnase everis 900L aqueous solution with a mass ratio of 0.5-1.5% for 10-20 minutes for protease hydrolysis treatment, wherein the volume of the protease aqueous solution is 3-10 times the volume of the pepper seeds, and the pepper seeds are stirred 1-2 times during the soaking process; after the soaking is completed, the pepper seeds are washed with clean water for 3-5 times, and finally the pepper seeds are soaked in clean water for 15 minutes, and the clean water is discarded;

[0040] Preferably, the room temperature is 20-30°C; preferably, the mass ratio of the savinnase everis 900L aqueous solution is 1%; preferably, the soaking time of the savinnase everis 900L aqueous solution is 20 minutes; the volume of the savinnase everis 900L aqueous solution is 5 times the volume of the pepper seeds.

[0041] (2) Photosensitizer and light irradiation treatment:

[0042] At room temperature, the pepper seeds treated in step (1) are soaked in a methylene blue aqueous solution with a concentration of 150-300 μg / mL for 16-18 hours, wherein the volume of the methylene blue aqueous solution is 3-10 times the volume of the pepper seeds; after the soaking is completed, a portion of the methylene blue aqueous solution is removed so that the volume of the remaining methylene blue aqueous solution is 1-4 times the volume of the pepper seeds, an H2O2 aqueous solution (concentration: 840 mM) is added so that the concentration of H2O2 in the photosensitizer aqueous solution is 1-10 mM, and light of 650-670 nm is used for 2-6 hours, and stirring is performed 2-3 times; after the light irradiation, the pepper seeds are washed with clean water for 3-5 times, and finally the pepper seeds are soaked in clean water for 15 minutes, and the clean water is discarded;

[0043] Preferably, the room temperature is 20-30°C; preferably, the concentration of the methylene blue aqueous solution (MB solution) is 250 μg / mL; the volume of the methylene blue aqueous solution is 5 times the volume of the pepper seeds, and part of the methylene blue aqueous solution is removed so that the ratio of the volume of the remaining methylene blue aqueous solution to the volume of the pepper seeds is 2 times; preferably, the concentration of H2O2 in the methylene blue aqueous solution is 10 mM; preferably, the light irradiation is 4 hours.

[0044] (3) Drying:

[0045] The pepper seeds treated in step (2) are placed on absorbent paper, initially dried at room temperature, and then placed in a disposable culture dish and dried in an electric blast drying oven at 45°C for 72 hours.

[0046] According to some embodiments of the present invention, the method further comprises performing virus detection on pepper seeds free of pepper mild mottle virus, wherein the virus detection is performed using a reverse transcription-loop-mediated isothermal amplification method.

[0047] According to some embodiments of the present invention, the virus detection comprises:

[0048] S1: extracting total RNA from non-virus-free pepper seeds and virus-free pepper seeds, and / or total RNA from seedlings obtained from non-virus-free pepper seeds and seedlings obtained from virus-free pepper seeds;

[0049] S2: Use the reverse transcription-loop-mediated isothermal amplification method to determine whether PMMoV virus exists in non-virus-free pepper seeds, virus-free pepper seeds, and / or seedlings obtained from non-virus-free pepper seeds and pepper seedlings obtained from virus-free pepper seeds.

[0050] According to some preferred embodiments of the present invention, the nucleotide sequences of the primers used in the reverse transcription-loop-mediated isothermal amplification method are shown as SEQ ID NO. 1 to SEQ ID NO. 6, respectively.

[0051] According to some embodiments of the present invention, the method further comprises conducting a germination test on pepper seeds free of pepper mild mottle virus.

[0052] According to some embodiments of the invention, the germination detection comprises:

[0053] The virus-free pepper seeds were germinated and the germination potential and germination rate of the pepper seeds were calculated.

[0054] In the present invention, the calculation formula of germination rate is:

[0055] Germination rate (%) = total number of pepper seeds germinated within 14 days / total number of pepper seeds × 100;

[0056] The calculation formula for germination potential is:

[0057] Germination potential (%) = total number of pepper seeds germinated within 7 days / total number of pepper seeds × 100.

[0058] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0059] 1. Compared with the existing pepper seed virus removal method, the protease hydrolysis treatment and photosensitizer and light irradiation treatment provided by the present invention can remove PMMoV virus from pepper seeds more thoroughly;

[0060] 2. The pepper seed virus removal method of the present invention has low requirements on the environment, temperature, equipment and professional personnel, can be used in laboratories or major production enterprises all year round, has strong practicality, and provides technical support for the long-term development of the pepper industry;

[0061] 3. The method for removing virus from pepper seeds of the present invention uses a photosensitizer whose excitation light is visible light, such as methylene blue. After research, it was found that the excitation light with a wavelength range of 400-750nm can remove the PMMoV virus in pepper seeds to the maximum extent through the photosensitizer, and the light of this wavelength will not have an adverse effect on the pepper seeds; further, the water-soluble photosensitizer is more conducive to the removal of the agent after the virus removal treatment is completed, will not affect the quality of the pepper seeds, will not reduce the germination rate and germination potential of the seeds, and solves the problem of incomplete virus removal and reduced pepper seed vitality in the prior art;

[0062] 4. Compared with the serological technology and RT-PCR detection methods commonly used in the prior art, the pepper seed virus removal method of the present invention can realize the combination of virus detection and germination detection by RT-LAMP, which can not only confirm whether the seeds are successfully virus-free and the virus removal effect with high sensitivity and high specificity, but also confirm the impact of virus removal on the germination rate and germination potential of seeds and whether the germinated leaves are completely virus-free, thus solving the problem that traditional detection methods (such as enzyme-linked immunosorbent assay, fluorescent quantitative PCR, etc.) cannot effectively judge virus activity and seed vitality by directly detecting seeds;

[0063] 5. The pepper seed virus removal method provided by the present invention is a more efficient, economical, green, environmentally friendly and easy-to-promote means of combating PMMoV virus disease, which can promote the prevention and control of pepper virus disease and the breeding of non-toxic pepper seedlings, and escort the long-distance transportation of seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0065] Figure 1 The photochemical reaction principle diagram of the light irradiation treatment of the present invention; wherein S0 represents the singlet methylene blue in the ground state, S1 represents the singlet methylene blue in the excited state, and T1 represents the triplet methylene blue in the excited state;

[0066] Figure 2 The PMMoV virus detection results of pepper seeds treated by four treatment methods in Example 1 of the present invention are shown; wherein curve a represents 10% Na3PO4 aqueous solution treatment for 30 min; curve b represents 10% Na3PO4 aqueous solution treatment for 1 h; curve c represents 10% Na3PO4 aqueous solution treatment for 1.5 h; curve d represents 10% Na3PO4 aqueous solution treatment for 2 h; curve NEG represents negative control; curve C represents untreated seeds (positive control);

[0067] Figure 3The PMMoV virus detection results of pepper seeds treated by six treatment methods in Example 2 of the present invention; wherein, curve a represents 0.5% savinnase everis 900L aqueous solution treatment for 10 min; curve b represents 0.5% savinnase everis 900L aqueous solution treatment for 20 min; curve c represents 1% savinnase everis 900L aqueous solution treatment for 10 min; curve d represents 1% savinnase everis 900L aqueous solution treatment for 20 min; curve e represents 1.5% savinnase everis 900L aqueous solution treatment for 10 min; curve f represents 1.5% savinnase everis 900L aqueous solution treatment for 20 min; curve NEG represents negative control; curve C represents untreated seeds (positive control);

[0068] Figure 4 The PMMoV virus detection results of pepper seeds treated by four treatment methods in Example 3 of the present invention are shown; wherein, curve a represents the treatment with 10% Na3PO4+150 μg / mL MB+10 mM H2O2 aqueous solution; curve b represents the treatment with 10% Na3PO4+200 μg / mL MB+10 mM H2O2 aqueous solution; curve c represents the treatment with 10% Na3PO4+250 μg / mL MB+10 mM H2O2 aqueous solution; curve d represents the treatment with 10% Na3PO4+300 μg / mL MB+10 mM H2O2 aqueous solution; curve NEG represents the negative control; curve C represents the untreated seeds (positive control);

[0069] Figure 5 The PMMoV virus detection results of pepper seeds treated by four treatment methods in Example 4 of the present invention; wherein, curve a represents 1% savinnase everis 900L+150 μg / mL MB+10 mM H2O2 aqueous solution treatment; curve b represents 1% savinnase everis 900L+200 μg / mL MB+10 mM H2O2 aqueous solution treatment; curve c represents 1% savinnase everis 900L+250 μg / mL MB+10 mM H2O2 aqueous solution treatment; curve d represents 1% savinnase everis 900L+300 μg / mL MB+10 mM H2O2 aqueous solution treatment; curve NEG represents the negative control; curve C represents untreated seeds (positive control);

[0070] Figure 6The PMMoV virus detection results of pepper seeds treated by three treatment methods in Example 5 of the present invention; wherein, curve a represents 1% savinnase everis 900L+250 μg / mL MB+10 mM H2O2 aqueous solution treatment for 2 hours; curve b represents 1% savinnase everis 900L+250 μg / mL MB+10 mM H2O2 aqueous solution treatment for 4 hours; curve c represents 1% savinnase everis 900L+250 μg / mL MB+10 mM H2O2 aqueous solution treatment for 6 hours; curve NEG represents the negative control; curve C represents the untreated seeds (positive control);

[0071] Figure 7 The PMMoV virus detection results in the germinated seedlings of the same batch of pepper seeds treated with various treatment methods in Example 6 of the present invention; wherein, curves a1-a4 represent the detection results of the control group; curves b1-b4 represent the detection results of 10% Na3PO4+250 μg / mL MB+10 mM H2O2 aqueous solution treatment; curves c1-c4 represent the detection results of 1% savinnaseeveris 900L+250μg / mL MB+10 mM H2O2 aqueous solution treatment; NEG represents the negative control;

[0072] Figure 8 The PMMoV virus detection results of virus-carrying pepper seeds No. 1-14 treated by treatment method 20 of Example 5 in Example 7 of the present invention; wherein, curves a1-a14 represent the detection results of 14 batches of pepper seeds that have not been treated; curves b1-b14 represent the detection results of 14 batches of pepper seeds treated by treatment method 20 of Example 5; NEG represents a negative control;

[0073] Fig. 9 These are the PMMoV virus detection results of virus-carrying pepper seeds numbered 15-28 that were treated with treatment method 20 of Example 5 in Example 7 of the present invention; wherein curves a1-a14 represent the detection results of 14 untreated batches of pepper seeds; curves b1-b14 represent the detection results of 14 batches of pepper seeds that were treated with treatment method 20 of Example 5; and NEG represents a negative control. DETAILED DESCRIPTION

[0074] The present invention is described in further detail below in conjunction with specific embodiments, and the following examples enable those skilled in the art to better understand the present invention. However, it should also be readily understood by those skilled in the art that the following examples are merely illustrative and are not intended to limit the present invention to these specific embodiments. It should be appreciated by those skilled in the art that the present invention will encompass all improvements, alternatives, and equivalents included within the scope of the claims.

[0075] Approximate terms used in the specification and claims of this application are used to modify quantities, which means that the present invention is not limited to the specific quantity, but also includes the modified parts that are close to the quantity and acceptable but do not cause the relevant basic functions to change. In the specification and claims of this application, range limits can be combined and / or interchanged. If not otherwise specified, these ranges also include all values ​​and sub-ranges contained therein.

[0076] In the following examples, unless otherwise specified, the concentrations are all mass percentage concentrations, and the solvents of the solutions are all water. Total RNA extracted from pepper seeds carrying PMMoV virus was used as a positive control.

[0077] In the following examples, the plant total RNA extraction kit used is a commercial kit, the product name is RNAEasy Fast Plant Tissue RNA Rapid Extraction Kit (Catalog Number: DP452), purchased from Root Biochemical Technology (Beijing) Co., Ltd.; RT-LAMP reaction components were purchased from NEB, and the kit name is: WarmStart LAMP Kit (DNA & RNA) Protocol (E1700); savinnase everis 900L was purchased from NOVOZYMES; pepper seed samples were provided by Hunan Xiangyan Seed Co., Ltd. and Changsha Customs Plant Inspection Department; other materials, reagents, etc., unless otherwise specified, can be obtained from commercial channels.

[0078] In the following examples, the primer mixture consists of 6 specific primers and nuclease-free water. The sequences of the 6 specific primers are obtained as follows:

[0079] The PMMoV virus target sequence retrieved from the US gene database was analyzed for homology using the NCBI BLAST software to obtain a specific conserved sequence, and then the primer combination for LAMP detection of PMMoV virus was designed using the Primer design V5 software based on the conserved target sequence. Among them, the GenBank number of the PMMoV virus-specific conserved gene sequence is: LC082099.1, and its position is 5685-6158. These 6 specific primers were synthesized by General Bio (Anhui) Co., Ltd., and their specific primer sequences and concentrations in the RT-LAMP reaction system are shown in Table 1 below.

[0080] Table 1 Primer composition and sequence of PMMoV virus

[0081]

[0082] Example 1 Using Na3PO4 aqueous solution to remove viruses for different time periods

[0083] a. At room temperature, 100 pepper seeds with virus were taken for each treatment, pre-soaked in clean water for 5 hours, the clean water was removed after the pre-soaking, and then soaked in 5 times the volume of 10% Na3PO4 aqueous solution, stirring 2 to 3 times during the soaking period; the soaking time was 30 minutes, 1 hour, 1.5 hours, and 2 hours respectively;

[0084] b. After soaking, remove the Na3PO4 aqueous solution and wash the seeds with clean water for 3 to 5 times. Soak the seeds in clean water for 15 minutes for the last time. After discarding the clean water, immediately place the seeds on absorbent paper and dry them at room temperature.

[0085] c. Virus detection:

[0086] (1) Extraction of total RNA from pepper seeds: Fifty pepper seeds that had been virus-free and 50 pepper seeds that had not been virus-free were randomly selected and ground into powder using a mortar. RNA was extracted from the ground samples using a plant tissue RNA extraction kit and stored at -80°C for later use.

[0087] (2) Prepare the RT-LAMP reaction system: 12.5 μL of WarmStart LAMP 2X Master Mix, 1 μL of fluorescent dye LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, and 5 μL of template (total RNA extracted from four different types of pepper seeds that have been treated with different virus removal methods in step (1)). The total RNA extracted from pepper seeds that have not been treated with virus removal methods in step (1) was used as the positive control, and nuclease-free water was used as the negative control.

[0088] (3) Instrument detection:

[0089] Reaction procedure: 25 μL reaction system, 63°C for 50 min, and fluorescence signal acquisition once per minute.

[0090] The processing method of Example 1 is summarized in Table 2 below.

[0091] Table 2 Treatment method of Example 1

[0092] serial number Treatment Treatment method 1 <![CDATA[10% aqueous Na3PO4 solution treatment for 30 min]]> Treatment method 2 <![CDATA[1h treatment with 10% aqueous Na3PO4 solution]]> Treatment method three <![CDATA[1.5 h treatment with 10% aqueous Na3PO4 solution]]> Treatment method 4 <![CDATA[Treatment with 10% aqueous Na3PO4 solution for 2 h]]>

[0093] Figure 2 The virus detection results of the seeds using four treatment methods to remove PMMoV virus in Example 1 are shown in FIG. Figure 2 The test results show that treatment with 10% Na3PO4 aqueous solution for 30 minutes, 1 hour, 1.5 hours and 2 hours respectively causes the peak time to be postponed as the treatment time increases, indicating that treatment with 10% Na3PO4 aqueous solution alone has a certain effect, but it is difficult to completely remove the virus in pepper seeds.

[0094] Example 2 Using different concentrations of subtilisin to remove viruses for different periods of time

[0095] a. Take 100 virus-infected pepper seeds at room temperature, pre-soak them in clean water for 5 hours, remove the clean water after pre-soaking, and then soak them in 5 times the volume of Savinnase Everis 900L aqueous solution, stirring 1 to 2 times during soaking. The mass concentrations of Savinnase Everis 900L aqueous solution are 0.5%, 1%, and 1.5%, respectively, and the three concentrations are treated for 10 minutes and 20 minutes, respectively.

[0096] a. After soaking, remove the Savinnase Everis 900L aqueous solution, wash the seeds with clean water for 3 to 5 times, and soak them in clean water for 15 minutes for the last time. After discarding the clean water, immediately place the seeds on absorbent paper and dry them at room temperature.

[0097] c. Virus detection:

[0098] (1) Extraction of total RNA from pepper seeds: Fifty pepper seeds that had been virus-free and 50 pepper seeds that had not been virus-free were randomly selected and ground into powder using a mortar. RNA was extracted from the ground samples using a plant tissue RNA extraction kit and stored at -80°C for later use.

[0099] (2) Prepare the RT-LAMP reaction system: 12.5 μL of WarmStart LAMP 2X Master Mix, 1 μL of fluorescent dye LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, and 5 μL of template (total RNA extracted from 6 different types of pepper seeds that have been treated with different virus removal methods in step (1)). The total RNA extracted from pepper seeds that have not been treated with virus removal methods in step (1) was used as the positive control, and nuclease-free water was used as the negative control.

[0100] (3) Instrument detection:

[0101] Reaction procedure: 25 μL reaction system, 63°C for 50 min, and fluorescence signal acquisition once per minute.

[0102] The processing method of Example 2 is summarized in Table 3 below.

[0103] Table 3 Treatment method of Example 2

[0104] serial number Treatment Treatment method five 0.5% savinnase everis 900L aqueous solution treatment for 10 minutes Treatment method six 0.5% savinnase everis 900L aqueous solution treatment for 20 minutes Treatment method seven 1% savinnase everis 900L aqueous solution treatment for 10 minutes Treatment method eight 1% savinnase everis 900L aqueous solution treatment for 20 minutes Treatment method nine 1.5% savinnase everis 900L aqueous solution treatment for 10 minutes Treatment method 10 1.5% savinnase everis 900L aqueous solution treatment for 20 minutes

[0105] Figure 3 The virus detection results of the seeds from which PMMoV virus was removed using six treatment methods in Example 2 are shown in FIG. Figure 3 The test results show that when treated with 0.5%~1.5% savinnase everis 900L aqueous solution for 10min or 20min, the peak time is postponed with the increase of treatment concentration and the extension of treatment time, indicating that treatment with protease solution alone has a certain effect.

[0106] Example 3 Virus removal using Na3PO4 aqueous solution and MB aqueous solution of different concentrations

[0107] a. Na3PO4 aqueous solution treatment: At room temperature, take 100 pepper seeds with virus at a time, pre-soak them in clean water for 5 hours, remove the clean water after pre-soaking, and then soak them in 5 times the volume of 10% Na3PO4 aqueous solution for 2 hours, stirring 2-3 times during soaking. After soaking, remove the Na3PO4 aqueous solution, wash with clean water 3-5 times, soak in clean water for 15 minutes for the last time, and discard the clean water.

[0108] b. MB solution treatment: Take the seeds cleaned in step a above, soak them in MB aqueous solution of different concentrations for 16 hours, discard part of the MB solution after soaking, make the ratio of MB solution to seed volume 2: 1, further add a certain volume of H2O2 solution (concentration: 840mM) to this system, make the final concentration of H2O2 10mM, irradiate for 6 hours with a spotlight (power 30w) with a wavelength range of 655-665nm, stir the seeds 2-3 times during irradiation; after irradiation, wash with clean water 3-5 times, soak in clean water for the last time for 15 minutes, discard the clean water and immediately place the seeds on absorbent paper and dry at room temperature. The concentrations of MB aqueous solution used are 150μg / mL, 200μg / mL, 250μg / mL, and 300μg / mL, respectively. These 4 concentrations must be verified separately according to the above method to confirm their virus removal effect.

[0109] c. Virus detection:

[0110] (1) Extraction of total RNA from pepper seeds: Fifty pepper seeds that had been virus-free and 50 pepper seeds that had not been virus-free were randomly selected and ground into powder using a mortar. RNA was extracted from the ground samples using a plant tissue RNA extraction kit and stored at -80°C for later use.

[0111] (2) Prepare the RT-LAMP reaction system: 12.5 μL of WarmStart LAMP 2X Master Mix, 1 μL of fluorescent dye LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, and 5 μL of template (total RNA extracted from four different types of pepper seeds that have been treated with different virus removal methods in step (1)). The total RNA extracted from pepper seeds that have not been treated with virus removal methods in step (1) was used as the positive control, and nuclease-free water was used as the negative control.

[0112] (3) Instrument detection:

[0113] Reaction procedure: 25 μL reaction system, 63°C for 50 min, and fluorescence signal acquisition once per minute.

[0114] The processing methods of Example 3 are summarized in Table 4 below.

[0115] Table 4 Treatment method of Example 3

[0116] serial number Treatment Treatment method 11 Treatment with an aqueous solution of 10% Na3PO4 + 150 μg / mL MB + 10 mM H2O2 Treatment method 12 Treatment with an aqueous solution of 10% Na3PO4 + 200 μg / mL MB + 10 mM H2O2 Treatment Method Thirteen Treatment with an aqueous solution of 10% Na3PO4 + 250 μg / mL MB + 10 mM H2O2 Treatment method 14 Treatment with an aqueous solution of 10% Na3PO4 + 300 μg / mL MB + 10 mM H2O2

[0117] Figure 4 The virus detection results of the seeds using four treatment methods to remove PMMoV virus in Example 3 are shown in Table 3. Figure 4The test results show that when the treatment time of 10% Na3PO4 aqueous solution is fixed, as the concentration of MB solution gradually increases, the peak time is gradually postponed, but the difference is small, indicating that the detoxification efficiency of PMMOV virus is still low and the detoxification scheme needs to be further optimized.

[0118] Example 4 Virus Removal Using 1% Subtilisin Aqueous Solution and Different Concentrations of MB Aqueous Solution

[0119] a. Subtilisin treatment: At room temperature, take 100 poisonous pepper seeds for each treatment, pre-soak them in clean water for 5 hours, remove the clean water after pre-soaking, and then soak them in 5 times the volume of 1% savinnase everis 900L aqueous solution for 20 minutes, stirring 1 to 2 times during the soaking period. After soaking, remove the savinnase everis 900L aqueous solution, wash with clean water 3-5 times, and soak them in clean water for 15 minutes for the last time, and discard the clean water.

[0120] b. MB aqueous solution treatment: take the seeds cleaned in step a above, and soak them in MB aqueous solution of different concentrations for 16 hours. After soaking, discard part of the MB aqueous solution to make the ratio of MB aqueous solution to seed volume 2: 1, further add a certain volume of H2O2 aqueous solution (concentration: 840mM) to this system to make the final concentration of H2O2 10mM, and irradiate them with a spotlight with a wavelength range of 655-665nm (power 30w) for 6 hours. Stir the seeds 2~3 times during the irradiation period; after irradiation, wash them with clean water 3-5 times, and soak them in clean water for the last time for 15 minutes. After discarding the clean water, immediately place the seeds on absorbent paper and dry them at room temperature.

[0121] The concentrations of the MB aqueous solutions used were 150 μg / mL, 200 μg / mL, 250 μg / mL, and 300 μg / mL, respectively. These four concentrations must be verified separately according to the above method to confirm their virus removal effects.

[0122] c. Virus detection

[0123] (1) Extraction of total RNA from pepper seeds: Fifty pepper seeds that had been virus-free and 50 pepper seeds that had not been virus-free were randomly selected and ground into powder using a mortar. RNA was extracted from the ground samples using a plant tissue RNA extraction kit and stored at -80°C for later use.

[0124] 2) Prepare the RT-LAMP reaction system: 12.5 μL of WarmStart LAMP 2X Master Mix, 1 μL of fluorescent dye LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, and 5 μL of template (total RNA extracted from four different types of pepper seeds that have been treated with different virus removal methods in step (1)). The total RNA extracted from pepper seeds that have not been treated with virus removal methods in step (1) was used as the positive control, and nuclease-free water was used as the negative control.

[0125] (3) Instrument detection:

[0126] Reaction procedure: 25 μL reaction system, 63°C for 50 min, and fluorescence signal acquisition once per minute.

[0127] The processing method of Example 4 is summarized in Table 5 below.

[0128] Table 5 Treatment method of Example 4

[0129] serial number Treatment Treatment method 15 <![CDATA[Treatment with 1% savinnase everis 900L + 150 μg / mL MB + 10 mM H2O2 aqueous solution]]> Treatment method 16 <![CDATA[Treatment with 1% savinnase everis 900L + 200 μg / mL MB + 10 mM H2O2 aqueous solution]]> Treatment method 17 <![CDATA[Treatment with 1% savinnase everis 900L + 250 μg / mL MB + 10 mM H2O2 aqueous solution]]> Treatment method 18 <![CDATA[Treatment with 1% savinnase everis 900L + 300 μg / mL MB + 10 mM H2O2 aqueous solution]]>

[0130] Figure 5 The virus detection results of the seeds using four treatment methods to remove PMMoV virus in Example 4 are shown in Table 4. Figure 5 The test results show that the peak time of curve a is less than 35 minutes, the peak time of curve b is greater than 35 minutes, and the gene tests of curves c and d are both negative. The peak time of curve b is greater than 35 minutes, but it cannot be ruled out that there is still a very small amount of inactivated virus in the seeds due to the reason that it is below the detection limit. It can be seen that the 1% savinnaseeveris 900L+250 μg / mL MB+10 mM H2O2 aqueous solution corresponding to curve c is the lowest dose chemical agent combination that can achieve complete removal of PMMoV virus.

[0131] Example 5 Effect of light irradiation treatment duration

[0132] a. Subtilisin treatment: At room temperature, take 100 virus-carrying pepper seeds for each treatment, pre-soak them in clean water for 5 hours, remove the clean water after pre-soaking, and then soak them in 5 times the volume of 1% savinnase everis 900L aqueous solution for 20 minutes, stirring 1 to 2 times during the soaking period. After soaking, remove the savinnase everis 900L aqueous solution, wash with clean water 3-5 times, and soak them in clean water for 15 minutes for the last time, and discard the clean water.

[0133] b. MB aqueous solution treatment: take the seeds cleaned in step a above, soak them in 250 μg / mL MB solution for 16 hours, discard part of the MB aqueous solution after soaking, so that the ratio of MB aqueous solution to seed volume is 2:1, further add a certain volume of H2O2 aqueous solution (concentration: 840mM) to this system, so that the final concentration of H2O2 is 10mM, use a spotlight with a wavelength range of 655-665nm (power 30w) for different lengths of time to irradiate, and stir the seeds 2~3 times during the irradiation period; after irradiation, wash with clean water 3-5 times, and soak in clean water for the last time for 15min. After discarding the clean water, immediately place the seeds on absorbent paper and dry them at room temperature.

[0134] The different durations of spotlight irradiation are 2h, 4h, and 6h respectively. These three irradiation durations must be verified separately according to the above method to confirm their virus removal effects.

[0135] c. Virus detection

[0136] (1) Extraction of total RNA from pepper seeds: Fifty pepper seeds that had been virus-free and 50 pepper seeds that had not been virus-free were randomly selected and ground into powder using a mortar. RNA was extracted from the ground samples using a plant tissue RNA extraction kit and stored at -80°C for later use.

[0137] 2) Prepare the RT-LAMP reaction system: 12.5 μL of WarmStart LAMP 2X Master Mix, 1 μL of fluorescent dye LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, and 5 μL of template (total RNA extracted from three different types of pepper seeds that have been treated with different virus removal methods in step (1)). The total RNA extracted from pepper seeds that have not been treated with virus removal methods in step (1) was used as the positive control, and nuclease-free water was used as the negative control.

[0138] (3) Instrument detection: Reaction procedure: 25 μL reaction system, 63°C reaction for 50 min, fluorescence signal acquisition once per minute.

[0139] The processing method of Example 5 is summarized in Table 6 below.

[0140] Table 6 Treatment method of Example 5

[0141] serial number Treatment Treatment method 19 <![CDATA[Treatment with 1% savinnase everis 900L + 250 μg / mL MB + 10 mM H2O2 aqueous solution for 2 hours]]> Treatment Method 20 <![CDATA[Treatment with an aqueous solution of 1% savinnase everis 900L + 250 μg / mL MB + 10 mM H2O2 for 4 hours]]> Treatment Method 21 <![CDATA[6-hour treatment with 1% savinnase everis 900L + 250 μg / mL MB + 10 mM H2O2 aqueous solution]]>

[0142] Figure 6 The results of virus detection of seeds using three treatment methods to remove PMMoV virus in Example 5 are shown in Table 2. Figure 6The test results show that PMMoV gene detection is positive after 2 hours of light irradiation, while PMMoV gene detection is negative after 4 and 6 hours of light irradiation, indicating that light irradiation for 4 hours and 6 hours can achieve the purpose of completely removing PMMoV virus, and the shorter light irradiation time of 4 hours is a more preferred detoxification method.

[0143] Example 6 Effects of different virus removal methods on pepper seed vitality and virus removal effects on leaves

[0144] In this example, the germination rate and germination potential of seeds were measured according to the method described in the national standard GB / T 3543.4-1995 "Regulations for Inspection of Crop Seeds: Germination Test", and the seed vigor was evaluated by calculating the germination potential and germination rate to determine the effects of the various virus removal methods in Examples 1-5 on seed vigor.

[0145] (1) Seed virus removal and germination

[0146] Virus-carrying pepper seeds were taken at a number of 200 seeds per treatment, and treatment method 4, treatment method 5, treatment method 6, treatment method 7, treatment method 8, treatment method 9, treatment method 10, modified treatment method 13 (the only difference from treatment method 13 is that the light irradiation time in step b is changed from 6 h to 4 h), and treatment method 20 in Examples 1 to 5 were selected. The seeds treated by the above treatment methods were pre-soaked in clean water at room temperature for 5 h, divided into 4 groups, 40 to 60 seeds / group, and placed on absorbent paper for germination at room temperature. During the germination process, attention was paid to replenishing water.

[0147] 2) Recording and comparison of germination conditions

[0148] The germination was counted on the 7th day after the radicle broke through the seed coat by 2 mm, and the proportion was the seed germination potential. The second count was made on the 14th day after germination, and the proportion was the germination rate.

[0149] The germination rate and germination potential were calculated according to the following formula:

[0150] Germination rate (%) = total number of test seeds germinated within 14 days / total number of test seeds × 100;

[0151] Germination potential (%) = total number of test seeds germinated within 7 days / total number of test seeds × 100.

[0152] 3) Detection of PMMoV in seedlings

[0153] In addition, the same batch of seedlings without virus removal were used as controls, and the seeds treated with the modified treatment method 13 and treatment method 20 were tested for PMMoV virus. The specific testing steps are as follows:

[0154] a. For nucleic acid extraction, 40 to 60 seedlings were grouped together and ground into powder using a mortar. RNA was extracted from the ground samples using a plant tissue RNA extraction kit and stored at -80°C for later use.

[0155] b. Prepare RT-LAMP reaction system: WarmStart LAMP 2X Master Mix 12.5μL, fluorescent dye LAMP Fluorescent Dye (10X) 1μL, primer mixture (2x) 2.5μL, nuclease-free water 4μL, template 5μL (total RNA extracted from two different devirus-treated pepper seeds in step (a)). The total RNA extracted from the pepper seeds without devirus-treatment in step (a) was used as the positive control, and the negative control was nuclease-free water.

[0156] c. Instrument detection:

[0157] Reaction procedure: 25 μL reaction system, 63°C for 50 min, and fluorescence signal acquisition once per minute.

[0158] Result analysis:

[0159] 1. The results of germination rate and germination potential of each treatment group are shown in Table 7:

[0160] Table 7 Germination rate and germination potential results of each treatment method

[0161] serial number Treatment Germination potential (%) Germination rate (%) 1 Control (no virus removal) 74 94 2 Treatment method 4 74 94 3 Treatment method five 74 95 4 Treatment method six 75 95 5 Treatment method seven 75 95 6 Treatment method eight 76 96 7 Treatment method nine 75 94 8 Treatment method 10 74 94 9 Modification method 13 76 96 10 Treatment Method 20 78 97

[0162] As shown in Table 7, the germination potential and germination rate of the control seeds were 74% and 94%, respectively. There was no significant difference in the germination potential and germination rate of the pepper seeds treated with 10% Na3PO4 +250μg / mLMB+10 mM H2O2 aqueous solution (modified treatment method thirteen) or 1% savinnase everis900L solution +250μg / mL MB+10 mM H2O2 solution (treatment method twenty), which were slightly higher than those of the control seeds, indicating that the modified treatment method thirteen and treatment method twenty's virus removal method treatment will not only have no adverse effects on the germination potential and germination rate of pepper seeds, but also promote the germination of pepper seeds to a certain extent. In addition, the germination condition was slightly better when treated with 1% savinnase everis 900L aqueous solution (treatment methods 7 and 8) than when treated with 1.5% savinnase everis 900L aqueous solution (treatment methods 9 and 10), so the treatment concentration of savinnase everis 900L aqueous solution can be further preferably 1%.

[0163] 2. Figure 7 The results of the removal of PMMoV virus from the germinated seedlings of the same batch of pepper seeds after the various treatment methods in Example 6 were used. After the leaves germinate, even if there is only a very small amount of active virus in the seeds, it can cause infection of a large number of leaves. That is, the activity of PMMoV virus in the seeds can be further confirmed by virus detection after germination, ensuring that PMMoV virus is completely removed. Figure 7 The test results show that compared with the PMMoV virus content in the control group, the PMMoV virus content in the leaves was reduced after treatment with 10% Na3PO4+250μg / mL MB+10 mM H2O2 aqueous solution (modified treatment method thirteen), but the virus in the pepper seeds could not be completely removed; after using 1% savinnase everis 900L+250μg / mL MB+10mM H2O2 combined treatment (treatment method twentieth), the PMMoV content in the leaves was 0, that is, the virus removal rate for the seeds was 100%.

[0164] Example 7 Effect of the preferred virus removal method on virus removal of different batches of pepper seeds

[0165] In this example, the treatment method 20 in Example 5 was used to remove viruses from 28 types of virus-carrying pepper seeds (see Table 8 below). After the virus removal was completed, nucleic acid was extracted from the virus-removed seeds and the seeds that were not treated for virus removal simultaneously, and the one-step RT-LAMP method was used to perform qualitative detection on the extracted pepper seed nucleic acid to evaluate the virus removal effect of the method on different pepper seeds, so as to confirm the feasibility of the pepper seed virus removal method.

[0166] Table 8 28 seed batch numbers and sources

[0167] Serial number Seed Number source 1 XXZY01 Hunan Xiangyan Seed Co., Ltd. 2 XXZY02 Hunan Xiangyan Seed Co., Ltd. 3 XXZY03 Hunan Xiangyan Seed Co., Ltd. 4 XXZY04 Hunan Xiangyan Seed Co., Ltd. 5 XXZY05 Hunan Xiangyan Seed Co., Ltd. 6 XXZY06 Hunan Xiangyan Seed Co., Ltd. 7 XXZY07 Hunan Xiangyan Seed Co., Ltd. 8 XXZY08 Hunan Xiangyan Seed Co., Ltd. 9 XXZY09 Hunan Xiangyan Seed Co., Ltd. 10 XXZY10 Hunan Xiangyan Seed Co., Ltd. 11 XXZY11 Hunan Xiangyan Seed Co., Ltd. 12 XXZY12 Hunan Xiangyan Seed Co., Ltd. 13 XXZY13 Hunan Xiangyan Seed Co., Ltd. 14 XXZY14 Hunan Xiangyan Seed Co., Ltd. 15 XXZY15 Hunan Xiangyan Seed Co., Ltd. 16 XXZY16 Hunan Xiangyan Seed Co., Ltd. 17 XXZY17 Hunan Xiangyan Seed Co., Ltd. 18 XXZY18 Hunan Xiangyan Seed Co., Ltd. 19 XXZY19 Hunan Xiangyan Seed Co., Ltd. 20 XXZY20 Hunan Xiangyan Seed Co., Ltd. 21 XY03 Changsha Customs Plant Inspection Department 22 1302 Changsha Customs Plant Inspection Department 23 1402 Changsha Customs Plant Inspection Department 24 1501 Changsha Customs Plant Inspection Department 25 1502 Changsha Customs Plant Inspection Department 26 XY13 Changsha Customs Plant Inspection Department 27 XY14 Changsha Customs Plant Inspection Department 28 XY15 Changsha Customs Plant Inspection Department

[0168] Figure 8 The PMMoV virus detection results of the virus-carrying pepper seeds Nos. 1-14 after being treated by the treatment method 20 of Example 5 in Example 7; Fig. 9 The PMMoV virus detection results of virus-carrying pepper seeds No. 15-28 processed by the processing method 20 of Example 5 in Example 7 of the present invention are shown in FIG. Figure 8 and Fig. 9 The test results show that:

[0169] The PMMoV virus detection of 28 batches of pepper seeds treated by the treatment method of the present invention was negative, and the PMMoV virus detection of 28 batches of pepper seeds that were not treated was positive. It can be seen that the treatment method 20 of Example 5 can completely remove the PMMoV virus in the 28 virus-carrying pepper seeds used, and the virus removal efficiency is 100%. In addition, this method can not only effectively remove the PMMoV virus in pepper seeds, but also will not have an adverse effect on the germination potential and germination rate of pepper seeds.

[0170] Although various aspects and embodiments of the present invention are disclosed herein, other aspects and embodiments of the present invention will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and not for limiting purposes. The scope and spirit of the present invention are determined solely by the appended claims.

Claims

1. A method for removing pepper mild mottle virus from pepper seeds, characterized in that: The removal method comprises: (1) using a protease aqueous solution capable of hydrolyzing the viral protein shell to hydrolyze the pepper seeds; (2) Irradiating the pepper seeds treated in step (1) in a solution containing a photosensitizer to excite the photosensitizer, thereby obtaining pepper seeds free of pepper mild mottle virus, wherein the photosensitizer is a photosensitizer excited by visible light.

2. The removal method according to claim 1, characterized in that: The protease is subtilisin.

3. The removal method according to claim 1, characterized in that: The mass concentration of the protease aqueous solution is 0.5-1.5%.

4. The removal method according to claim 1, characterized in that: The volume of the protease aqueous solution is 3 to 10 times the volume of the pepper seeds.

5. The removal method according to claim 1, characterized in that: The protease hydrolysis treatment comprises soaking the pepper seeds in an aqueous solution of protease for 10 to 20 minutes.

6. The removal method according to claim 1, characterized in that: The photosensitizer excited by visible light is selected from one or more of methylene blue, dihydrochlorin E6, tetraphenylporphyrin and 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine zinc.

7. The removal method according to claim 1, characterized in that: The wavelength range of the visible light is 400-750 nm, or light of the maximum absorption wavelength of the photosensitizer excited by the visible light is used for light irradiation.

8. The removal method according to claim 1, characterized in that: The concentration of the photosensitizer in the photosensitizer-containing solution is 150-300 μg / mL.

9. The removal method according to claim 1, characterized in that: The volume of the solution containing the photosensitizer is 3 to 10 times the volume of the pepper seeds.

10. The removal method according to claim 1, characterized in that: Prior to light irradiation, H2O2 was added to the photosensitizer-containing solution.

11. The removal method according to claim 10, characterized in that: The concentration of H2O2 in the photosensitizer-containing solution is 1-10 mM.

12. The removal method according to claim 1, characterized in that: The light irradiation is performed for 2 to 6 hours.

13. The removal method according to any one of claims 1 to 12, characterized in that: The removal method also includes performing virus detection on pepper seeds from which pepper mild mottle virus has been removed, wherein the virus detection is performed using a reverse transcription-loop-mediated isothermal amplification method.

14. The removal method according to any one of claims 1 to 12, characterized in that: The removal method also includes conducting a germination test on pepper seeds free of pepper mild mottle virus.

15. The removal method according to claim 14, characterized in that: The germination detection comprises: germinating the virus-free pepper seeds, and calculating the germination potential and germination rate of the pepper seeds.

Images