A method for removing pepper mild mottle virus from pepper seeds

Abstract

The present invention provides a method for removing pepper mild mottle virus from pepper seeds, which comprises: (1) performing protease hydrolysis treatment on pepper seeds with an aqueous protease solution capable of hydrolyzing the viral protein coat; (2) irradiating the pepper seeds treated in step (1) with light in a solution containing a photosensitizer to excite the photosensitizer, thereby obtaining pepper seeds from which the pepper mild mottle virus has been removed, wherein the photosensitizer is a photosensitizer excited by visible light. The virus removal method provided by the present invention can not only effectively remove the PMMoV virus from pepper seeds compared with the existing methods for combating PMMoV virus disease, but also does not reduce the germination potential and germination rate of pepper seeds, and is more efficient, economical, environmentally friendly and easy to promote; it can promote the prevention and control of pepper virus disease and the breeding of virus-free pepper seedlings, and escort the long-distance transportation of seeds.

Description

Technical Field

[0001] The present 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] Virus diseases are an important type of diseases restricting the production of hot (sweet) peppers in China. Among them, pepper mild mottle virus (PMMoV) has become the main virus species in many regions of China.

[0003] PMMoV virus belongs to the genus Tobamovirus of the family Virgaviridae, which is an 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. When it is severe, mottling or yellow-green mosaic symptoms will appear; the growth of the plant is significantly slow, and the earlier the disease occurs, the more severe the dwarfing; the symptoms of the diseased fruits are relatively obvious. Diseased fruits are often flat, smaller, deformed, showing light brown to dark brown necrotic spots, stripes or light yellow mottles. Sometimes, sunken necrotic parts are produced, and the fruits lose their commercial and edible values.

[0004] PMMoV virus is widely distributed in tissues such as leaves, petals, anthers, pollen grains, fruits, seeds, and roots. As a seed-borne virus, it is extremely easy to spread rapidly along with the transportation of pepper seeds and virus-infected seedlings. Moreover, this virus can also be transmitted over long distances through human feces, water sources, pepper products, etc. Therefore, it has a very large potential for transmission. Therefore, strengthening the quarantine of pepper seeds and pepper products, and treating the seeds to remove the virus, and controlling the source of infection are the most effective measures for controlling and preventing the risk of pepper seed virus transmission.

[0005] Currently, the most commonly used method for dealing with pepper virus diseases is the trisodium phosphate (TSP) method. However, treating seeds with TSP can only inactivate the virus and cannot eliminate the infectivity of the virus. Moreover, although long-term TSP treatment can remove PMMoV from some seeds, it will also reduce the germination rate of pepper seeds and significantly increase the number of abnormal seedlings.

[0006] Although the shoot tip virus removal technology can effectively remove the virus from pepper seeds and does not affect the seed quality, due to its complex procedures, high requirements for operators and the environment, and high economic and time costs, it is not widely applicable and is difficult to promote.

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

[0008] At present, the methods for detecting PMMoV at home and abroad include biological methods, electron microscopy techniques, enzyme - linked immunosorbent assay, and molecular biology assay methods. The enzyme - linked immunosorbent assay (ELISA) and real - time fluorescence quantitative PCR method (RT - PCR) are the most commonly used. However, if only ELISA or RT - PCR is used to detect the seeds after virus removal, it cannot confirm the activity of the virus carried by the seeds, and it is impossible to evaluate the impact of virus removal on seed quality.

[0009] Based on the current situation 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 has better effects. Summary of the Invention

[0010] The purpose of the present invention is to overcome the deficiencies existing in 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. It can not only effectively remove PMMoV virus from pepper seeds, but also does not reduce the germination potential and germination rate of pepper seeds, solving the problems of incomplete virus removal and reduced pepper seed vitality existing in the prior art.

[0011] The purpose of the present invention is achieved by the following technical solutions:

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

[0013] (1) Performing protease hydrolysis treatment on pepper seeds with an aqueous solution of protease capable of hydrolyzing the viral protein coat;

[0014] (2) Irradiating the pepper seeds treated in step (1) with light in a solution containing a photosensitizer to excite the photosensitizer, thereby obtaining pepper seeds from which the pepper mild mottle virus has been removed, 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 derived from Bacillus subtilis with high-quality broad-spectrum endo-functional ability, which can deeply hydrolyze the protein coat of the virus.

[0016] According to some embodiments of the present invention, the mass concentration of the 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 - 10 times the volume of the pepper seeds.

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

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

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

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

[0022] According to some embodiments of the present invention, the photosensitizer excited by visible light is selected from one or more of methylene blue (MB), chlorin e6, tetraphenylporphyrin, and zinc 29H,31H-phthalocyanine octabutoxy derivative, 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 carried out with light having the maximum absorption wavelength of the photosensitizer excited by the 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 3-10 times the volume of the pepper seeds.

[0028] According to some embodiments of the present invention, before the light irradiation, H2O2 is added to the photosensitizer-containing solution; preferably, before adding H2O2, the pepper seeds are soaked in the photosensitizer-containing solution for 16-18 hours, and then part of the photosensitizer-containing solution is removed; preferably, the concentration of H2O2 in the photosensitizer-containing solution is 1-10 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 carried out for 2-6 hours, preferably 4-6 hours.

[0030] According to some embodiments of the present invention, after the light irradiation, the pepper seeds are washed with clean water, preferably 3-5 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, the photosensitizer-containing solution containing the pepper seeds is stirred, for example, 2-3 times.

[0032] Figure 1 Show the photochemical reaction principle diagram of the light irradiation treatment of the present invention. As can be seen from Figure 1 It can be seen that after the photosensitizer-containing solution selectively absorbs light energy by using light of a specific wavelength, it will promote the occurrence of a photochemical reaction. For example, after methylene blue (MB) absorbs light in the wavelength range of 650-670 nm, it will enter the excited state, and then transfer the energy to the surrounding oxygen to generate highly reactive singlet oxygen. These reactive molecules can undergo oxidation reactions with nearby biological macromolecules (nucleic acids, proteins, lipids, etc.), damaging their structures and functions; at the same time, a certain amount of H2O2 is added to the solution, and under the participation of catalase in plants, it decomposes to produce O2, increasing the surrounding oxygen concentration, promoting the generation of singlet oxygen to a certain extent, and further enhancing the photochemical reaction.

[0033] Based on the principle of photodynamic therapy and through a large number of experiments and studies, the inventors of the present invention found that, in view of the characteristics of the easy widespread transmission of PMMoV virus, by using the combined treatment of protease and photosensitizer, after the protease destroys the protein coat of the PMMoV virus, the photosensitizer can enter the virus interior more quickly, and then under light irradiation, it can destroy nucleic acids and proteins, making the virus unable to replicate and lose its infectivity. Further, adding a certain concentration of H2O2 can generate oxygen through the catalysis of catalase in pepper seeds, promoting the generation of singlet oxygen, so as to achieve the purpose of further enhancing the virus removal effect. 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 includes the steps of:

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

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

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

[0038] (1) Hydrolytic enzyme treatment:

[0039] At room temperature, after presoaking pepper seeds in clean water for 5 hours, removing the clean water, then soaking them in an aqueous solution of savinnase everis 900L with a mass ratio of 0.5 - 1.5% for 10 - 20 minutes for protease hydrolysis treatment, wherein the volume of the aqueous protease solution is 3 - 10 times the volume of the pepper seeds, and stirring 1 - 2 times during the soaking process; after the soaking is completed, washing the pepper seeds with clean water 3 - 5 times, and finally soaking the pepper seeds in clean water for 15 minutes, and discarding the clean water;

[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, soak the pepper seeds treated in step (1) in an aqueous methylene blue solution with a concentration of 150 - 300 μg / mL for 16 - 18 hours. The volume of the aqueous methylene blue solution is 3 - 10 times the volume of the pepper seeds; after the soaking is completed, remove part of the aqueous methylene blue solution so that the volume of the remaining aqueous methylene blue solution is 1 - 4 times the volume of the pepper seeds, and add an H2O2 aqueous solution (concentration: 840 mM) so that the concentration of H2O2 in the photosensitizer aqueous solution is 1 - 10 mM. Irradiate with light of 650 - 670 nm for 2 - 6 h and stir 2 - 3 times; after the light irradiation, wash the pepper seeds with clean water 3 - 5 times, and finally soak the pepper seeds in clean water for 15 minutes and discard the clean water;

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

[0044] (3) Drying:

[0045] Place the pepper seeds treated in step (2) on absorbent paper, initially air-dry at room temperature, and then place them in a disposable petri dish and dry them in an electric blast drying oven at 45°C for 72 h.

[0046] According to some embodiments of the present invention, the method further includes virus detection of the pepper seeds from which the pepper mild mottle virus has been removed. Among them, the virus detection is carried out by using the reverse transcription-loop-mediated isothermal amplification method.

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

[0048] S1: Extract the total RNA of the pepper seeds without virus removal and the pepper seeds with virus removal, and / or the total RNA of the seedlings obtained from the pepper seeds without virus removal and the seedlings obtained from the pepper seeds with virus removal;

[0049] S2: Use reverse transcription-loop-mediated isothermal amplification method to determine whether PMMoV virus exists in pepper seeds without virus removal, pepper seeds with virus removal, and / or pepper seedlings obtained from pepper seeds without virus removal and pepper seedlings obtained from pepper seeds with virus removal.

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

[0051] According to some embodiments of the present invention, the method further includes performing germination detection on pepper seeds with pepper mild mottle virus removed.

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

[0053] Germinate the pepper seeds with virus removed, and calculate the germination potential and germination rate of the pepper seeds.

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

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

[0056] The calculation formula for the germination potential is:

[0057] Germination potential (%) = total number of germinated pepper seeds within 7 days / total number of tested 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 methods for removing virus from pepper seeds, the protease hydrolysis treatment, photosensitizer and light irradiation treatment provided by the present invention can remove PMMoV virus from pepper seeds more thoroughly.

[0060] 2. The method for removing virus from pepper seeds of the present invention has low requirements for the environment, temperature, equipment and personnel expertise, and can be used in laboratories or major production enterprises throughout the year, with strong practicability, providing technical support for the long-term development of the pepper industry.

[0061] 3. The method for removing viruses from pepper seeds of the present invention uses a photosensitizer with excitation light being visible light, such as methylene blue. After research, it is found that the excitation light with a wavelength range of 400 - 750 nm can maximally remove PMMoV virus from pepper seeds through the photosensitizer, and this wavelength of light will not have an adverse effect on 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 pepper seeds, will not reduce the germination rate and germination potential of the seeds, and solves the problems of incomplete virus removal and reduced vitality of pepper seeds existing in the prior art;

[0062] 4. Compared with the commonly used serological techniques and RT-PCR detection methods in the prior art, the method for removing viruses from pepper seeds of the present invention can combine RT-LAMP for virus detection and germination detection. It can not only confirm with high sensitivity and high specificity whether the seeds have successfully removed the virus and the virus removal effect, but also confirm the impact on the germination rate and germination potential of the seeds after virus removal and whether the germinated leaves have completely removed the virus, solving the problem that traditional detection methods (such as enzyme-linked immunosorbent assay, fluorescence quantitative PCR, etc.) cannot effectively judge the virus activity and seed vitality when directly detecting seeds;

[0063] 5. The method for removing viruses from pepper seeds provided by the present invention, as a more efficient, economical, green and environmentally friendly means for combating PMMoV virus disease and facilitating popularization, can promote the prevention and control of pepper virus disease and the breeding of virus-free pepper seedlings, and escort the long-distance transportation of seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, where:

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

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

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

[0068] Figure 4 Detection results of PMMoV virus in pepper seeds treated with four treatment methods in Example 3 of the present invention; among them, curve a represents treatment with 10% Na3PO4 + 150 μg / mL MB + 10 mM H2O2 aqueous solution; curve b represents treatment with 10% Na3PO4 + 200 μg / mL MB + 10 mM H2O2 aqueous solution; curve c represents treatment with 10% Na3PO4 + 250 μg / mL MB + 10 mM H2O2 aqueous solution; curve d represents 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 Detection results of PMMoV virus in pepper seeds treated with four treatment methods in Example 4 of the present invention; among them, curve a represents treatment with 1% savinnase everis 900L + 150 μg / mL MB + 10 mM H2O2 aqueous solution; curve b represents treatment with 1% savinnase everis 900L + 200 μg / mL MB + 10 mM H2O2 aqueous solution; curve c represents treatment with 1% savinnase everis 900L + 250 μg / mL MB + 10 mM H2O2 aqueous solution; curve d represents treatment with 1% savinnase everis 900L + 300 μg / mL MB + 10 mM H2O2 aqueous solution; curve NEG represents the negative control; curve C represents the untreated seeds (positive control);

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

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

[0072] Figure 8 Detection results of PMMoV virus in virus - infected pepper seeds numbered 1 - 14 treated with the treatment method XX in Example 5 of the present invention in Example 7; among them, curves a1 - a14 represent the detection results of 14 batches of untreated pepper seeds; curves b1 - b14 represent the detection results of 14 batches of pepper seeds treated with the treatment method XX in Example 5; NEG represents the negative control;

[0073] Figure 9 Detection results of PMMoV virus in virus - infected pepper seeds numbered 15 - 28 treated with the treatment method XX in Example 5 of the present invention in Example 7; among them, curves a1 - a14 represent the detection results of 14 batches of untreated pepper seeds; curves b1 - b14 represent the detection results of 14 batches of pepper seeds treated with the treatment method XX in Example 5; NEG represents the negative control. Detailed implementation manners

[0074] The present invention will be further described in detail in conjunction with specific embodiments below. The following embodiments enable those skilled in the art to better understand the present invention. However, it should also be easily understood by those skilled in the art that the following embodiments are only descriptive and do not mean to limit the present invention to these specific embodiments. Those skilled in the art should realize that the present invention will cover all improvement schemes, alternative schemes and equivalent schemes within the scope of the claims.

[0075] The approximate terms used in the specification and claims of this application are used to modify quantities, indicating that the present invention is not limited to the specific quantity, but also includes modified parts that are close to and acceptable for this quantity and do not cause changes in related basic functions. In the specification and claims of this application, range limitations can be combined and / or interchanged. If not otherwise stated, these ranges also include all numerical values and sub-ranges contained therein.

[0076] In the following embodiments, unless otherwise specified, the concentrations are all mass percentage concentrations, and the solvent of the solution is water. The total RNA extracted from chili seeds carrying PMMoV virus is used as a positive control.

[0077] In the following embodiments, the plant total RNA extraction kit used is a commercial kit, with the product name being RNAEasy Fast Plant Tissue RNA Rapid Extraction Kit (Catalog No.: DP452), purchased from Genebase Biotech Co., Ltd. (Beijing); the RT-LAMP reaction components are purchased from NEB, and the kit name is: WarmStart LAMP Kit (DNA & RNA) Protocol (E1700); savinnase everis 900L is purchased from NOVOZYMES; the chili seed samples are provided by Hunan Xiangyan Seed Industry Co., Ltd. and the Plant Quarantine Department of Changsha Customs; the rest of the materials, reagents, etc., unless otherwise specified, can be obtained from commercial channels.

[0078] In the following embodiments, the primer mixture is composed of 6 specific primers and nuclease-free water. The acquisition routes of the sequences of these 6 specific primers are as follows:

[0079] The target sequence of the PMMoV virus retrieved from the American gene database was subjected to homology analysis using the NCBI BLAST software to obtain a specific conserved sequence. Then, based on this conserved target sequence, a primer combination for LAMP detection of the PMMoV virus was designed using the software Primer design V5. Among them, the GenBank number of the specific conserved gene sequence of the PMMoV virus is: LC082099.1, and its position is at 5685 - 6158. These 6 specific primers were synthesized by General Biology (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 sequences of the PMMoV virus

[0081]

[0082] Example 1 Virus removal treatment with an aqueous solution of Na3PO4 for different times

[0083] a. At room temperature, take virus-infected pepper seeds at a rate of 100 per treatment, pre-soak them in clear water for 5 h. After the pre-soaking is completed, remove the clear water, and then soak them in a 5-fold volume of 10% aqueous solution of Na3PO4. Stir 2 - 3 times during the soaking period; the soaking times are 30 min, 1 h, 1.5 h, and 2 h respectively.

[0084] b. After the soaking is completed, remove the aqueous solution of Na3PO4, wash the seeds 3 - 5 times with clear water, soak them in clear water for the last time for 15 min, and immediately place the seeds on absorbent paper and dry them at room temperature after discarding the clear water.

[0085] c. Virus detection:

[0086] (1) Extract total RNA from pepper seeds: Randomly select 50 pepper seeds treated with virus removal and 50 pepper seeds not treated with virus removal, grind them into powder respectively using a mortar, and extract the RNA in the above-ground samples using a plant tissue RNA extraction kit. The extracted samples are 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, 5 μL of template (total RNA of 4 kinds of pepper seeds treated with different virus removal methods extracted in step (1)). The total RNA of pepper seeds not treated with virus removal extracted in step (1) is used as a positive control, and nuclease-free water is used as a negative control.

[0088] (3) Instrument detection:

[0089] Reaction procedure: 25 μL reaction system, react at 63 °C for 50 min, collect fluorescence signals once every 1 min.

[0090] The treatment methods of Example 1 are summarized in Table 2 below.

[0091] Table 2 Treatment methods of Example 1

[0092] Number Processing method Processing method 1 <![CDATA[10% aqueous solution of Na3PO4 for 30 min]]> Processing method 2 <![CDATA[1h treatment with 10% aqueous Na3PO4 solution]]> Processing method 3 <![CDATA[1.5 h treatment with 10% aqueous Na3PO4 solution]]> Processing method 4 <![CDATA[Treatment with 10% aqueous Na3PO4 solution for 2 h]]>

[0093] Figure 2 It is the virus detection results of the seeds treated with four treatment methods to remove PMMoV virus in Example 1. From Figure 2 the detection results, it can be seen that treating with 10% Na3PO4 aqueous solution for 30 min, 1 h, 1.5 h, and 2 h respectively causes the peak emergence time to be postponed as the treatment time prolongs, indicating that treating only with 10% Na3PO4 aqueous solution has a certain effect, but it is difficult to completely remove the virus in pepper seeds.

[0094] Example 2 uses different concentrations of subtilisin for virus removal treatment for different times

[0095] a. At room temperature, take virus-infected pepper seeds at a quantity of 100 per treatment, pre-soak them in water for 5 h. After the pre-soaking is completed, remove the water, and then soak them in an aqueous solution of savinnase everis 900L with a volume 5 times that of the seeds. Stir 1 - 2 times during the soaking period. Among them, the mass concentrations of the aqueous solution of savinnase everis 900L are 0.5%, 1%, and 1.5% respectively, and the three concentrations are treated for 10 min and 20 min respectively.

[0096] a. After the soaking is completed, remove the aqueous solution of savinnase everis 900L, wash the seeds with water 3 - 5 times, soak them in water for the last time for 15 min, discard the water, and immediately place the seeds on absorbent paper and dry them at room temperature.

[0097] c. Virus detection:

[0098] (1) Extract total RNA from pepper seeds: Randomly select 50 pepper seeds treated with virus removal and 50 pepper seeds not treated with virus removal, grind them into powder with a mortar respectively, and extract the RNA in the above-ground samples using a plant tissue RNA extraction kit. The extracted samples are stored at -80 °C for standby.

[0099] (2) Prepare the RT-LAMP reaction system: 12.5 μL of WarmStart LAMP 2X Master Mix, 1 μL of LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, and 5 μL of template (total RNA of 6 kinds of virus-free pepper seeds treated by different virus removal methods extracted in step (1)). The total RNA of pepper seeds without virus removal treatment extracted in step (1) is used as the positive control, and the negative control is nuclease-free water.

[0100] (3) Instrument detection:

[0101] Reaction procedure: For a 25 μL reaction system, react at 63 °C for 50 min, and collect fluorescence signals once every 1 min.

[0102] The treatment methods of Example 2 are summarized in Table 3 below.

[0103] Table 3 Treatment methods of Example 2

[0104] Number Processing method Processing method 5 Treat with 0.5% savinnase everis 900L aqueous solution for 10 min Processing method 6 Treat with 0.5% savinnase everis 900L aqueous solution for 20 min Processing method 7 Treat with 1% savinnase everis 900L aqueous solution for 10 min Processing method 8 Treat with 1% savinnase everis 900L aqueous solution for 20 min Processing method 9 Treat with 1.5% savinnase everis 900L aqueous solution for 10 min Processing method 10 Treat with 1.5% savinnase everis 900L aqueous solution for 20 min

[0105] Figure 3 For the virus detection results of the seeds treated with six treatment methods to remove PMMoV virus in Example 2. From Figure 3 the detection results, it can be seen that when treated with 0.5% - 1.5% savinnase everis 900L aqueous solution for 10 min or 20 min, the peak time is postponed with the increase of treatment concentration and the extension of time, indicating that there is a certain effect only by protease solution treatment.

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

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

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

[0109] c. Virus detection:

[0110] (1) Extract total RNA from pepper seeds: Randomly select 50 pepper seeds treated for virus removal and 50 pepper seeds not treated for virus removal, grind them into powder separately in a mortar, and extract the RNA in the above-ground samples using a plant tissue RNA extraction kit. The extracted samples are 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 LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, 5 μL of template (total RNA of 4 kinds of pepper seeds treated with different virus removal methods extracted in step (1)). The total RNA of pepper seeds not treated for virus removal extracted in step (1) is used as a positive control, and nuclease-free water is used as a negative control.

[0112] (3) Instrument detection:

[0113] Reaction procedure: 25 μL reaction system, react at 63 °C for 50 min, and collect fluorescence signals once every 1 min.

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

[0115] Table 4 Treatment methods of Example 3

[0116] Number Processing method Processing method 11 <![CDATA[Treatment with 10% Na3PO4 + 150 μg / mL MB + 10 mM H2O2 aqueous solution]]> Processing method 12 Treatment with an aqueous solution of 10% Na3PO4 + 200 μg / mL MB + 10 mM H2O2 Processing method 13 Treatment with an aqueous solution of 10% Na3PO4 + 250 μg / mL MB + 10 mM H2O2 Processing method 14 Treatment with an aqueous solution of 10% Na3PO4 + 300 μg / mL MB + 10 mM H2O2

[0117] Figure 4 For the virus detection results of the seeds treated with four treatment methods to remove PMMoV virus in Example 3. From Figure 4From the detection results, it can be seen that when the treatment time of the fixed 10% Na3PO4 aqueous solution is maintained, as the concentration of the MB solution gradually increases, the peak emergence time is gradually postponed, but the difference is relatively small, indicating that the detoxification efficiency of the PMMOV virus is still relatively low, and the detoxification plan needs to be further optimized.

[0118] Example 4 Virus removal treatment using 1% aqueous solution of subtilisin and MB aqueous solutions with different concentrations

[0119] a. Subtilisin treatment: At room temperature, take virus-infected pepper seeds in batches of 100 each time, pre-soak them in clear water for 5 h. After the pre-soaking is completed, remove the clear water, and then soak them in 5 times the volume of 1% savinnase everis 900L aqueous solution for 20 min, with stirring 1 - 2 times during the soaking period. After the soaking is completed, remove the savinnase everis 900L aqueous solution, wash with clear water 3 - 5 times, and soak in the last clear water for 15 min, then discard the clear water.

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

[0121] The concentrations of the MB aqueous solutions used are 150 μg / mL, 200 μg / mL, 250 μg / mL, and 300 μg / mL respectively. These 4 concentrations must be verified separately according to the above method to confirm their virus removal effects.

[0122] c. Virus detection

[0123] (1) Extract total RNA from pepper seeds: Randomly select 50 pepper seeds treated with virus removal and 50 pepper seeds untreated with virus removal, grind them into powder in a mortar respectively, and extract the RNA in the above-ground samples using a plant tissue RNA extraction kit. The extracted samples are stored at -80 °C for standby.

[0124] 2) Prepare the RT-LAMP reaction system: 12.5 μL of WarmStart LAMP 2X Master Mix, 1 μL of LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, and 5 μL of template (total RNA of 4 kinds of virus-free pepper seeds treated by different virus removal methods in step (1)). The total RNA of the untreated virus-free pepper seeds extracted in step (1) is used as a positive control, and the negative control is nuclease-free water.

[0125] (3) Instrument detection:

[0126] Reaction procedure: For a 25 μL reaction system, react at 63 °C for 50 min, and collect fluorescence signals once every 1 min.

[0127] The treatment methods of Example 4 are summarized in Table 5 below.

[0128] Table 5 Treatment methods of Example 4

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

[0130] Figure 5 It is the virus detection results of the seeds treated by four treatment methods to remove PMMoV virus in Example 4. From Figure 5 the detection results, it can be seen that: the peak emergence time of curve a is less than 35 min, the peak emergence time of curve b is greater than 35 min, and the gene detections of curves c and d are both negative. The peak emergence time of curve b is greater than 35 min, but it cannot be excluded whether it is due to being below the detection limit, so there is a possibility that there is still a very small amount of inactivated virus in the seeds. It can be seen that the 1% savinnase everis 900L + 250 μg / mL MB + 10 mM H2O2 aqueous solution corresponding to curve c is the lowest-dose chemical agent combination that can completely remove PMMoV virus.

[0131] Example 5 Influence of light irradiation treatment duration

[0132] a. Subtilisin treatment: At room temperature, take virus-infected pepper seeds at a quantity of 100 per treatment, pre-soak them in clear water for 5 h. After the pre-soaking is completed, remove the clear water, and then soak them in 5 times the volume of 1% savinnase everis 900L aqueous solution for 20 min, and stir 1 - 2 times during the soaking. After the soaking is completed, remove the savinnase everis 900L aqueous solution, wash them with clear water 3 - 5 times, and soak them in clear water for the last time for 15 min, then discard the clear water.

[0133] b. Treatment with MB aqueous solution: Take the seeds cleaned in step a above, soak them in a 250 μg / mL MB aqueous solution for 16 h. After soaking, discard part of the MB aqueous solution to make the volume ratio of the MB aqueous solution to the seeds 2:1. Further, add a certain volume of H2O2 aqueous solution (concentration: 840 mM) to this system to make the final concentration of H2O2 10 mM. Irradiate with a spotlight (power 30 w) in the wavelength range of 655 - 665 nm for different durations, and 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 min, discard the clean water, and immediately place the seeds on absorbent paper and dry at room temperature.

[0134] The different irradiation durations of the spotlight are 2 h, 4 h, and 6 h respectively. These 3 irradiation durations must be verified separately according to the above method to confirm their virus removal effects.

[0135] c. Virus detection

[0136] (1) Extract total RNA from pepper seeds: Randomly select 50 pepper seeds treated to remove viruses and 50 pepper seeds not treated to remove viruses, grind them into powder with a mortar respectively, and extract the RNA in the above-ground samples using a plant tissue RNA extraction kit. The extracted samples are 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 LAMP Fluorescent Dye (10X), 2.5 μL of primer mixture (2x), 4 μL of nuclease-free water, 5 μL of template (total RNA of 3 kinds of pepper seeds treated with different virus removal methods extracted in step (1)). The total RNA of pepper seeds not treated to remove viruses extracted in step (1) is used as a positive control, and the negative control is nuclease-free water.

[0138] (3) Instrument detection: Reaction procedure: 25 μL reaction system, react at 63 °C for 50 min, and collect fluorescence signals once every 1 min.

[0139] The treatment methods of Example 5 are summarized in Table 6 below.

[0140] Table 6 Treatment methods of Example 5

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

[0142] Figure 6 For the virus detection results of the seeds treated with three treatment methods to remove PMMoV virus in Example 5. From Figure 6The detection results show that the PMMoV gene was detected as positive after 2 hours of light irradiation treatment, while it was detected as negative after 4 hours and 6 hours of light irradiation treatment, indicating that both 4 hours and 6 hours of light irradiation can achieve the purpose of completely removing the PMMoV virus, and a shorter light irradiation time of 4 hours is a more preferred virus removal method.

[0143] Example 6 Effects of Different Virus Removal Methods on the Vigor of Pepper Seeds and the Virus Removal Effect on Leaves

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

[0145] (1)Seed virus removal treatment and germination

[0146] Take virus-infected pepper seeds in the amount of 200 per treatment, and select Treatment Method Four, Treatment Method Five, Treatment Method Six, Treatment Method Seven, Treatment Method Eight, Treatment Method Nine, Treatment Method Ten, Modified Treatment Method Thirteen (the only difference from Treatment Method Thirteen is that the light irradiation time in step b is changed from 6 hours to 4 hours), and Treatment Method Twenty in Examples 1 to 5. The seeds treated by the above treatment methods were pre-soaked in clear water at room temperature for 5 hours, then divided into 4 groups, 40-60 seeds per group, and placed on absorbent paper to germinate at room temperature, and pay attention to replenishing water during the germination process.

[0147] 2)Recording and comparison of germination conditions

[0148] Taking the radicle breaking through the seed coat by 2 mm as the standard, count the germination on the 7th day of seed germination, and the ratio is the seed germination potential. Conduct a second count on the 14th day of germination, and the ratio is the germination rate.

[0149] Calculate the germination rate and germination potential according to the following formulas:

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

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

[0152] 3)Detection of PMMoV virus in seedlings

[0153] In addition, using the same batch of seedlings without virus removal treatment as a control, the seeds in the two treatment groups of Modified Treatment Method Thirteen and Treatment Method Twenty were tested for PMMoV virus. The specific detection steps are as follows:

[0154] a. Nucleic acid extraction: Using 40 - 60 seedlings as a group, grind them into powder with a mortar respectively, and extract the RNA in the above - ground samples using a plant tissue RNA extraction kit. The extracted samples are stored at - 80 °C for future use.

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

[0156] c. Instrument detection:

[0157] Reaction procedure: For a 25 - μL reaction system, react at 63 °C for 50 min, and collect fluorescence signals once every 1 min.

[0158] Result analysis:

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

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

[0161] Number Processing method Germination potential (%) Germination rate (%) 1 Control (without virus removal treatment) 74 94 2 Processing method 4 74 94 3 Processing method 5 74 95 4 Processing method 6 75 95 5 Processing method 7 75 95 6 Processing method 8 76 96 7 Processing method 9 75 94 8 Processing method 10 74 94 9 Modified processing method 13 76 96 10 Processing method 20 78 97

[0162] As shown in Table 7, the germination potential and germination rate of the control seeds are 74% and 94% respectively. There is no significant difference in the germination potential and germination rate between the pepper seeds treated with 10% Na3PO4 + 250 μg / mL MB + 10 mM H2O2 aqueous solution (modified treatment method thirteen) or 1% savinnase everis900L solution + 250 μg / mL MB + 10 mM H2O2 solution (treatment method twenty), and both are slightly higher than those of the control seeds. This indicates that after being treated with the virus - removal methods of modified treatment method thirteen and treatment method twenty, it will not only have no adverse effects on the germination potential and germination rate of pepper seeds, but also can promote the germination of pepper seeds to a certain extent. In addition, the germination situation of the treatment with 1% concentration of savinnase everis 900L aqueous solution (treatment methods seven and eight) is slightly better than that of the treatment with 1.5% concentration of savinnase everis 900L aqueous solution (treatment methods nine and ten). Therefore, the treatment concentration of savinnase everis 900L aqueous solution can be further preferably 1%.

[0163] 2、 Figure 7 The virus removal situation of PMMoV virus in the germinated seedlings after treating the same batch of pepper seeds with various treatment methods in Example 6. After the leaves germinate, even if there is only a very small amount of active virus in the seeds, it can cause the infection of a large number of leaves. That is, by detecting the virus after germination, the viability of PMMoV virus in the seeds can be further confirmed to ensure the complete removal of PMMoV virus. From Figure 7 the detection results, it can be seen that: compared with the PMMoV virus content in the control group, although the PMMoV virus content in the leaves after treatment with 10% Na3PO4 + 250 μg / mL MB + 10 mM H2O2 aqueous solution (modified treatment method thirteen) decreased to some extent, the virus in the pepper seeds could not be completely removed; while after treatment with the combination of 1% savinnase everis 900L + 250 μg / mL MB + 10 mM H2O2 (treatment method twenty), the PMMoV content in the leaves was 0, that is, the virus removal rate for the seeds was 100%.

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

[0165] In this example, the treatment method twenty in Example 5 was used to treat 28 kinds of virus-infected pepper seeds (see Table 8 below) for virus removal. After the virus removal was completed, the nucleic acids of the virus-removed seeds and the seeds without virus removal treatment were extracted synchronously, and one-step RT-LAMP was used to qualitatively detect the nucleic acids of the extracted pepper seeds to evaluate the virus removal effect of this method on different pepper seeds and confirm the feasibility of the virus removal method for pepper seeds.

[0166] Table 8 Numbers and sources of 28 different batches of seeds

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

[0168] Figure 8 The PMMoV virus detection results after treating the virus-infected pepper seeds numbered 1-14 in Example 7 with the treatment method twenty in Example 5; Figure 9 The PMMoV virus detection results of the virus-infected pepper seeds numbered 15-28 treated with the treatment method twenty in Example 5 in Example 7 of the present invention. From Figure 8 and Figure 9 the detection results, it can be seen that:

[0169] The PMMoV virus detection of 28 batches of pepper seeds treated by the treatment method of the present invention was all negative, and the PMMoV virus detection of 28 batches of untreated pepper seeds was all positive. It can be seen that the treatment method 20 of Example 5 can completely remove the PMMoV virus in the 28 virus-infected 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 has no 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 gist of the present invention are determined only by the appended claims.

Claims

1. A method for removing pepper mild mottle virus from pepper seeds, characterized in that, The removal method includes: (1) subjecting pepper seeds to protease hydrolysis treatment with an aqueous protease solution capable of hydrolyzing the viral protein coat; the protease is savinnase everis 900L; (2) irradiating the pepper seeds treated in step (1) with light in a solution containing a photosensitizer to excite the photosensitizer, thereby obtaining pepper seeds from which the pepper mild mottle virus has been removed, wherein the photosensitizer is a photosensitizer excited by visible light; wherein, the solution containing the photosensitizer is an aqueous methylene blue solution, and the concentration of the photosensitizer in the solution containing the photosensitizer is 250 - 300 μg / mL; wherein, before the light irradiation, H2O2 is added to the solution containing the photosensitizer, and the concentration of H2O2 in the solution containing the photosensitizer is 1 - 10 mM.

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

3. The removal method according to claim 1, wherein The volume of the aqueous protease solution is 3 - 10 times the volume of the pepper seeds.

4. The removal method according to claim 1, wherein The protease hydrolysis treatment includes soaking the pepper seeds in the aqueous protease solution for 10 - 20 minutes.

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

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

7. The removal method according to claim 1, characterized in that, The light irradiation is carried out for 2 - 6 hours.

8. The removal method according to any one of claims 1 to 7, characterized in that, The removal method further includes virus detection on the pepper seeds from which the pepper mild mottle virus has been removed, wherein, the reverse transcription-loop-mediated isothermal amplification method is used for the virus detection.

9. The removal method according to any one of claims 1 to 7, characterized in that, The removal method further includes germination detection on the pepper seeds from which the pepper mild mottle virus has been removed.

10. The removal method according to claim 9, wherein The germination detection includes: germinating the pepper seeds from which the virus has been removed, and calculating the germination potential and germination rate of the pepper seeds.

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