Method for removing cucumber mosaic virus in bitter gourd seeds

Abstract

The invention relates to the technical field of seed treatment, in particular to a method for removing cucumber mosaic virus in bitter gourd seeds, which comprises the following steps: step 1, cellulase pretreatment; step 2, irradiation treatment of nano particles and light: soaking the seeds treated in the step 1 in a mixed solution of a nano particle solution coated with a photosensitizer, protease K and lauryl sodium sulfate, then adding a hydrogen peroxide solution for soaking, irradiating with light matched with the maximum absorption wavelength of the photosensitizer, and cleaning after irradiation is completed; step 3, drying: drying the seeds cleaned in the step 2; step 4, germination detection: taking part of the seeds obtained in the step 3 for germination test, and observing and recording the germination rate of the seeds; and 5, RT-LAMP detection: carrying out cucumber mosaic virus detection on the seedlings obtained in the step 4 by adopting an RT-LAMP method, and carrying out multiple treatment, so that the cucumber mosaic virus in the bitter gourd seeds can be effectively removed.

Description

Technical Field

[0001] This invention relates to the field of seed treatment technology, and more specifically, to a method for removing cucumber mosaic virus from bitter gourd seeds. The method involves pretreatment with cellulase combined with the delivery of a photosensitizer via a nanocarrier. Under irradiation with light of a specific wavelength, reactive oxygen species (ROS) are generated. These ROS are then used in a step-by-step process with multiple chemical reagents, working synergistically to efficiently destroy proteins and nucleic acids. This achieves highly efficient inactivation of the virus within the seed while maintaining seed viability. Background Technology

[0002] Bitter melon, an important specialty vegetable crop in my country, possesses both nutritional and medicinal value, and its production and demand have been steadily increasing in recent years. However, viral diseases have become a key bottleneck restricting the development of the bitter melon industry, with cucumber mosaic virus (CCV) being particularly severe. CCV belongs to the Cucumber Mosaic Virus genus of the Bromeliidae family and is a triadic, single-stranded, positive-sense RNA virus. Infection with this virus can cause typical symptoms such as stunted growth, leaf deformities, and mosaic patterns, leading to poor fruit development, loss of commercial value, and in severe cases, total crop failure.

[0003] Existing research indicates that cucumber mosaic virus is primarily transmitted through aphids, spreading extremely rapidly in the field. It also occurs through mechanical transmission and seed-borne transmission. Seed-borne infection is a significant source of initial viral infection, making source control crucial for reducing field morbidity. Currently, virus removal treatments for bitter gourd seeds mainly employ hot water soaking and chemical disinfection, but these methods have significant limitations.

[0004] The warm water soaking method typically involves soaking seeds in water at 50℃ to 55℃, but this method only inactivates pathogens on the seed surface and has limited effectiveness against internal viruses. Trisodium phosphate treatment is currently the mainstream chemical method; while it has some inactivation effect on viruses, it cannot completely eliminate viral infectivity, and residual trisodium phosphate causes seed germination rates to decrease with prolonged storage. Sodium hypochlorite and dilute hydrochloric acid treatments also cannot completely block virus transmission, and the strong corrosiveness of dilute hydrochloric acid limits its application. While shoot tip virus removal technology can completely eliminate viruses, the process is complex and costly, making large-scale implementation difficult.

[0005] Photochemotherapy, as an emerging technology, uses photosensitizers and specific wavelengths of light to trigger photochemical reactions and has been widely applied in the medical field. However, its application in seeds with thick seed coats faces several challenges. Bitter gourd seed coats are highly lignified and have a thick waxy layer, forming a physical barrier that hinders photosensitizer penetration. The effective radius of singlet oxygen is only about 200 nm, making it difficult for photosensitizers to penetrate the hydrophobic seed coat to reach the internal embryonic tissue. Furthermore, reactive oxygen species are rapidly quenched in the shallow layers of the seed coat, failing to effectively target deeper viruses.

[0006] Therefore, developing a simple and efficient method for virus removal from bitter gourd seeds, while ensuring seed quality, is a key issue that urgently needs to be addressed in current seed treatment technology. Establishing a scientific and reasonable virus removal program is of great significance for ensuring the safety of bitter gourd seeds used in production and promoting the large-scale development of the industry. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a highly efficient and safe method for detoxifying bitter gourd seeds based on photochemical treatment, thereby solving problems such as incomplete detoxification, impact on seed quality, complex operation, and high cost associated with traditional methods.

[0008] To achieve the above objectives, the technical solution adopted by the present invention includes the following steps:

[0009] First, cellulase pretreatment is performed. Bitter gourd seeds are pre-soaked in warm water, then immersed in a mixed solution of cellulase and surfactant. Cellulase specifically degrades the cellulose components in the seed coat, forming a microporous structure on the seed coat surface, creating channels for subsequent nanoparticle penetration. Compared to traditional acid-base or high-temperature treatments, this method has stronger targeting and avoids damage to seed viability. The addition of surfactant further reduces surface tension and enhances the penetration ability of the treatment solution.

[0010] Subsequently, nanoparticle and light irradiation treatment was performed. The pretreated seeds were immersed in a solution of nanoparticles coated with a photosensitizer, along with proteinase K and sodium dodecyl sulfate. The nanoparticles acted directly on the viral protein capsid through surface effects, while sodium dodecyl sulfate assisted in disrupting the viral capsid and enhancing the dispersibility of the nanoparticles. Proteinase K degraded the viral nucleic acid-binding protein; the three worked synergistically to achieve initial viral inactivation. Hydrogen peroxide solution was then added. On one hand, hydrogen peroxide degraded the chitosan capsid, enabling controlled release of the photosensitizer; on the other hand, the oxygen generated by its decomposition provided sufficient substrate for subsequent photochemical reactions, avoiding the limitation of singlet oxygen production in an anaerobic environment. Finally, irradiation was performed using light matching the maximum absorption wavelength of the photosensitizer. The photosensitizer was excited to produce reactive oxygen species such as singlet oxygen, which oxidatively damaged the viral nucleic acid and capsid proteins, achieving precise removal of residual virus.

[0011] After treatment, the seeds were dried, and a portion of the seeds were then used for germination tests. The effect of the treatment on seed vigor was assessed by observing the germination rate. Simultaneously, the RT-LAMP method was used to detect viruses in the germinated seedlings. This method uses specific primers designed for viral target genes to amplify nucleic acids under isothermal conditions, offering advantages such as high sensitivity, strong specificity, and ease of operation. By combining germination rate detection and viral nucleic acid detection, both the effectiveness of virus elimination and the impact on seed quality can be confirmed.

[0012] The beneficial effects of this invention are as follows:

[0013] First, a multi-stage delivery system was established. Pretreatment with cellulase expands the micropores of the seed coat, allowing nanoparticles to easily penetrate into the seed interior. Hydrogen peroxide triggers the release of photosensitizers, and light excitation generates reactive oxygen species that target the virus, forming a progressively advancing virus clearance pathway. This design overcomes the technical challenges of virus removal from thick-coated seeds and significantly improves the delivery efficiency and depth of action of the photosensitizers.

[0014] Second, dynamic oxygen regulation was achieved. The synergistic oxygen supply mechanism formed by hydrogen peroxide decomposition and photodynamic reaction overcomes the bottleneck of insufficient oxygen in traditional photodynamic technology. Sufficient oxygen supply ensures the continuous generation of reactive oxygen species, enhancing the virus-killing effect.

[0015] Third, water-soluble chitosan was used as a nanocarrier. Chitosan carries a positive charge and readily binds to negatively charged seed coat cellulose, enhancing its adsorption and permeation capabilities. Under the action of hydrogen peroxide, chitosan degrades to release photosensitizers, achieving controlled release and avoiding efficiency loss due to premature leakage.

[0016] Fourth, dihydroporphyrin E6 was selected as the photosensitizer. This photosensitizer has maximum absorption wavelength in the red light region, and red light has stronger tissue penetration than other wavelengths, allowing it to reach the seed interior to exert its effect to the maximum extent. At the same time, this wavelength of light has minimal impact on the physiological state of the seed, avoiding the decrease in germination rate caused by traditional heat treatment.

[0017] Fifth, both the nanomaterials and hydrogen peroxide used can be degraded by microorganisms or photodegraded in the natural environment, making it more environmentally friendly than chemical treatment. The entire process does not use toxic or harmful substances, meeting the requirements of sustainable development in modern agriculture.

[0018] Sixth, it is easy to operate and highly adaptable to different environments. This method requires no specific temperature control conditions or specialized equipment, has low technical requirements for operators, and can be stably applied year-round in laboratories and production enterprises. The processing cost is far lower than that of stem tip detoxification technology, making it suitable for large-scale promotion and use.

[0019] Seventh, a verification method combining RT-LAMP and germination detection is employed. This method not only provides highly sensitive and specific confirmation of successful seed devirulence but also confirms the impact of treatment on seed vigor through germination rate statistics. This dual verification approach solves the problem that traditional detection methods cannot simultaneously evaluate virus activity and seed quality.

[0020] Experimental results show that after treatment with the method of this invention, cucumber mosaic virus was completely eliminated from 20 kinds of bitter gourd seeds, while the seed germination rate remained at around 96%, which was not significantly different from the 95.6% germination rate of untreated seeds. This method provides a reliable technical guarantee for the sustainable development of the bitter gourd industry, helps to establish a virus-free seedling propagation technology system for bitter gourd, and improves the biosafety of seed transportation across regions. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the photochemical reaction principle, showing the complete process of cellulase pretreatment, nanoparticle delivery, hydrogen peroxide-triggered release, and photosensitizer excitation to generate reactive oxygen species.

[0022] Figure 2 The RT-LAMP curves for detecting cucumber mosaic virus in germinating leaves of a bitter gourd seed after treatment with 10% trisodium phosphate solution for different time periods are shown. In the figure, a, b, and c represent the results after 1 h, 2 h, and 3 h of treatment, respectively. NEG is the negative control, and C is the positive control of untreated seeds.

[0023] Figure 3 This study presents the statistical results of the peak emergence time of cucumber mosaic virus in the buds and leaves of 20 bitter gourd seeds after treatment with 10% trisodium phosphate solution for different durations. I, II, and III represent statistical data for 1 h, 2 h, and 3 h of treatment, respectively.

[0024] Figure 4 To statistically analyze the detection results of cucumber mosaic virus in the buds and leaves of 20 kinds of bitter gourd seeds after treatment with the method of the present invention, T represents the treatment group and C represents the untreated control group.

[0025] Figure 5 The results of the detection of cucumber mosaic virus in the buds and leaves of 20 kinds of bitter gourd seeds using the method of the present invention but without the photosensitizer being encapsulated by chitosan are shown. a, b, and c represent statistical data of dihydroporphyrin E6 concentrations of 50 μg / mL, 100 μg / mL, and 150 μg / mL, respectively.

[0026] Figure 6 The results are for the detection of dihydroporphyrin E6, sodium dodecyl sulfate, proteinase K, and hydrogen peroxide encapsulated only by chitosan, without cellulase pretreatment. N1 represents 7 seeds that failed to be detoxified, and N2 represents 13 seeds that were successfully detoxified.

[0027] Figure 7 To compare the detoxification effects of encapsulating dihydroporphyrin e6 with different nanoparticles, P1 and P2 were polylactic acid-glycolic acid copolymers, Z1 and Z2 were liposomes, I1 and I2 were silica, and S1 and S2 were polystyrene. The test results were statistically analyzed.

[0028] Figure 8The results are for the detection when the method of the present invention is used but without hydrogen peroxide treatment. H1 represents the 5 seeds that failed to be detoxified, and H2 represents the 15 seeds that were successfully detoxified.

[0029] Figure 9 The results are for the detection when the method of the present invention is used but without the addition of sodium dodecyl sulfate and proteinase K. D1 represents the 3 seeds that failed to be detoxified, and D2 represents the 17 seeds that were successfully detoxified.

[0030] Figure 10 To statistically analyze the germination rate of 20 bitter gourd seeds treated with the method of this invention, T represents the treatment group and C represents the untreated control group. Detailed Implementation

[0031] The present invention will be described in detail below through specific embodiments. These embodiments are only for illustrating the present invention and do not limit the scope of protection of the present invention. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the materials and reagents used can be obtained commercially.

[0032] The main materials and reagents used in the examples included: cellulase purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 10000 U / g; Tween 80 purchased from Sinopharm Chemical Reagent Co., Ltd., analytical grade; water-soluble chitosan purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd., with a degree of deacetylation of 90% and a molecular weight of 2 kDa; dihydroporphyrin e6 purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of 98%; proteinase K purchased from Beijing Solarbio Science & Technology Co., Ltd.; sodium dodecyl sulfate purchased from Sinopharm Chemical Reagent Co., Ltd., analytical grade; and hydrogen peroxide solution purchased from Sinopharm Chemical Reagent Co., Ltd., with a mass fraction of 30%. The RT-LAMP detection kit was purchased from NEB Corporation, USA, under the product name WarmStart LAMP Kit. The plant total RNA extraction kit was purchased from Wuhan Baisai Biotechnology Co., Ltd. The bitter gourd seed samples were provided by Hunan Xiangyan Seed Industry Co., Ltd., and all were seeds carrying cucumber mosaic virus.

[0033] In the following examples, all concentrations are mass percentages, and all solutions are in water as the solvent.

[0034] The primer mixture consists of six specific primers and nuclease-free water. The sequences of these six specific primers were obtained as follows:

[0035] CMV virus target sequences were obtained from US gene databases. Homology analysis using NCBI BLAST software was performed to identify specific conserved sequences. Based on these conserved target sequences, primer combinations for LAMP detection of CMV virus were designed using Primer Design V5 software. The GenBank accession number for the CMV virus-specific conserved gene sequence is X89652.1. These six specific primers were synthesized by General Biotechnology (Anhui) Co., Ltd., and their specific primer sequences and concentrations in the RT-LAMP reaction system are shown in Table 1.

[0036] Table 1 Primer composition and sequence of CMV virus

[0037]

[0038] Total RNA extracted from bitter gourd seeds carrying CMV virus was used as a positive control.

[0039] The following examples evaluate the feasibility of bitter gourd seed detoxification methods from five main dimensions: reagent composition, reagent concentration, treatment time, detoxification effect, and seed quality. In this example, untreated seeds are used as a control. After detoxification, the detoxified seeds and untreated seeds germinate simultaneously, and the buds and leaves are taken for nucleic acid extraction. The nucleic acid extracted from the germinated bitter gourd seed leaves is qualitatively detected using a one-step RT-LAMP method, and the detoxification effects of each method are compared.

[0040] Example 1

[0041] This embodiment uses a cellulase concentration of 10 g / L and a soaking time of 30 min to verify the detoxification effect of a lower cellulase concentration and a shorter treatment time.

[0042] One hundred bitter gourd seeds carrying cucumber mosaic virus were pre-soaked in 50℃ warm water for 5 hours, stirring once every hour to ensure even heating. After pre-soaking, the warm water was discarded, and a cellulase solution with a pH of 5.0 and a cellulase concentration of 10 g / L was prepared using citrate buffer, with Tween 80 added to bring the final concentration to 1 g / L. The pre-soaked seeds were then immersed in this mixed solution, with the solution volume being 5 times the seed volume. The soaking temperature was controlled at 42℃, and the soaking time was 30 minutes, with stirring twice during the soaking process. After soaking, the seeds were rinsed three times with clean water, soaking in clean water for 5 minutes after each rinse, and finally soaking for 15 minutes before discarding the clean water.

[0043] Prepare 5 g / L water-soluble chitosan nanoparticles (with a dihydroporphyrin E6 concentration of 50 μg / mL). Add proteinase K to this solution to a final concentration of 300 μg / mL, and add sodium dodecyl sulfate to a final concentration of 5 g / L, then mix thoroughly. Immerse the washed seeds in this mixture, using 5 times the volume of the seeds, and soak at room temperature for 2 hours.

[0044] After soaking, discard part of the mixture, maintaining a 2:1 volume ratio of mixture to seeds. Add 30% hydrogen peroxide solution to a final concentration of 10 g / L, gently shake, and soak at room temperature for 30 minutes. After soaking, irradiate with an LED lamp with a peak wavelength of 660±5 nm, a light source power of 30 W, an irradiation distance of approximately 30 cm, and an irradiation time of 4 hours, stirring the seeds every hour during irradiation. After irradiation, discard all treatment solution, rinse the seeds four times with clean water, soaking for 10 minutes after each rinse, and finally soaking for 15 minutes. Discard the clean water and place the seeds on absorbent paper to air dry at room temperature.

[0045] One hundred virus-free seeds and one hundred untreated control seeds were soaked in 50℃ warm water for 5 hours, then divided into four groups and placed on absorbent paper for germination testing. The absorbent paper was kept moist during the germination process. Germination rate was recorded after 14 days, with the radicle breaking through the seed coat by 2 mm as the germination standard. Germinated seedlings were randomly selected, ground into powder using a mortar and pestle, and RNA was extracted using a plant total RNA extraction kit. The reaction system was prepared according to the RT-LAMP kit instructions for detection.

[0046] Example 2

[0047] This embodiment uses the optimal treatment parameters of 20 g / L cellulase concentration and 60 min soaking time to verify the detoxification effect under the best process conditions.

[0048] The basic operating steps are the same as in Example 1, with the main difference being that the cellulase concentration is increased to 20 g / L and the soaking time is extended to 60 min. Specifically, 100 bitter gourd seeds carrying cucumber mosaic virus were pre-soaked in 50°C warm water for 5 hours, and then treated with a 20 g / L cellulase solution plus a 1 g / L Tween 80 solution. Soaking was performed for 60 min at pH 5.0 and 42°C, with stirring every 20 min during the soaking process. The remaining steps, including nanoparticle treatment, hydrogen peroxide soaking, light irradiation, washing, and drying, are the same as in Example 1. Germination tests and RT-LAMP assays were then performed after treatment.

[0049] Example 3

[0050] This embodiment uses a cellulase concentration of 30 g / L and a soaking time of 90 min to verify the effect of higher cellulase concentration and longer treatment time on detoxification.

[0051] One hundred bitter gourd seeds carrying cucumber mosaic virus were pre-soaked in 50°C water for 5 hours, then treated with a 30 g / L cellulase solution plus a final concentration of 1 g / L Tween 80. Soaking was performed at pH 5.0 and 42°C for 90 minutes, with stirring every 30 minutes during the soaking process. It is worth noting that higher concentrations of cellulase can more thoroughly degrade seed coat cellulose, but the treatment time needs to be controlled to avoid excessive damage to the seeds. The remaining treatment steps were the same as in Example 1.

[0052] Example 4

[0053] This embodiment verifies the effect of different photosensitizer concentrations on the detoxification effect, using a dihydroporphyrin E6 concentration of 20 μg / mL as the treatment parameter.

[0054] The cellulase pretreatment step was performed under preferred conditions: a cellulase concentration of 20 g / L and soaking at 42°C for 60 min. When preparing the nanoparticle mixture, the concentration of dihydroporphyrin E6 was reduced to 20 μg / mL, while the concentrations of other components remained unchanged. The lower photosensitizer concentration may result in a reduction in reactive oxygen species production, but it helps to lower processing costs. The remaining steps were the same as in Example 2.

[0055] Example 5

[0056] This embodiment verifies the treatment effect of higher photosensitizer concentrations, using a dihydroporphyrin E6 concentration of 80 μg / mL.

[0057] Pretreatment with cellulase was performed under optimized conditions. When preparing the nanoparticle mixture, the concentration of dihydroporphyrin E6 was increased to 80 μg / mL, which will generate more singlet oxygen and may enhance the detoxification effect. However, it is necessary to monitor whether a higher photosensitizer concentration will adversely affect seed vigor. The remaining steps are the same as in Example 2.

[0058] Example 6

[0059] This example verifies the effect of different proteinase K concentrations, using a proteinase K concentration of 200 μg / mL as the treatment parameter.

[0060] Cellulase pretreatment and nanoparticle preparation were performed under preferred conditions, but the proteinase K concentration was reduced to 200 μg / mL when preparing the mixture. Proteinase K primarily acts on viral nucleic acid-binding proteins, and a lower concentration may affect virus inactivation efficiency. The remaining steps were the same as in Example 2.

[0061] Example 7

[0062] This example verifies the treatment effect of a higher proteinase K concentration, using a proteinase K concentration of 400 μg / mL.

[0063] When preparing the nanoparticle mixture, the proteinase K concentration was increased to 400 μg / mL, which will more fully degrade viral proteins, but it is necessary to assess whether this will affect the seed's own proteins. The remaining steps are the same as in Example 2.

[0064] Example 8

[0065] This embodiment verifies the effect of different illumination times, using a illumination time of 2 hours as the processing parameter.

[0066] The cellulase pretreatment, nanoparticle treatment, and hydrogen peroxide immersion steps all used optimized conditions, but the light irradiation time was shortened to 2 hours. A shorter light irradiation time may result in insufficient reactive oxygen species production, affecting the clearance effect of deep-seated viruses. The remaining steps were the same as in Example 2.

[0067] Example 9

[0068] This embodiment verifies the processing effect of a longer illumination time, using a illumination time of 6 hours.

[0069] Extending the light irradiation time to 6 hours will generate more reactive oxygen species, potentially improving the detoxification effect, but the increased energy consumption and impact on production efficiency also need to be considered. The remaining steps are the same as in Example 2.

[0070] Comparative Example 1

[0071] This comparative example uses the traditional 10% trisodium phosphate solution treatment method to compare and verify the superiority of the present invention.

[0072] One hundred bitter gourd seeds carrying cucumber mosaic virus were pre-soaked in 50℃ warm water for 5 hours, then immersed in a 10% trisodium phosphate solution (5 times the seed volume) for 1 hour, 2 hours, and 3 hours at room temperature, stirring twice during the soaking process. After soaking, the seeds were rinsed repeatedly with clean water five times to remove residual trisodium phosphate. The final soaking in clean water for 20 minutes was followed by discarding the water, and the seeds were placed on absorbent paper to air dry at room temperature. Germination tests and RT-LAMP assays were then performed.

[0073] Comparative Example 2

[0074] This comparative example did not undergo cellulase pretreatment, but was directly treated with nanoparticles and light irradiation to verify the necessity of cellulase pretreatment.

[0075] One hundred bitter gourd seeds carrying cucumber mosaic virus were pre-soaked in 50°C water for 5 hours, then directly immersed in a nanoparticle mixture. The mixture was prepared in the same way as in Example 2, containing 50 μg / mL of dihydroporphyrin E6 encapsulated in water-soluble chitosan at a concentration of 5 g / L, 300 μg / mL of proteinase K, and 5 g / L of sodium dodecyl sulfate. The seeds were soaked at room temperature for 2 hours. Subsequent steps, including hydrogen peroxide soaking, light irradiation, washing, and drying, were the same as in Example 2.

[0076] Comparative Example 3

[0077] This comparative example uses a treatment method without photosensitizer encapsulation. The dihydroporphyrin E6 mother liquor is directly mixed with the treatment solution to verify the necessity of nanoparticle encapsulation.

[0078] The cellulase pretreatment steps were the same as in Example 2. When preparing the treatment solution, the dihydroporphyrin E6 stock solution was directly mixed with an aqueous solution containing proteinase K and sodium dodecyl sulfate, without using chitosan nanoparticles for encapsulation. The final concentrations of dihydroporphyrin E6 were 50 μg / mL, 100 μg / mL, and 150 μg / mL, respectively, and the concentrations of the other components were the same as in Example 2. Subsequent treatment steps were the same.

[0079] Comparative Example 4

[0080] This comparative example uses other nanoparticles to replace chitosan in encapsulating the photosensitizer to verify the substitutability of water-soluble chitosan.

[0081] The cellulase pretreatment steps were the same as in Example 2. When preparing the treatment solution, 5 g / L polylactic acid-glycolic acid copolymer, liposomes, silica, and a 50 μg / mL dihydroporphyrin E6 solution encapsulated in polystyrene were prepared. Subsequently, proteinase K was added to these solutions to a final concentration of 300 μg / mL, and sodium dodecyl sulfate was added to a final concentration of 5 g / L, and the solutions were thoroughly mixed. The washed seeds were then immersed in this mixture, with the volume of the mixture being 5 times the volume of the seeds, and soaked at room temperature for 2 hours. Subsequent hydrogen peroxide soaking, light irradiation, washing, and drying steps were the same as in Example 2.

[0082] Comparative Example 5

[0083] This comparative example does not contain hydrogen peroxide and is used to verify the role of hydrogen peroxide in the detoxification process.

[0084] The cellulase pretreatment and nanoparticle soaking steps were the same as in Example 2. After soaking the nanoparticles for 2 hours, part of the mixture was discarded to achieve a liquid-to-solid ratio of 2:1, but no hydrogen peroxide solution was added, and the nanoparticles were directly irradiated with light. Due to the lack of hydrogen peroxide, the process of chitosan degradation and release of photosensitizer was hindered, and the oxygen supply for the photodynamic reaction was insufficient. The remaining steps were the same as in Example 2.

[0085] Comparative Example 6

[0086] This comparative example does not contain sodium dodecyl sulfate or proteinase K, and is used to verify the synergistic effect of these two components.

[0087] The cellulase pretreatment steps were the same as in Example 2. When preparing the nanoparticle mixture, only 50 μg / mL of dihydroporphyrin E6 encapsulated in water-soluble chitosan at a concentration of 5 g / L was included; proteinase K and sodium dodecyl sulfate were not added. The absence of these two components may result in insufficient disruption of the viral protein coat, affecting the effect of the photosensitizer on the viral core. The remaining steps were the same as in Example 2.

[0088] RT-LAMP detection method: After extracting total RNA from germinating seedlings, a 25 μL reaction system was prepared, containing 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL fluorescent dye, 2.5 μL primer mixture, 4 μL nuclease-free water, and 5 μL RNA template. The primer mixture contained six specific primers designed against conserved sequences of cucumber mosaic virus, with the following sequences and concentrations: F3 and B3 0.2 μM each, FIP and BIP 1.6 μM each, and LF and LB 0.8 μM each. The reaction system was placed in a real-time PCR instrument and incubated at 63℃ for 45 min, with fluorescence signals collected every 1 min. After multiple experiments, a peak of 40+ indicated the presence of a small amount of viral RNA.

[0089] Germination rate test method: conducted according to national standard GB / T3543.4-1995. Germination standard is defined as the radicle breaking through the seed coat by 2mm. Germination is counted 14 days after germination, and the germination rate is calculated according to the following formula: Germination rate = total number of germinated seeds in 14 days ÷ total number of seeds tested × 100%.

[0090] Table 2 lists the RT-LAMP detection results and germination rate statistics for each example and comparative example. As can be seen from the table, the optimized process parameters used in Example 2 achieved the best detoxification effect; cucumber mosaic virus was not detected in the germinating leaves of all 20 bitter gourd seeds, and the germination rate reached 96.3%. Examples 1 and 3 differed in cellulase concentration and treatment time. Example 1 achieved a detoxification rate of 90%, while Example 3 also achieved complete detoxification, with germination rates of 95.8% and 96.0%, respectively, indicating that the method of the present invention has good stability within a certain parameter range.

[0091] Example 4 used a lower photosensitizer concentration, and 2 out of 20 seeds were not completely devirulent, resulting in a devirulence rate of 90% and a germination rate of 95.5%. This indicates that when the photosensitizer concentration is below 20 μg / mL, the reactive oxygen species production may be insufficient to completely inactivate the virus. Example 5 used a higher photosensitizer concentration, achieving a 100% devirulence rate and a germination rate of 95.9%, demonstrating that increasing the photosensitizer concentration to 80 μg / mL does not negatively affect seed vigor.

[0092] Examples 6 and 7 verified the effect of proteinase K concentration. Example 6 used 200 μg / mL proteinase K, achieving a detoxification rate of 95%, while Example 7 used 400 μg / mL proteinase K, achieving a detoxification rate of 100%. This indicates that detoxification combinations with proteinase K concentrations in the range of 300 μg / mL to 400 μg / mL are effective in removing the virus.

[0093] Examples 8 and 9 verified the effect of illumination time. In Example 8, the detoxification rate was 90% after 2 hours of illumination, indicating that insufficient illumination time led to inadequate reactive oxygen species production. In Example 9, 100% detoxification was achieved after 6 hours of illumination, but considering energy consumption and efficiency, 4 hours of illumination is a more reasonable choice.

[0094] Comparative Example 1, using the traditional trisodium phosphate method, failed to completely detoxify any of the 20 seeds. Viral nucleic acid was still detectable in the germinating leaves, with a peak elution time ranging from 23.66 min to 26.15 min. Although this was significantly later than the treatment group of this invention, it still did not meet the detoxification requirements. This verifies the deficiency of traditional methods in completely eliminating viruses inside seeds.

[0095] In Comparative Example 2, without cellulase pretreatment, 7 out of 20 seeds failed to be detoxified, resulting in a detoxification rate of only 65%. This indicates that cellulase pretreatment plays a crucial role in expanding seed coat micropores and promoting nanoparticle penetration. The absence of this step significantly reduces detoxification efficiency.

[0096] In Comparative Example 3, without nanoparticle encapsulation of the photosensitizer, even with the dihydroporphyrin E6 concentration increased to 150 μg / mL, some seeds still failed to be completely detoxified. This indicates that nanoparticle encapsulation is crucial for protecting the photosensitizer and promoting its delivery into the seed interior.

[0097] Comparative Example 4 used other nanoparticles to encapsulate the photosensitizer. These other nanoparticles included polylactic-co-glycolic acid copolymer, liposomes, silica, and polystyrene, with detoxification rates of 85%, 85%, 80%, and 70%, respectively. Experimental data show that selecting a suitable encapsulation material is crucial for improving detoxification performance. Water-soluble chitosan, as an encapsulation material, exhibits a significant advantage in detoxification efficiency and is one of the key factors in achieving efficient detoxification.

[0098] In Comparative Example 5, without the addition of hydrogen peroxide, 5 out of 20 seeds failed to be detoxified, resulting in a detoxification rate of 75%. This verifies the important role of hydrogen peroxide in both degrading chitosan to release photosensitizers and providing oxygen substrates.

[0099] Comparative Example 6, without the addition of sodium dodecyl sulfate and proteinase K, achieved a detoxification rate of 85%. Although partial detoxification was still achieved, the effect was significantly less than that of the complete process. This indicates that the two components play a synergistic role in disrupting the viral protein coat.

[0100] The germination rate of all examples and comparative examples remained above 95%, indicating that the method of the present invention and its variations do not significantly affect seed vigor. The germination rate of the untreated control group was 95.6%, which was not significantly different from that of the treatment groups.

[0101] Table 2 shows the test results for each embodiment and comparative example.

[0102]

[0103] The technical effect of this invention stems from the synergistic effect of multiple processing steps. Cellulase specifically hydrolyzes the β-1,4-glycosidic bonds of cellulose in the seed coat, loosening the dense cellulose network structure and forming nanoscale microporous channels on the seed coat surface. These micropores provide pathways for the subsequent penetration of nanoparticles and are the foundation of the entire processing technology. Tween 80, as a nonionic surfactant, reduces the surface tension of the liquid, enhancing the wettability and penetration ability of the treatment solution on the hydrophobic seed coat surface.

[0104] Water-soluble chitosan offers several advantages as a nanocarrier. First, the amino groups on the chitosan molecular chain carry a positive charge under neutral or weakly acidic conditions, while the hydroxyl groups on the surface of seed coat cellulose exhibit a negative charge in aqueous solution. The electrostatic interaction between these two groups enhances the adsorption of nanoparticles on the seed coat surface. Second, chitosan possesses excellent biocompatibility and biodegradability, posing no toxicity to seeds or the environment. Third, chitosan can stably load hydrophobic dihydroporphyrin E6 within nanoparticles, preventing the photosensitizer from agglomerating and precipitating in the aqueous phase, thus ensuring the stability and bioavailability of the photosensitizer.

[0105] Dihydroporphyrin e6, as a second-generation photosensitizer, possesses excellent photophysical properties. Its maximum absorption wavelength is in the red light region of 655 nm to 665 nm. Light at this wavelength can penetrate biological tissues to a depth of several millimeters, far exceeding visible and ultraviolet light. When dihydroporphyrin e6 absorbs photon energy, electrons transition from the ground state to an excited state. The excited-state photosensitizer then transforms into a triplet state through intersystem crossing. The triplet-state photosensitizer transfers energy to surrounding oxygen molecules, transforming ground-state oxygen into singlet oxygen. Singlet oxygen has extremely strong oxidizing activity, capable of attacking guanine bases in viral nucleic acids, leading to DNA or RNA chain breaks. Simultaneously, it oxidizes amino acid residues such as tryptophan, histidine, and methionine in viral capsid proteins, disrupting the three-dimensional structure of the protein and ultimately causing viral inactivation.

[0106] Sodium dodecyl sulfate (SOS), as an anionic surfactant, can insert its hydrophobic tail into the viral lipid envelope or interact with hydrophobic protein regions, disrupting the hydrophobic core of proteins and leading to capsid protein denaturation. Simultaneously, SOS enhances the dispersibility of nanoparticles in aqueous solutions, preventing particle aggregation. Proteinase K, a serine protease, can hydrolyze peptide bonds at their carbon ends, exhibiting broad-spectrum degradation activity against viral nucleocapsid proteins and replication-related proteins. The synergistic effect of proteinase K and SOS can cause initial damage to the virus before light exposure, creating favorable conditions for subsequent photodynamic reactions.

[0107] Hydrogen peroxide plays a dual role in this invention. First, as an oxidant, hydrogen peroxide can break the β-1,4-glycosidic bonds in the chitosan molecular chain, reducing the degree of polymerization and shortening the molecular chain, leading to the degradation of chitosan nanoparticles and the release of encapsulated dihydroporphyrin E6. This controlled release mechanism ensures that the photosensitizer works at the appropriate time and location, avoiding efficiency losses caused by large-scale leakage in the early stages of immersion. Second, hydrogen peroxide decomposes under light and the catalysis of the photosensitizer to produce oxygen, continuously replenishing the oxygen substrate required for the photodynamic reaction. Traditional photodynamic technology often suffers from insufficient singlet oxygen production due to tissue hypoxia, but this invention overcomes this limitation through the in-situ decomposition of hydrogen peroxide, significantly increasing singlet oxygen production and greatly enhancing the virus inactivation effect.

[0108] The entire treatment process forms a multi-stage, progressive virus elimination pathway. Cellulase pretreatment breaks through the physical barrier of the seed coat, nanoparticles carrying photosensitizers penetrate deep into the seed interior, hydrogen peroxide triggers the release of the photosensitizers and provides oxygen, and light excitation generates reactive oxygen species to precisely attack the virus. This design fully considers the characteristics of the thick seed coat of bitter gourd seeds and the biological characteristics of cucumber mosaic virus, achieving highly efficient detoxification through the organic combination of physical, chemical, and biological methods.

[0109] It is worth emphasizing that all reagents used in this invention have good environmental compatibility. Cellulase, proteinase K, and other biological enzymes can be rapidly degraded by microorganisms in the environment; chitosan can be degraded into oligosaccharides and monosaccharides by chitosanases secreted by fungi and bacteria; sodium dodecyl sulfate can be degraded through the β-oxidation pathway of microorganisms; hydrogen peroxide decomposes into water and oxygen; and dihydroporphyrin E6 can undergo photodegradation under light. Therefore, the method of this invention does not cause environmental pollution and meets the requirements of green agriculture development.

[0110] The results of the above embodiments and comparative examples demonstrate that the bitter gourd seed virus removal method provided by this invention achieves complete elimination of cucumber mosaic virus while preserving seed viability, significantly outperforming traditional methods. This method is simple to operate, cost-effective, and environmentally friendly, making it suitable for widespread application in seed companies and research institutions, and providing strong technical support for the healthy development of the bitter gourd industry.

[0111] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the inventive concept of the present invention and the contents of this specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for removing cucumber mosaic virus from bitter gourd seeds, characterized by The method comprises the following steps: Step one, cellulase pretreatment: after the balsam pear seeds are pre-soaked in warm water, the seeds are soaked in a mixed solution of cellulase and surfactant, and then cleaned for standby; Step two, nanoparticle and light irradiation treatment: the seeds treated in step one are soaked in a mixed solution of nanoparticle solution coated with photosensitizer, proteinase K and sodium dodecyl sulfate, and then soaked in hydrogen peroxide solution, and then irradiated with light matching the maximum absorption wavelength of the photosensitizer, and then cleaned after irradiation; Step three, drying: the seeds cleaned in step two are dried; Step four, germination detection: part of the seeds obtained in step three are taken for germination test, and the seed germination rate is observed and recorded; Step five, RT-LAMP detection: the seedlings obtained in step four are detected for cucumber mosaic virus by RT-LAMP method.

2. The method of claim 1, wherein, The temperature of the warm water in step one is 45-55 DEG C, and the pre-soaking time is 4-6 hours; the concentration of the cellulase is 10-30 g / L, the surfactant is Tween 80 and the concentration is 1 g / L, the pH value of the mixed solution is 4.5-5.5, the soaking temperature is 40-44 DEG C, the soaking time is 30-90 min, and the amount of the mixed solution is 3-10 times the volume of the seeds.

3. The method of claim 2, wherein, In step one, the concentration of the cellulase is 20 g / L, the cellulase solution is prepared by citrate buffer, the pH value is 5.0, the soaking temperature is 42 DEG C, the soaking time is 60 min, and the amount of the mixed solution is 5 times the volume of the seeds.

4. The method of claim 1, wherein, In step two, the nanoparticle is selected from any one of water-soluble chitosan, polylactic acid-glycolic acid copolymer, silicon dioxide, polystyrene or liposome, and the concentration of the nanoparticle is 5-10 g / L.

5. The method of claim 4, wherein, In step two, the nanoparticle is water-soluble chitosan and the concentration is 5 g / L.

6. The method of claim 1, wherein, In step two, the photosensitizer is selected from any one of methylene blue, zinc phthalocyanine, chlorin e6 or tetraphenyl porphyrin tetrasulfonic acid, the concentration of the photosensitizer is 20-80 μg / mL, the concentration of the proteinase K is 200-400 μg / mL, and the concentration of the sodium dodecyl sulfate is 2-10 g / L.

7. The method of claim 6, wherein, In step two, the photosensitizer is chlorin e6 and the concentration is 50 μg / mL, the concentration of the proteinase K is 300 μg / mL, the concentration of the sodium dodecyl sulfate is 5 g / L, the soaking time of the mixed solution is 2 hours, and the amount of the mixed solution is 3-10 times the volume of the seeds.

8. The method of claim 1, wherein, In step two, before the hydrogen peroxide solution is added, part of the treatment solution is discarded so that the volume ratio of the treatment solution to the seeds is 1:1-3:1, the final concentration of the hydrogen peroxide is 5-15 g / L, and the soaking time is 30-60 min; the wavelength of the light irradiation is 655-665 nm, and the irradiation time is 2-6 hours.

9. The method of claim 8, wherein, In step two, after part of the treatment solution is discarded, the volume ratio of the treatment solution to the seeds is 2:1, the final concentration of the hydrogen peroxide is 10 g / L, and the soaking time is 30 min; the wavelength of the light irradiation is 655-665 nm, and the irradiation time is 4 hours.

10. The method of claim 1, wherein, The specific operation of the RT-LAMP detection in step five is as follows: total RNA of the seedling in step four is extracted, an RT-LAMP reaction system is prepared to perform isothermal amplification reaction, the reaction temperature is 63 DEG C, the reaction time is 45 min, the fluorescence signal is collected every 1 min, and whether the positive signal appears is judged to determine whether the cucumber mosaic virus is removed; the RT-LAMP reaction system comprises LAMP premix, fluorescent dye, primer mixture designed for the cucumber mosaic virus, nuclease-free water and RNA template.

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