A specific non-destructive pest removal process for tomato germplasm resources based on pathogen transmission mechanism

Abstract

The application belongs to the field of agricultural biotechnology and germplasm resource protection, and particularly relates to a specific non-destructive pest removal process for tomato germplasm resources based on pathogen transmission mechanism, which comprises the following steps: (1) placing tomato seeds in an environment with a temperature of 40-42 DEG C and a relative humidity of 80-90%, while introducing ozone gas with a concentration of 20-30 ppm, and treating for 1-2 hours; (2) placing the seeds treated in step (1) in dry hot air with a temperature of 48-52 DEG C and a wind speed of 1.0-1.5 m / s for dehydration treatment until the water content of the seeds is reduced to 5-6%; (3) heating the seeds after dehydration in step (2) to 70-72 DEG C for constant-temperature hot air circulation dry heat treatment for 36-48 hours, thereby completing the pest removal. The process of the application overcomes the technical problems of long time, damage to germination rate and easy scalding of seeds by wet heat in traditional dry heat, and realizes the dual effects of specific protection of seed embryo activity and specific inactivation of pathogens.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and germplasm resource protection, specifically involving a specific non-destructive pest control process for tomato germplasm resources based on the pathogen transmission mechanism. Background Technology

[0002] Tomato germplasm resources are the material basis for breeding and germplasm innovation. However, tomato seeds are highly susceptible to carrying a variety of persistent seed-borne pathogens, such as Tomato Brown Fruit Curl Virus (ToBRFV), Tomato Yellow Leaf Curl Virus (TYLCV), and Clavibacter michiganensis. These pathogens can not only survive during seed storage and transportation but may also remain latent inside the seed coat and even in the embryo tissue, seriously threatening seed health and subsequent production.

[0003] Currently, the commonly used harmless treatment method in germplasm resource banks is traditional high-temperature dry heat treatment (such as constant temperature dry heat at 72~75℃ for 72~96 hours). Although this method leaves no chemical residues, it has significant drawbacks: prolonged dry heat causes cumulative heat damage to the embryo, resulting in a sharp decline in germination rate; at the same time, pathogens are dehydrated and embedded in the dry seed coat, exhibiting extremely strong heat resistance, making it difficult for pure dry heat to penetrate and completely inactivate latent pathogens in a short time. If the treatment humidity is simply increased (such as soaking seeds in warm water), tomato seeds are very prone to complete loss of vitality due to heat and moisture denaturation under high temperature and humidity, i.e., the so-called "steaming" phenomenon occurs.

[0004] Therefore, how to shorten the treatment time and lower the inactivation temperature threshold without damaging the embryo viability, so as to achieve specific and thorough elimination of deep pathogens in tomato germplasm resources, is a technical problem that urgently needs to be solved in the field. Summary of the Invention

[0005] The purpose of this invention is to provide a specific non-destructive pest control process for tomato germplasm resources based on the pathogen transmission mechanism. By combining gas-phase hydration with ozone sensitization and dry heat treatment, this process overcomes the technical problems of long dry heat treatment time, damage to germination rate, and easy scalding of seed embryos by wet heat.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A non-destructive pest control process for tomato germplasm resources includes the following steps:

[0008] (1) Place tomato seeds in an environment with a temperature of 40~42℃ and a relative humidity of 80%~90%, and simultaneously introduce ozone gas with a concentration of 20~30 ppm for 1~2 hours;

[0009] (2) Place the seeds treated in step (1) in a dry hot air at 48~52℃ and a wind speed of 1.0~1.5 m / s for dehydration until the seed moisture content drops to 5%~6%;

[0010] (3) Heat the seeds after dehydration in step (2) to 70~72℃ and perform constant temperature hot air circulation dry heat treatment for 36~48 hours to complete the pest control.

[0011] Further, in step (1), the relative humidity is 85%, the temperature is 41°C, the ozone concentration is 25 ppm, and the treatment time is 1.5 hours.

[0012] Furthermore, in step (2), the temperature of the drying hot air is 50°C, the wind speed is 1.3 m / s, and the dehydration time is 1.5 to 2 hours.

[0013] Furthermore, in step (3), the temperature of the constant temperature hot air circulation dry heat treatment is 72°C, and the treatment time is 36 hours.

[0014] Furthermore, the process is used to inactivate tomato brown wrinkled fruit virus, tomato yellow leaf curl virus and / or tomato canker bacteria carried on tomato seeds.

[0015] Furthermore, in step (1), the ozone gas is prepared on-site by a pure oxygen corona discharge generator and is uniformly fed into the treatment chamber by a gas phase circulating fan.

[0016] Beneficial effects of the present invention

[0017] Based on the colonization characteristics of tomato seed-borne pathogens, which are hidden in the seed coat villi, micropores, and microcracks, this invention first utilizes vapor-phase hydration to induce the seed coat micropores to swell and crack, forcibly exposing the pathogens and transforming them from a dense, heat-resistant glassy state in a dry state to a loose, hydrated state. Simultaneously, the water film formed by vapor-phase hydration on the seed coat surface and within the micropores dissolves ozone to generate active free radicals, which oxidize and sensitize the protein capsids and nucleic acids of the pathogens on the surface and within the micropores, while the inner embryo is not eroded by free radicals due to the seed coat barrier. Subsequently, the pre-sensitized pathogens can be mildly inactivated by dry heat under conditions that reduce the temperature from the traditional 75°C to 70-72°C and shorten the treatment time from 72-96 hours to 36-48 hours, achieving the dual effect of specific protection of the embryo's activity and specific inactivation of the pathogen. Attached Figure Description

[0018] Figure 1 A schematic diagram of the process flow of the method of the present invention is shown. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0020] Detection and evaluation methods:

[0021] Moisture content determination: Refer to GB / T 3543.6 Crop Seed Inspection Procedure for Moisture Determination, and adopt the high temperature drying method (dry at 130℃ to constant weight).

[0022] Pathogen inactivation rate evaluation: PMA-RT-qPCR (for viruses ToBRFV and TYLCV) and PMA-qPCR (for ulcer pathogens) were used to specifically detect the number of live pathogens with intact biofilms / capsids. Virus inactivation was verified by biological friction inoculation test of Tobacco denudata / Tobacco japonica to confirm the incidence of necrotic spots; ulcer pathogen colony formation was verified by streak culture on semi-selective medium. The overall assessment determined whether the pathogen had completely lost its infectivity / reproductive capacity.

[0023] Germination rate and germination potential determination: In accordance with GB / T 3543.4, the paper germination test was carried out in a constant temperature and light incubator at 25℃. Germination potential was calculated after 4 days and germination rate was calculated after 7 days. Each treatment was set up with 3 biological replicates. The results are expressed as "mean ± standard deviation".

[0024] Accelerated aging test: The treated seeds were placed in an environment of 45℃ and 85% relative humidity for 7 days, and then a standard germination test was performed to evaluate the seeds’ anti-aging ability and vigor retention.

[0025] Seed raw materials to be treated: Selected "Zhongshu No. 4" tomato seeds that were confirmed to be infected with ToBRFV, TYLCV and tomato canker fungus, with an initial moisture content of 6.0% and an initial germination rate of 95.8%.

[0026] Example 1

[0027] (1) Spread 100g of seeds evenly on a porous tray (thickness ≤1cm) and place it in a temperature and humidity test chamber. Set the temperature to 40℃ and the relative humidity to 80%, turn on the pure oxygen corona discharge ozone generator, and stabilize the ozone concentration in the chamber at 20 ppm. Circulate the gas phase fan. Treat for 2.0 hours.

[0028] (2) The seeds were quickly transferred to a convection drying oven, the temperature was set at 48℃, the wind speed at 1.0 m / s, and the drying time was 2.0 hours. The seed moisture content was measured to be 6.0%.

[0029] (3) Raise the temperature of the drying oven to 70°C and continuously circulate clean hot air for 48 hours. After the drying is completed, cool it to room temperature with sterile air.

[0030] Example 2

[0031] (1) Take 100g of seeds and place them in the test chamber. Set the temperature to 41℃, relative humidity to 85%, ozone concentration to 25 ppm, and circulate the gas phase for 1.5 hours.

[0032] (2) The seeds were placed in a drying oven, the temperature was set at 50℃, the wind speed was 1.3 m / s, and the drying time was 1.5 hours. The moisture content of the seeds was measured to be 5.5%.

[0033] (3) Raise the temperature to 72°C and circulate hot air at a constant temperature for 36 hours. After the process, cool the temperature to room temperature.

[0034] Example 3

[0035] (1) Take 100g of seeds and place them in the test chamber. Set the temperature to 42℃, relative humidity to 90%, ozone concentration to 30 ppm, and gas phase circulation treatment for 1.0 hour.

[0036] (2) The seeds were placed in a drying oven, the temperature was set at 52℃, the wind speed was 1.5 m / s, and the drying time was 1.5 hours. The moisture content of the seeds was measured to be 5.2%.

[0037] (3) Raise the temperature to 72°C and circulate hot air at a constant temperature for 36 hours. After the process, cool the temperature to room temperature.

[0038] Comparative Example 1 (Traditional Dry Heat Treatment)

[0039] Seeds from the same batch were placed directly in a 75℃ forced-air drying oven for 72 hours without pre-hydration or ozone treatment.

[0040] Comparative Example 2 (Moist heat damage treatment without dehydration step)

[0041] After processing according to step (1) of Example 2, without performing step (2) drying and dehydration, the temperature inside the chamber is directly raised from 41°C to 72°C and kept at a constant temperature for 36 hours.

[0042] Comparative Example 3 (Ozone-free pretreatment)

[0043] Replace the ozone gas in step (1) of Example 2 with clean air of the same flow rate, and keep the other conditions the same as in Example 2.

[0044] Comparative Example 4 (without gas phase hydration and ozone pretreatment)

[0045] The seeds were not subjected to the gas phase hydration and ozone treatment in step (1) of Example 2, but were directly subjected to dehydration at 50°C for 1.5 hours in step (2), followed by constant temperature dry heat treatment at 72°C for 36 hours.

[0046] Comparative Example 5 (without dry heat inactivation)

[0047] After processing for 1.5 hours according to step (1) of Example 2, and then dehydrating at 50°C for 1.5 hours in step (2), the high-temperature dry heat treatment at 72°C in step (3) is not performed, and the product is directly cooled for testing.

[0048] Comparative Example 6 (Pretreatment by immersion in liquid ozone water)

[0049] Tomato seeds from the same batch were placed in a prepared 25 ppm ozone solution and soaked at 41°C for 1.5 hours. They were then removed and quickly placed in a dry hot air at 50°C and a wind speed of 1.3 m / s for 1.5 hours to dehydrate until the moisture content dropped to about 5-6%. Finally, the temperature was raised to 72°C and circulated with hot air for 36 hours for dry heat treatment.

[0050] Test Example 1: Comparison of Pathogen Inactivation and Germination Rate

[0051] Seeds from each treatment group and the untreated control group were sampled and subjected to PMA-RT-qPCR / qPCR detection, biological inoculation verification, and germination test. The results are shown in Table 1.

[0052] Table 1: Pathogen inactivation rate and germination performance test results for each treatment group (mean ± SD, n=3)

[0053] Processing group ToBRFV inactivation rate (%) TYLCV inactivation rate (%) Inactivation rate of peptic ulcer bacteria (%) Biological inoculation live detection Germination rate (%) Germination potential (%) Untreated control group - - - Positive (pathogenic) 95.8±1.6 90.8±1.9 Example 1 >99.99 >99.99 >99.999 Not detected (negative) 94.8±1.5 89.5±1.8 Example 2 >99.999 >99.999 >99.999 Not detected (negative) 95.5±1.2 90.2±1.5 Example 3 >99.999 >99.999 >99.999 Not detected (negative) 95.0±1.3 89.8±1.6 Comparative Example 1 99.2 98.8 99.5 Positive (residual) 82.3±2.1 71.5±2.5 Comparative Example 2 >99.999 >99.999 >99.999 Not detected (negative) 0 0 Comparative Example 3 99.8 99.1 99.7 Positive (residual) 95.2±1.4 90.1±1.8 Comparative Example 4 98.5 97.9 98.8 Positive (residual) 94.5±1.6 88.2±2.0 Comparative Example 5 88.5 85.2 92 Positive (residual) 95.6±1.3 90.5±1.7 Comparative Example 6 99.8 99.2 99.9 Positive (residual) 78.5±2.8 65.0±3.2

[0054] Table 1 shows that Examples 1-3, while protecting the germination rate (94.8%~95.5%, with no significant difference from the untreated control group of 95.8%), also achieved complete inactivation of the three pathogens (extremely low molecular detection rate and negative biological inoculation), confirming the feasibility and stability of the process parameter range. Comparative Example 1 showed that traditional high-temperature, long-term dry heat resulted in a sharp decrease in germination rate and incomplete inactivation of pathogens such as ToBRFV lurking deep within the layers, indicating that pure dry heat is insufficient to overcome the heat-resistant glassy state of the pathogens. Comparative Example 2 verified the irreplaceable nature of step (2) of tiered dehydration protection; direct high-temperature treatment without dehydration leads to thermal denaturation and death of the embryo. Comparative Examples 3 and 4 show that without ozone pre-sensitization or gas-phase hydration induction, complete inactivation of the pathogens could not be achieved under mild conditions of 72℃ / 36h, demonstrating the synergistic effect of "hydration swelling + ozone sensitization". Comparative Example 5 shows that relying solely on room temperature ozone hydration treatment can only inactivate some surface pathogens; a combined mild dry heat treatment is necessary to achieve deep pest control. Comparative Example 6 uses liquid-phase ozone water soaking instead of the gas-phase hydration synergistic ozone pretreatment of this invention. The results show that liquid-phase soaking is difficult to efficiently penetrate into the micropores and microcracks deep within the seed coat, thus failing to remove ToBRFV and TYLCV viruses lurking deep within the micropores. Furthermore, liquid soaking causes excessive water absorption by the seeds, with water entering the embryo and damaging the protective barrier of the seed coat on the inner embryo. Even after a short period of dehydration, the embryo cells have already undergone physiological germination / hydration damage, resulting in a significant decrease in embryo viability during subsequent dry heat treatment at 72°C.

[0055] Test Example 2: Assessment of Accelerated Seed Aging and Long-Term Viability Maintenance

[0056] Seeds treated in Examples 1-3 and the main comparative examples were subjected to accelerated aging tests (45℃, 85% RH, 7 days) to evaluate their anti-aging ability and latent heat damage. The results are shown in Table 2.

[0057] Table 2: Comparison of seed germination vigor after accelerated aging (mean ± SD, n=3)

[0058] Processing group Germination rate after aging (%) Germination potential after aging (%) Simplified Vitality Index (Sprout Length × Germination Rate) Untreated control group 86.3±2.0 80.5±2.4 7.4 Example 1 84.5±2.2 78.5±2.3 7.1 Example 2 85.8±1.8 79.8±2.1 7.3 Example 3 85.2±1.9 79.2±2.2 7.2 Comparative Example 1 64.1±3.5 50.3±4.2 5.1 Comparative Example 3 84.8±2.1 78.8±2.4 7.1 Comparative Example 4 81.2±2.5 74.5±2.8 6.5 Comparative Example 6 58.2±3.6 42.1±4.0 4.5

[0059] Table 2 shows that the germination rate of seeds treated with the processes of Examples 1-3 of this invention remained at 84.5%~85.8% under harsh aging conditions, which was the same as that of the untreated control group (86.3%), with no significant difference. However, the germination rate of the traditional dry heat treatment group (Comparative Example 1) dropped sharply to 64.1% after aging, indicating that traditional dry heat caused serious latent heat damage. The test data confirms that the process of this invention can achieve truly specific and non-destructive pest removal, fully meeting the requirements for long-term safe preservation of germplasm resources.

[0060] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A non-destructive pest control process for tomato germplasm resources, characterized in that, Includes the following steps: (1) Place tomato seeds in an environment with a temperature of 40~42℃ and a relative humidity of 80%~90%, and simultaneously introduce ozone gas with a concentration of 20~30ppm for 1~2 hours; (2) Place the seeds treated in step (1) in a dry hot air at 48~52℃ and a wind speed of 1.0~1.5 m / s for dehydration until the seed moisture content drops to 5%~6%; (3) Heat the seeds after dehydration in step (2) to 70~72℃ and perform constant temperature hot air circulation dry heat treatment for 36~48 hours to complete the pest control.

2. The process according to claim 1, characterized in that, In step (1), the relative humidity is 85%, the temperature is 41°C, the ozone concentration is 25 ppm, and the treatment time is 1.5 hours.

3. The process according to claim 1, characterized in that, In step (2), the temperature of the drying hot air is 50°C, the wind speed is 1.3 m / s, and the dehydration time is 1.5 to 2 hours.

4. The process according to claim 1, characterized in that, In step (3), the temperature of the constant temperature hot air circulation dry heat treatment is 72°C and the treatment time is 36 hours.

5. The process according to any one of claims 1-4, characterized in that, The process is used to inactivate tomato brown wrinkled fruit virus, tomato yellow leaf curl virus and / or tomato canker bacteria carried on tomato seeds.

6. The process according to claim 1, characterized in that, In step (1), the ozone gas is prepared on-site by a pure oxygen corona discharge generator and is evenly fed into the treatment chamber by a gas phase circulation fan.

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