Method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting appliance

Abstract

The invention discloses a method for inactivating and disinfecting cucumber green mottle mosaic virus on a grafting appliance. A disinfectant composed of trisodium phosphate and ribonuclease is used for soaking and disinfecting a grafting appliance, and the cucumber green mottle mosaic virus on the surface of the appliance can be effectively inactivated. In the process, trisodium phosphate can split cucumber green mottle mosaic virus particles to release virus genome RNA, ribonuclease degrades virus RNA, the cucumber green mottle mosaic virus can be completely inactivated through the synergistic effect of trisodium phosphate and ribonuclease, and thorough disinfection of grafting tools is achieved. The method is applied to the disinfection of the grafting appliance, can effectively block the propagation of the cucumber green mottle mosaic virus through a grafting path, achieves the purpose of controlling diseases, is harmless to operators and plants, and is suitable for popularization and application in the melon grafting process.

Description

A technical field

[0001] The invention relates to a disinfection technology for agricultural tools, in particular to a method for inactivating cucumber green mottle mosaic virus by synergistically utilizing trisodium phosphate and ribonuclease, and the method is suitable for surface disinfection of grafting tools. 2. Background Technology

[0002] Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobaccomovirus in the family Bacillusviridae, causes severe damage to cucurbit crops worldwide. Symptoms vary depending on the host and environmental conditions, often manifesting as mosaic and mottling, often accompanied by dark green, blister-like protrusions. CGMMV can also cause discoloration, deformity, and deterioration of fruit, severely impacting crop quality and yield, leading to significant economic losses.

[0003] CGMMV once broke out in the Soviet Union, resulting in a watermelon production reduction of over 30%. In Japan, it caused direct economic losses of 1 billion yen to cantaloupes and watermelons. In 2005, a large-scale outbreak of CGMMV also occurred on watermelons in Gaizhou City, Liaoning Province, my country, with direct losses of 333 hectares. 2 In 2007, the virus also occurred in Gaozhou City, Guangdong Province, my country, and the cucumber loss area was about 3hm 2 In view of the severe impact of CGMMV on cucurbit crops worldwide, my country listed CGMMV as a national agricultural plant quarantine pest and an imported plant quarantine pest in 2006.

[0004] CGMMV can be spread through agricultural practices such as pruning, pruning, and grafting. Watermelon grafting is an effective technical measure to improve watermelon growth performance, but the grafting process is also a key route for CGMMV transmission and spread. Currently, grafting tools are commonly disinfected with 10% trisodium phosphate during the grafting process, but there is no data demonstrating whether this method effectively inactivates CGMMV in practice. Furthermore, tools coated with 10% trisodium phosphate can cause burns to the grafting wound if used without cleaning. During research and development, the inventors discovered that treating CGMMV particles with 10% trisodium phosphate causes the capsid protein of CGMMV particles to dissociate and release viral genomic RNA, but does not destroy the viral RNA. Previous studies have reported that tobacco mosaic virus particles are particularly stable in the environment. At room temperature, the capsid protein can persist in water for two weeks, but the viral RNA can remain infectious for over 14 weeks. Therefore, disinfecting grafting tools with only 10% trisodium phosphate cannot completely inactivate CGMMV and prevent its transmission. 3. Summary of the invention

[0005] Based on the existing mechanism research of the effect of trisodium phosphate on CGMMV particles, the application provides a method for inactivating CGMMV by using trisodium phosphate and ribonuclease in cooperation, which is suitable for surface disinfection of grafting tools. Four technical solutions

[0006] Through the mechanism research of the effect of trisodium phosphate on CGMMV, it is found that trisodium phosphate can destroy and dissociate the shell protein polymer on the CGMMV virus particle, and release the complete genome RNA originally wrapped in the virus particle; when a certain concentration of ribonuclease exists in the reaction solution, the ribonuclease can rapidly degrade the released CGMMV genome RNA, and the complete elimination of CGMMV can be achieved, therefore, the combination of trisodium phosphate and ribonuclease can inactivate CGMMV in cooperation. As a preferred technical solution, trisodium phosphate and ribonuclease inactivate CGMMV in cooperation, by soaking the CGMMV-contaminated tools in 0.1%-10% trisodium phosphate and ribonuclease (20-100 μg / mL) for 10-15 minutes, the CGMMV on the tools can be effectively inactivated. Preferably, the concentration range of 0.1%-5% trisodium phosphate and ribonuclease (20-100 μg / mL) can inactivate CGMMV on the tools in cooperation. Further preferably, 0.1%-1% trisodium phosphate and ribonuclease (20-100 μg / mL) are used in combination, which has no burning effect on the plant wound. Five beneficial effects of the application

[0007] The application discloses a method for completely inactivating CGMMV on grafting tools, which can effectively inhibit the spread of CGMMV, and the disinfection method is harmless to the operators and plants. Six description of drawings

[0008] Figure 1 : Inactivation effect of different disinfection treatments on CGMMV on the surface of grafting tools; wherein 1-5 is 10% trisodium phosphate solution treatment, 6-10 is 10% trisodium phosphate and 50 μg / mL ribonuclease mixed solution treatment, 11-15 is water treatment, 16 is a blank control, 17 is a negative control, and 18 is a positive control.

[0009] Figure 2 : RNA content in the tool washing solution treated by different concentrations of trisodium phosphate.

[0010] Figure 3: Different concentrations of trisodium phosphate treatment on the lysis of virus on the surface of the tool; wherein 1-5 is 10% trisodium phosphate solution treatment, 6-10 is 5% trisodium phosphate solution treatment, 11-15 is 1% trisodium phosphate solution treatment, 16-20 is 0.1% trisodium phosphate solution treatment, 21-25 is 0.01% trisodium phosphate solution treatment, 26-30 is water treatment, 31 is blank control, 32 is negative control, and 33 is positive control. Seven DETAILED DESCRIPTION

[0011] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or substitutions of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, are within the scope of the present application.

[0012] Unless otherwise specified, the experimental materials, reagents and instruments used in the examples of the present application are commercially available. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art.

[0013] Example 1: Inactivation of CGMMV on grafting tools by combined use of trisodium phosphate and ribonuclease

[0014] 1. Preparation of CGMMV virus solution

[0015] Take 100 g of CGMMV-infected leaves, add 10 mL of distilled water, grind and homogenize thoroughly, centrifuge at 3000 rpm for 10 minutes, take the upper liquid, and use it as the virus solution for infecting the grafting tools.

[0016] 2. Virus-contaminated grafting tools

[0017] Use a grafting blade as a representative grafting tool to carry out experimental verification. Immerse the blade in the virus solution, then take out the blade, and attach the blade tip to the blotting paper to absorb the residual liquid on the surface of the blade. At this time, the blade surface has been contaminated with viruses.

[0018] 3. Disinfection of tools

[0019] Soak the blade in 10% trisodium phosphate solution, 10% trisodium phosphate and 50 μg / mL ribonuclease (RNase A) mixed solution, and water for 10-15 minutes, respectively. Set 5 replicates for each treatment. Take out the blade, attach the blade tip to the blotting paper to absorb the residual liquid on the surface of the blade, and use a pipette to rinse the blade with 100 μL of distilled water repeatedly to obtain the washing liquid.

[0020] 4. Virus detection

[0021] The utensil washing liquid was directly used for RT-PCR detection of CGMMV virus. The detection primers used were CGMMV1: 5'-CGTGGTAAGCGGCATTCTAAACCTC-3', CGMMV2: 5'-CCGCAAACCAATGAGCAAACCG-3'.

[0022] Reverse transcription reaction system:

[0023] Using a reverse transcription kit, add each component to an RNase-free PCR tube on ice as follows, mix well and centrifuge briefly;

[0024]

[0025] Reverse transcription reaction conditions:

[0026] Perform reverse transcription reaction on PCR instrument according to the following reaction program;

[0027]

[0028] PCR reaction system:

[0029]

[0030] The reaction conditions are 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 30 s, 35 cycles, 72°C maintenance for 5 min, and 4°C storage. Take 8 μL of the PCR product for 1.0% agarose gel electrophoresis detection, and observe the electrophoresis results on an ultraviolet gel imaging system.

[0031] 5. Inactivation results

[0032] RT-PCR detection can obtain the residual CGMMV on the surface of the utensil after disinfection treatment. The results are shown in Figure 1 As shown, the blade treated with water can detect virus-specific fragments, indicating that the blade is contaminated with virus; compared with the water control group, the 10% trisodium phosphate solution treatment group also detects virus-specific fragments, and the fragment brightness is higher, indicating that the blade is contaminated with higher viral RNA load, indicating that trisodium phosphate can destroy and dissociate CGMMV particles to release genomic RNA; and the trisodium phosphate and ribonuclease mixed solution treatment group cannot detect virus-specific fragments, indicating that this treatment method can completely eliminate the virus contaminated on the blade, and the principle of elimination is that ribonuclease degrades the viral RNA released by trisodium phosphate.

[0033] From the above results, it can be seen that soaking the grafting utensil in a mixed solution of 10% trisodium phosphate and 50 μg / mL ribonuclease can effectively eliminate CGMMV contaminated on the surface of the utensil, including viral particles and genomic RNA.

[0034] Example 2: Effective concentration of trisodium phosphate to lyse CGMMV particles and plant safety

[0035] 1. Preparation of CGMMV virus solution and virus-contaminated grafting tools

[0036] The same as Example 1.

[0037] 2. Trisodium phosphate disinfection treatment

[0038] To determine the effective concentration of trisodium phosphate to lyse CGMMV particles, blades were immersed in trisodium phosphate solutions with gradient concentrations (10%, 5%, 1%, 0.1%, and 0.01%) for 10-15 minutes, and water treatment was used as a control, with 5 replicates for each treatment. The blades were removed, and the blade tips were gently attached to absorbent paper to absorb the residual liquid on the blade surface. A pipette was used to rinse the blade repeatedly with 100 μL of distilled water to obtain the washing liquid.

[0039] 3. Detection of RNA content in washing liquid

[0040] The RNA concentration in the washing liquid of different treatment groups was determined using a Nanodrop 2000c ultramicro spectrophotometer.

[0041] 4. Virus detection

[0042] RT-PCR was used to detect the residual CGMMV on the surface of tools after different disinfection treatments, following the same method as in Example 1.

[0043] 5. Safety evaluation of trisodium phosphate on plants

[0044] Blades were immersed in trisodium phosphate solutions with gradient concentrations (10%, 5%, 1%, 0.1%, and 0.01%), and after removal, the blades were used to gently cut tobacco stems. The burn situation of the wounds was observed after 4 hours.

[0045] 6. Results of the lytic effect of different concentrations of trisodium phosphate on CGMMV

[0046] The content of the washing liquid of tools treated with different concentrations of trisodium phosphate was determined, and the results are shown in Table 2. Figure 2 The RNA concentration in the washing liquid of the 10%, 5%, 1%, and 0.1% trisodium phosphate treatment groups was higher than 50 ng / μL, with no significant difference between them. However, the RNA concentration in the 4 treatment groups was significantly higher than that in the 0.01% trisodium phosphate treatment group and the water control group, indicating that when the trisodium phosphate solution is higher than 0.1%, it can lyse CGMMV particles to release RNA.

[0047] The virus detection results are shown in Table 3. Figure 3As shown, the blade treated with water can detect the virus-specific fragments, indicating that the blade is contaminated with viruses; compared with the water control group, the 10%, 5%, 1%, 0.1% trisodium phosphate solution treatment group detects higher brightness of virus fragments, indicating that the blade is contaminated with higher viral RNA load, indicating that when the trisodium phosphate solution is higher than 0.1%, the CGMMV particles can be lysed; the brightness of the virus fragments detected by the 0.01% trisodium phosphate treatment group is similar to that of the water control group, indicating that the trisodium phosphate solution at this concentration cannot lyse the CGMMV particles.

[0048] 7. Safety results of different concentrations of trisodium phosphate to plants

[0049] Observing the burning of the tobacco wound cut by the blade of different treatment groups, it is found that 10% and 5% trisodium phosphate can cause corrosion to the plant wound, while the 1%, 0.1%, 0.01% trisodium phosphate treatment groups and the water treatment group have no corrosion to the wound.

[0050] From the above results, when the trisodium phosphate solution is higher than 0.1%, the CGMMV particles can be lysed to release RNA, and at the same time, the 0.1%-1% trisodium phosphate solution is safe to the plant.

[0051] The specific embodiments of the present application are described in detail above, but they are only examples, and the present application is not limited to the specific embodiments described above, and the implementation and protection scope of the present application are not limited by the above description. For those skilled in the art, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be included in the protection scope of the present application.

Claims

1. A method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting equipment, characterized in that: Soak grafting instruments in a disinfectant solution consisting of trisodium phosphate and ribonuclease.

2. The method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting instruments according to claim 1, wherein: The concentration of trisodium phosphate in the disinfectant is 0.1-10%, and the concentration of ribonuclease is 20-100 μg / mL.

3. The method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting instruments according to claims 1-2, characterized in that: The ribonuclease is a degradative RNase, including but not limited to RNase A.

4. The method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting tools according to claims 1-2, characterized in that: The disinfectant solution needs to be prepared and used immediately, that is, the two components are mixed in proportion before use.

5. The method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting instruments according to claim 1, wherein: The grafting apparatus is soaked for no less than 15-20 minutes.

6. The method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting instruments according to claim 1, wherein: The grafting tools are all tools required for the grafting process of melon crops, including but not limited to blades, bamboo sticks, grafting needles, steel drills, grafting clamps, grafting rings, trays, etc.

7. The method for inactivating and disinfecting cucumber green mottle mosaic virus on grafting tools according to claims 1-2, characterized in that: After disinfection, the instruments must be rinsed with clean water.

8. Use of the method according to any one of claims 1 to 7 in the grafting process of melon crops.

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