Preparation method of prodenia litura nuclear polyhedrosis virus parent drug

Abstract

The invention belongs to the technical field of biological control, and relates to a preparation method of prodenia litura nuclear polyhedrosis virus concentrate, which comprises the following steps: cultivating and hatching prodenia litura eggs at constant temperature, culturing the prodenia litura eggs in a dark place into 4-day-old larvae, performing virus infection and proliferation on the 4-day-old larvae, collecting and screening dead insects with strong toxicity and early death as virus semi-finished products, and refrigerating and storing the virus semi-finished products. And finally, preparing the virus semi-finished product into mother powder to obtain the prodenia litura nuclear polyhedrosis virus mother drug. The method comprises the following steps: extracting a parent drug sample to obtain a virus polyhedrosis, extracting a DNA comparison gene sequence of the virus polyhedrosis, prodenia litura nuclear polyhedrosis virus which is identified as prodenia litura nuclear polyhedrosis virus when the similarity between the virus polyhedrosis and Spodoptera litura Nucleoplydrovirus reaches 80% or above, and measuring various performance indexes, so that the prodenia litura nuclear polyhedrosis virus can be identified as prodenia litura nuclear polyhedrosis virus. The prodenia litura nuclear polyhedrosis virus mother drug prepared by the preparation method is used for preventing and treating lepidoptera pests such as prodenia litura, has important significance, has good development and application prospects, and has application value in preparation of insecticides and prevention and treatment of flat thorn moths.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to a method for preparing a parent drug of Spodoptera litura nuclear polyhedrosis virus. Background Art

[0002] Virus stock drugs usually refer to virus particles or the original concentrated form of viruses, which contain the basic structure and genetic material of the virus. In the fields of scientific research and medicine, virus stock drugs are often used to study the characteristics of viruses, develop antiviral drugs and prepare vaccines.

[0003] Spodoptera litura is a worldwide agricultural pest that harms nearly 390 species of plants in 109 families, including vegetables, flowers, fruits and vegetables, and tobacco. At present, the control of Spodoptera litura mainly relies on chemical pesticides such as organophosphates, carbamates, pyrethroids, indoxacarb, and chlorantraniliprole. However, the use of chemical pesticides can easily cause ecological problems such as environmental pollution and food safety problems such as pesticide residues. Therefore, it is necessary to reduce the use of chemical pesticides in the control of Spodoptera litura and to find and develop biological pesticides that can replace chemical pesticides. Spodoptera litura nuclear polyhedrosis virus (Spodoptera lituramuhieapsid nucleopolyhedrovims, SpltMNPV) has a good control effect on Spodoptera litura, has the advantages of being safe from natural enemies, not polluting the environment, and not easy to develop drug resistance. In particular, it can form an epidemic in the pest population to achieve the purpose of long-term pest control, and has a good application prospect.

[0004] However, the Spodoptera litura nuclear polyhedrosis virus preparations in the city have unstable insecticidal effects when used to control local noctuid pests. The Spodoptera litura nuclear polyhedrosis virus mother drug prepared by the present invention has a high pathogenic effect on Spodoptera litura, a long lasting effect, and has good development and application prospects, and has application value in preparing insecticides and controlling flat moths.

[0005] Therefore, whether it is possible to provide a preparation method for preparing the above-mentioned Spodoptera litura nuclear polyhedrosis virus mother drug and overcome the above-mentioned technical deficiencies is a problem that technicians in this field urgently need to solve. Summary of the invention

[0006] In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a method for preparing a Spodoptera litura nuclear polyhedrosis virus mother drug, wherein the prepared virus mother drug has a high pathogenic effect on Spodoptera litura and a long lasting effect.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing a parent drug of Spodoptera litura nuclear polyhedrosis virus comprises the following steps:

[0009] S1. Collecting populations and pupal emergence: Collect 5- to 6-day-old pupae of Spodoptera litura from populations collected from the wild or from populations artificially reared indoors and place them in growth boxes, adding nutrients to transform them into adults;

[0010] S2. Adult egg laying: After the sex of the adults is identified, the Spodoptera litura is selected in a ratio of 1:1.2 and placed in an egg-laying box for mating and egg laying;

[0011] S3, egg collection: 48 hours after egg laying, the eggs in the egg-laying box are collected in batches into a refrigerator with egg foil, and collected every 12 hours until a total of 72 hours have passed;

[0012] S4. Egg culture: The egg foil in the refrigerator is transferred to a disinfection cabinet and fumigated with formaldehyde gas. After fumigation for 30 minutes, the egg foil is taken out and placed in a constant temperature incubator to culture brown eggs. Among the cultured eggs, 5% is retained for seed preservation by the optimization method, and 95% is used for larval culture.

[0013] S5, seed preservation: artificial feed is prepared and packed in insect boxes. After 5% of the eggs for seed preservation are hatched, they are placed in insect boxes in batches and cultured in the dark. After culturing to 4 days old, healthy larvae are picked out and placed in insect trays filled with artificial feed until pupation. When the pupae develop into brown, they are sent to the pupal emergence process;

[0014] S6, larval culture: prepare artificial feed and pack it in insect culture boxes, and after 95% of the eggs used for larval culture are hatched, put them into insect culture boxes in batches and culture them in the dark until 4-day-old larvae;

[0015] S7. Virus infection and proliferation: prepare infection solution and place it in the artificial feed in the insect box, culture and proliferate 4-day-old larvae in the dark, and track and check the larvae's disease status until the disease rate reaches 98%;

[0016] S8. Virus collection and storage: Collect dead insects from the insect box, select dead insects with strong virulence and early death as semi-finished virus products and store them in a refrigerator;

[0017] S9. Preparation of mother powder: dilute the refrigerated virus semi-finished product with purified water to form a slurry by colloid mill, crush and filter by colloid mill, concentrate by centrifugation, freeze-dry and test the content to obtain the mother drug of Spodoptera litura nuclear polyhedrosis virus.

[0018] As a further solution of the present invention: the temperature of the egg laying box in step S2 is 25-28°C and the humidity is 75-85%.

[0019] As a further solution of the present invention: the refrigerator temperature in step S3 is 8-10°C

[0020] As a further solution of the present invention: the temperature of the constant temperature incubator in step S4 is 25-28° C. and the humidity is 70-80%.

[0021] As a further embodiment of the present invention, the raw materials of the artificial feed include soybeans, barley, yeast powder, preservatives, agar, vitamin C, acetic acid and water. The production steps of the artificial feed are as follows: the soybeans and barley are roasted and crushed after maturity, agar and water are heated and dissolved, and finally the agar aqueous solution, yeast powder, preservatives, vitamin C, acetic acid and crushed soybeans and barley cooked dry materials are mixed and poured into a feed stirring pot, stirred for 3-5 minutes, and then poured into a cooling plate to cool until solidified.

[0022] As a further solution of the present invention: the packing amount of the artificial feed into the insect raising box in step S5 is 5g / box, the packing amount of the artificial feed into the insect raising tray in step S5 is 3g / hole, and the packing amount of the artificial feed into the insect raising box in step S6 is 20g / box.

[0023] As a further solution of the present invention: in step S5, each insect box receives 100 newly hatched larvae, each hole in the insect tray receives two healthy larvae, and in step S6, each insect box receives 100 newly hatched larvae.

[0024] As a further solution of the present invention: in step S5, the temperature of the dark culture is 25-28° C. and the humidity is 70-80%, and in step S6, the temperature of the dark culture is 25-28° C. and the humidity is 65-75%.

[0025] As a further solution of the present invention: the infection liquid preparation step in step S7 is to grind, filter, centrifuge and purify the selected typical dead insects and then put them into a container as a virus seed stock solution and store them in a refrigerator; the content of the infection liquid is selected by dilution multiple depending on the concentration of the stock solution in the container, and the liquid is dripped onto a blood cell counting plate and left to stand for 20 minutes before counting, and the calculation formula is the average number of each small grid × 400 × 10000 × dilution multiple, that is, the content of each milliliter of stock solution; determine an effective and accurate infection concentration to prepare the infection liquid, and the formula is stock solution concentration ÷ required concentration - 1 = dilution multiple.

[0026] As a further solution of the present invention: in step S7, the amount of the infection liquid is mixed with the artificial feed at a dosage of 0.05 ml per gram of feed, and the light-proof culture temperature in step S7 is 23-25° C. and the humidity is 60-70%.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention comprises the steps of laying eggs by adults of Spodoptera litura, culturing eggs at a constant temperature, culturing larvae in the dark and infecting and multiplying the larvae with viruses, collecting and screening dead insects as virus semi-finished products and preserving them, and making the virus semi-finished products into mother powder to obtain a virus mother drug, wherein a mother drug sample is extracted to obtain a virus polyhedrosis, and its DNA is extracted to compare the gene sequence, and the similarity between the virus polyhedrosis virus and Spodoptera litura Nucleopolyhedrovirus is proved to be 80% or more, that is, it is identified as Spodoptera litura nuclear polyhedrosis virus, and various performance indicators are measured.

[0029] (2) The Spodoptera litura nuclear polyhedrosis virus mother drug prepared by the present invention is used for the prevention and treatment of Lepidoptera pests such as Spodoptera litura, which is of great significance and has a good development and application prospect, and has application value in the preparation of insecticides and the prevention and treatment of flat moths. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The invention is further described below in conjunction with the accompanying drawings and embodiments:

[0031] Figure 1 This is the process flow for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug of this embodiment.

[0032] Figure 2 This is the electropherogram of the lef-8 gene of the Spodoptera litura nuclear polyhedrosis virus of this example. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments.

[0034] Example

[0035] The present embodiment provides a method for preparing a parent drug of nuclear polyhedrosis virus of Spodoptera litura, comprising the following steps: S1, collecting populations and pupae: collecting 5-6 day-old pupae of Spodoptera litura from populations collected in the wild or from populations artificially reared indoors to a growth box, adding nutrients to transform them into adults; S2, laying eggs by adults: after the sexes of the adults are identified, Spodoptera litura is selected in a ratio of 1:1.2 and placed in an egg-laying box for mating and laying eggs, and the egg-laying temperature and humidity are adjusted to 27°C and 80%; S3, collecting eggs: after 48 hours of egg-laying, the eggs in the egg-laying box are placed in batches in a refrigerator with egg foil. The eggs were collected every 12 hours until the total collection time was 72 hours. S4, egg culture: the egg foil in the refrigerator was transferred to the disinfection cabinet and fumigated with formaldehyde gas. After fumigation for 30 minutes, the eggs were taken out and placed in a constant temperature incubator at 27°C and 75% environment to culture brown eggs. Among the cultured eggs, 5% was retained for seed preservation by the optimization method, and 95% was used for larval culture. S5, seed preservation: artificial feed was prepared and packaged in insect boxes, with a packaging amount of 5g / box. After the 5% eggs to be used for seed preservation were hatched, they were placed in the insect boxes in batches at a temperature and humidity of 27°C. , 75% light-proof culture, after culturing to 4 days old, pick out the healthy larvae and place them in the insect tray filled with artificial feed, the filling amount is 3g / hole, until pupation, when the pupae develop to brown, send them to the pupal emergence process; S6, larval culture: prepare artificial feed and pack it in insect boxes, the filling amount is 20g / box, after 95% of the eggs to be used for larval culture are hatched, they are put into the insect boxes in batches, each box is filled with 100 newly hatched larvae, and cultured in the dark at a temperature and humidity of 27℃ and 75% light-proof culture until 4-day-old larvae; S7, virus infection and proliferation: prepare infection solution and infect the solution at a concentration of 1g / g human Spray 0.05 ml of the artificial feed into the artificial feed in the insect box, culture and proliferate 4-day-old larvae in the dark under the conditions of temperature and humidity of 24°C and 65%, and track and check the disease susceptibility of the larvae until the disease susceptibility rate reaches 98%; S8, virus collection and storage: collect diseased and dead insects in the insect box, select diseased and dead insects with strong virulence and early death as virus semi-finished products and store them in a refrigerator; S9, mother powder preparation: add purified water to the refrigerated virus semi-finished product to dilute and homogenize it into a colloid mill, crush and filter it with the colloid mill, concentrate it by centrifugation, freeze-dry it and detect its content to obtain the mother drug of Spodoptera litura nuclear polyhedrosis virus.

[0036] In this embodiment, the raw materials of the artificial feed include soybeans, barley, yeast powder, preservatives, agar, vitamin C, acetic acid and water. The steps of making the artificial feed are as follows: roasting the soybeans and barley until mature and then crushing them, then heating the agar and water to dissolve them, and finally mixing the agar aqueous solution, yeast powder, preservatives, vitamin C, acetic acid and crushed soybeans and cooked barley dry materials into a feed mixing pot, stirring for 5 minutes, and then pouring it into a cooling plate to cool until solidified.

[0037] In this embodiment, the steps of preparing the infection liquid are as follows: grinding, filtering, centrifuging and purifying the selected typical dead insects, and then putting them into a container as a virus seed stock solution and storing them in a refrigerator; the content of the infection liquid is selected according to the dilution multiple of the stock solution concentration in the container, and the liquid is dripped onto a blood cell counting plate and left to stand for 20 minutes before counting. The calculation formula is the average number of each small grid × 400 × 10000 × dilution multiple, which is the content of the stock solution per milliliter; an effective and accurate infection concentration is determined to prepare the infection liquid, and the formula is the stock solution concentration ÷ required concentration - 1 = dilution multiple.

[0038] Technical effect verification

[0039] Polymerase chain reaction method: extract the sample virus DNA, use polymerase chain reaction to amplify the DNA lef-8 gene fragment, and separate and compare the homology of the lef-8 gene fragment by agarose gel electrophoresis

[0040] 1) Test sample: Weigh 1 g of the Spodoptera litura nuclear polyhedrosis virus stock drug of the embodiment, dilute and suspend with sterile water, add 0.5% SDS to make the final SDS concentration 0.1%, keep at 37°C for 30 min, dilute 10 times, wash away SDS by centrifugation at 4000 rpm / min, dilute 10 times again, place on the top layer of 40-60% sucrose gradient, centrifuge at 8000 rpm / min for 30 min, collect the milky white layer, wash twice with sterile water, centrifuge at 4000 rpm / min to obtain pure virus polyhedrosis and store in a 4°C refrigerator for use;

[0041] Take 1×109pib / g virus polyhedron in a 2ml centrifuge tube, centrifuge at 8000rpm / min for 10min to precipitate the virus, remove the supernatant, suspend the precipitate in 600μLddH2O, add 300μL3×DAS, incubate at 37℃ water bath for 30min, shake every 5min during the period, centrifuge at 8000rpm for 5min, take the supernatant, add 9μL proteinase K, incubate at 37℃ water bath for 16h, add 100μL10% SDS, keep warm in 50℃ water bath for 30min, then add an equal volume of phenol, reverse extract on a 360° rotating shaker for 30min, centrifuge at 12000rpm for 10min, absorb the upper aqueous phase, transfer to another centrifuge tube, add an equal volume of phenol and chloroform mixture with a ratio of 1:1 to the centrifuge tube, and reverse extract on a 360° rotating shaker for 30min. 0min, centrifuge at 12000rpm for 10min, draw the upper aqueous phase, transfer it to another centrifuge tube, add an equal volume of a mixture of phenol, chloroform and isoamyl alcohol in a ratio of 25:24:1 to the centrifuge tube, and then invert and extract on a 360° rotating shaker for 30min, centrifuge at 12000rpm for 10min, draw the upper aqueous phase, transfer it to another centrifuge tube, add 2.5 times the volume of 95% ice ethanol to the centrifuge tube, precipitate at -20℃ for 30min, centrifuge at 12000rpm for 10min, discard the supernatant, wash the precipitate with 500μL pre-cooled ethanol, centrifuge at 12000rpm for 10min, discard the supernatant, evaporate the residual ethanol in the centrifuge tube at room temperature, obtain DNA after drying, dissolve the DNA in ddH2O, first dissolve it at room temperature for 3-4h, and then place it at 4℃ overnight to obtain the DNA sample for use.

[0042] 2) Lef-8 gene fragment amplification (PCR amplification) of DNA samples:

[0043] PCR amplification reaction system:

[0044]

[0045] Reaction conditions: first stage, denaturation at 95°C for 3 min, 1 cycle; second stage, 95°C for 30 sec, 40°C for 1 min, 70°C for 1 min, 36 cycles in total; third stage, extension at 72°C for 10 min; after the PCR, add 5 μL 10× loading buffer to the product.

[0046] 3) Horizontal gel electrophoresis test: 1% agarose was prepared with 1.0×TAE buffer and two gel tanks were filled to form; the gel was placed in a gel electrophoresis tank containing 1.0×TAE buffer with the buffer covering the gel, 5-6 μL of amplified DNA sample and 5 μL Mark3 were added to the first gel as a control, and the remaining amplified DNA sample and 5 μL Mark3 were added to the second gel as a control, and electrophoresed at 135V for 35 minutes; after electrophoresis, the gel was placed in EB for staining for 5-10 minutes, the first gel was used for taking pictures, and the second gel was used for recovering the target band gel, and the recovered gel was sent to a genetic company for sequencing.

[0047] 4) Verification results: The horizontal gel electrophoresis test sequencing results were compared with the gene sequence on NCBI blast. The result showed that the similarity with Spodoptera litura Nucleopolyhedrovirus reached more than 80%, which means it was identified as Spodoptera litura Nucleopolyhedrovirus. The electrophoresis map of the lef-8 gene is as follows: Figure 1 shown.

[0048] Quality indicators

[0049] The performance indicators of the Spodoptera litura nuclear polyhedrosis virus stock drug were determined according to relevant national standards.

[0050] a. The content of the active ingredient, Spodoptera litura nuclear polyhedrosis virus, is determined by the blood cell plate counting method:

[0051] Determination steps: accurately weigh 1g of the Spodoptera litura nuclear polyhedrosis virus mother drug of the embodiment into a 1000mL volumetric flask, add distilled water to the scale, mix by inversion, and set aside; when measuring, first cover the clean and dry blood cell counting plate with a cover glass, then drop the above-mentioned 1000-fold diluted virus polyhedron suspension from one side of the cover glass, let the suspension automatically enter the counting chamber along the gap capillary osmosis, absorb the excess liquid with absorbent paper, there should be no bubbles in the counting chamber, and let it stand for 5-10 minutes; observe and count the number of virus polyhedroses in 5 medium grids (i.e., 80 small grids) under a microscope, and calculate the number of virus polyhedroses by the following formula:

[0052] The number of Spodoptera litura nuclear polyhedrosis virus inclusion bodies X1 per gram is calculated according to formula (1):

[0053] X1=A / 80×400×10000×1000………………………………………………………………(1)

[0054] Where:

[0055] A—the number of Spodoptera litura nuclear polyhedrosis virus inclusion bodies in 5 large grids, in PIB / g;

[0056] 80—The number of small grids contained in 5 large grids;

[0057] 400—The number of small grids contained in the 25 large grids of the blood cell counting chamber;

[0058] 10000—the ratio between the actual sample volume remaining on the hemacytometer and 1 g;

[0059] 1000—Dilution factor.

[0060] b. Determination of pH value:

[0061] The measurement was carried out according to GB / T 1601-2023 method.

[0062] c. Determination of moisture content

[0063] Determine according to the “Karl Fischer method” in GB / T 1600-2021.

[0064] The results are shown in Table 1. The appearance of the Spodoptera litura nuclear polyhedrosis virus mother drug prepared in Example 1 is off-white or off-white powder, the number of virus inclusion bodies is 30.15 billion PIB / g, the pH value is 7.05, and the moisture content is 5.29%.

[0065] Table 1 Main index test results of Spodoptera litura nuclear polyhedrosis virus stock drug

[0066]

[0067] Since there is no national standard for the parent drug of Spodoptera litura nuclear polyhedrosis virus, the parent drug of Spodoptera litura nuclear polyhedrosis virus was evaluated by referring to the standard for pesticide suspension products. The test results showed that the main performance indicators of the parent drug of Spodoptera litura nuclear polyhedrosis virus met the standards for most pesticide suspension products, indicating that the agent is qualified and can be used for pest control.

[0068] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a parent drug of Spodoptera litura nuclear polyhedrosis virus, characterized in that: The following steps are involved: S1. Collecting populations and pupal emergence: Collect 5- to 6-day-old pupae of Spodoptera litura from populations collected from the wild or from populations artificially reared indoors and place them in growth boxes, adding nutrients to transform them into adults; S2. Adult egg laying: After the sex of the adults is identified, the Spodoptera litura is selected in a ratio of 1:1.2 and placed in an egg-laying box for mating and egg laying; S3, egg collection: 48 hours after egg laying, the eggs in the egg-laying box are collected in batches into a refrigerator with egg foil, and collected every 12 hours until a total of 72 hours have passed; S4. Egg culture: The egg foil in the refrigerator is transferred to a disinfection cabinet and fumigated with formaldehyde gas. After fumigation for 30 minutes, the egg foil is taken out and placed in a constant temperature incubator to culture brown eggs. Among the cultured eggs, 5% is retained for seed preservation by the optimization method, and 95% is used for larval culture. S5, seed preservation: artificial feed is prepared and packed in insect boxes. After 5% of the eggs for seed preservation are hatched, they are placed in insect boxes in batches and cultured in the dark. After culturing to 4 days old, healthy larvae are picked out and placed in insect trays filled with artificial feed until pupation. When the pupae develop into brown, they are sent to the pupal emergence process; S6, larval culture: prepare artificial feed and pack it in insect culture boxes, and after 95% of the eggs used for larval culture are hatched, put them into insect culture boxes in batches and culture them in the dark until 4-day-old larvae; S7. Virus infection and proliferation: prepare infection solution and place it in the artificial feed in the insect box, culture and proliferate 4-day-old larvae in the dark, and track and check the larvae's disease status until the disease rate reaches 98%; S8. Virus collection and storage: Collect dead insects from the insect box, select dead insects with strong virulence and early death as semi-finished virus products and store them in a refrigerator; S9. Preparation of mother powder: dilute the refrigerated virus semi-finished product with purified water to form a slurry by colloid mill, crush and filter by colloid mill, concentrate by centrifugation, freeze-dry and test the content to obtain the mother drug of Spodoptera litura nuclear polyhedrosis virus.

2. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: The temperature of the egg laying box in step S2 is 25-28° C. and the humidity is 75-85%.

3. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: The refrigerator temperature in step S3 is 8-10°C.

4. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: The temperature of the constant temperature incubator in step S4 is 25-28° C. and the humidity is 70-80%.

5. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: The raw materials of the artificial feed include soybean, barley, yeast powder, preservative, agar, vitamin C, acetic acid and water. The production steps of the artificial feed are as follows: roast the soybean and barley and then crush them, then heat the agar and water to melt, finally mix the agar aqueous solution, yeast powder, preservative, vitamin C, acetic acid and crushed soybean and barley cooked dry materials into a feed stirring pot, stir for 3-5 minutes, and then pour into a cooling plate to cool and solidify.

6. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: The amount of artificial feed packed into the insect box in step S5 is 5g / box, the amount of artificial feed packed into the insect tray in step S5 is 3g / hole, and the amount of artificial feed packed into the insect box in step S6 is 20g / box.

7. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: In step S5, each insect raising box receives 100 newly hatched larvae, and each hole in the insect raising tray receives two healthy larvae. In step S6, each insect raising box receives 100 newly hatched larvae.

8. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: In step S5, the temperature of the dark culture is 25-28° C. and the humidity is 70-80%. In step S6, the temperature of the dark culture is 25-28° C. and the humidity is 65-75%.

9. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: The preparation step of the infection liquid in step S7 is to grind, filter, centrifuge and purify the selected typical dead insects, put them into a container as a virus seed stock solution and store them in a refrigerator; the content of the infection liquid is selected according to the concentration of the stock solution in the container. The dilution multiple is selected, and the infection liquid is dripped on a blood cell counting plate and left to stand for 20 minutes before counting. The calculation formula is the average number of each small grid × 400 × 10000 × dilution multiple, which is the content of each milliliter of stock solution; an effective and accurate infection concentration is determined to prepare the infection liquid, and the formula is the stock solution concentration ÷ required concentration - 1 = dilution multiple.

10. The method for preparing the Spodoptera litura nuclear polyhedrosis virus mother drug according to claim 1, characterized in that: In step S7, the amount of the infection liquid is mixed with the artificial feed at a dosage of 0.05 ml per gram of feed. In step S7, the light-proof culture temperature is 23-25° C. and the humidity is 60-70%.

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