A method for producing a high-efficiency strain of the tea geometrid nuclear polyhedrosis virus

Abstract

The application discloses a production method of a high-efficiency strain of Bupalus piniarius nuclear polyhedrosis virus, and belongs to the technical field of biological pesticide production. 8 The virus yield of the high-efficiency strain of Bupalus piniarius nuclear polyhedrosis virus is 6*10 The virus loss rate is reduced by 1.4% by adding 0.1% detergent before centrifugal purification of the virus, and the high yield of the virus is further ensured.The insecticidal effect of the high-efficiency strain produced by the method is increased by more than 64.1% than that of an ordinary strain, and the high-efficiency property is continuously maintained.The method has a very beneficial effect on the production of the high-efficiency strain of Bupalus piniarius nuclear polyhedrosis virus, and provides a solid guarantee for popularization and application of the high-efficiency strain.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticide production technology, and in particular relates to a method for producing a highly efficient strain of tea geometrid moth nucleopolyhedrovirus. Background Technology

[0002] Tea geometrid moth ( Ectropis obliqua Prout) and gray tea inchworm ( Ectropis grisescens *Warren*, one of two closely related species, is the most significant inchworm pest on tea trees in my country. Due to its numerous generations and rapid reproduction, it often causes outbreaks and significant impacts on tea yield and quality. Tea inchworm nucleopolyhedrovirus (NTV) Ectropis obliqua nuclear polyhedrosis virus (EoNPV) is a parasitic insect (EoNovice moth). E. obliqua The virus is a dominant natural enemy of the tea geometrid moth (Prout), and is an important factor in controlling the tea geometrid moth population in the field. This virus can also infect the grey tea geometrid moth (Prout). E. grisescens Warren, but its pathogenicity against the tea geometrid moth is much lower than that against the tea geometrid moth. Therefore, researchers screened and isolated a highly effective strain, QF4, from tea geometrid moths infected with nucleopolyhedrovirus. This strain showed more than 50% higher virulence against the tea geometrid moth compared to the initial strain (Tang, Meijun; Guo, Huawei; Ge, Chaomei; Yin, Kunshan; Xiao, Qiang, 2017). Eo Pathogenicity of NPV against the tea geometrid moth and screening of highly effective strains. Zhejiang Journal of Agricultural Sciences, 29(10): 1686-1691. However, there is currently little research on the production of highly effective strains of tea geometrid moth nucleopolyhedrovirus both domestically and internationally, which hinders the application of this highly effective strain in biological control.

[0003] Traditionally, insect viruses have been produced using live insect propagation methods. The production of tea geometrid moth nucleopolyhedrovirus, however, utilizes the tea geometrid moth (…). Ectropis obliqua The live insect propagation method using *Prout* as the propagation host involves rearing large numbers of tea geometrid moths and then propagating the tea geometrid moth virus using live tea geometrid moth larvae for mass production (Yin Kunshan; Xiao Qiang; Tang Meijun; Guo Huawei, 2008. A collection method for the propagation of insect polyhedrosis viruses, CN100384989C.). Although the traditional live insect propagation method using tea geometrid moths can propagate the highly efficient strain QF4, the high-efficiency strain of tea geometrid moth nucleopolyhedrovirus is highly pathogenic to both tea geometrid moths and gray tea geometrid moth larvae compared to the ordinary strain. Therefore, using conventional production methods for ordinary strains results in very low virus yields, and the high efficiency of the strain is easily lost after propagation. Thus, this research proposes a high-yield virus production method that maintains the high efficiency of the strain, which is a pressing technical problem to be solved in the production of tea geometrid moth nucleopolyhedrovirus. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention aims to provide a high-yield production technology for a highly efficient strain of tea geometrid moth nucleopolyhedrovirus while maintaining its high efficiency. This technology utilizes the tea geometrid moth (… Ectropis grisescens Using Warren as the propagation host and the highly efficient strain QF4 of the tea geometrid moth nucleopolyhedrovirus as the inoculation source, a specific concentration of virus suspension was inoculated at specific instars of the tea geometrid moth. Then, a large amount of the high-efficiency strain of the tea geometrid moth nucleopolyhedrovirus was obtained through a specific purification method.

[0005] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a method for producing a highly efficient strain of tea geometrid moth nucleopolyhedrovirus, comprising the following steps: With the gray tea inchworm ( Ectropis grisescens The larvae of the tea geometrid moth (Warren) were used as the host; the highly effective strain QF4 of the tea geometrid moth nucleopolyhedrovirus was used as the inoculum; when the larvae of the tea geometrid moth reached the 3rd instar, a concentration of 1×10⁻⁶ was used. 7 PIB / mL ~5×10 7 Inoculation with a PIB / mL EoNPV-QF4 virus suspension was performed; finally, dead insect carcasses were collected and treated with detergent for virus purification.

[0006] Furthermore, the larvae of the gray tea geometrid moth are obtained through the following steps: 1) Collect adult gray tea geometrid moths to obtain their eggs; 2) Place the insect eggs in a container to hatch, and feed the newly hatched larvae with tender tea leaves; 3) After raising them for 1-2 days, transfer the larvae to tea tree branches for continued rearing.

[0007] Furthermore, after inoculation and infection, the larvae were continued to be raised for 5 to 7 days, and then supplemented with virus-free tea tree feed.

[0008] Furthermore, the feeding temperature in the production method is 22℃~24℃, and natural light conditions are used.

[0009] Furthermore, dead insect carcasses were collected starting 7–9 days after inoculation and continued until 13–15 days after inoculation.

[0010] Furthermore, the virus purification includes the following steps: 1) Soak the insect carcasses in water for 1-2 days, then grind and filter them to obtain the filtrate; 2) Allow the filtrate to settle naturally into layers, and collect the upper layer. 3) Add 0.05-0.15% detergent to the upper layer and centrifuge. 4) Wash away the polyhedral precipitate to obtain the stock solution of the highly efficient strain of the tea geometrid moth nucleopolyhedrovirus.

[0011] Furthermore, the centrifugation conditions are: centrifugation at 3000-4000 r / min for 25-35 minutes, and repeated 1-3 times.

[0012] The second objective of this invention is to provide a highly efficient stock solution of the tea geometrid moth nucleopolyhedrovirus produced using the aforementioned production method.

[0013] The third objective of this invention is to provide a product resistant to inchworm pests, which contains a highly effective strain of the tea inchworm nucleopolyhedrovirus.

[0014] The fourth objective of this invention is to provide the application of the highly effective strain of the tea geometrid moth nucleopolyhedrovirus or the anti-geometrid moth product in the control of geometrid moth pests.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) High virus yield. By using specific insect age and virus concentration matched to the host, the tea geometrid moth, and the QF4 strain, the high-yield strain of tea geometrid moth nucleopolyhedrovirus achieved a virus yield of 6 × 10⁻⁶. 8 (1) The virus yield is 122% higher than that of traditional methods. (2) Less virus loss during purification. Adding 0.1% detergent before centrifugation and purification reduces the virus loss rate by 1.4%, further ensuring high virus production. (3) Maintaining the high efficiency of the strain. Using the tea geometrid moth as the propagation host, the insecticidal effect of the high-efficiency strain produced is more than 64.1% higher than that of ordinary strains, ensuring the continuous and stable high efficiency. This invention has a very beneficial effect on the production of high-efficiency strains of tea geometrid moth nucleopolyhedrovirus, providing a solid guarantee for the promotion and application of high-efficiency strains. Attached Figure Description

[0016] Figure 1 The mortality dynamics of the gray tea geometrid moth after being fed a highly effective strain of tea geometrid moth virus in this invention; Figure 2 The polyhedral morphology of the highly efficient tea geometrid moth virus strain QF4 obtained in this invention; Figure 3 This invention demonstrates the pathogenicity of EoNPV-QF4 against second-instar larvae of the tea geometrid moth. Detailed Implementation

[0017] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0018] The main objective of this invention is to provide a high-yield production technology for a highly efficient strain of the tea geometrid moth nucleopolyhedrovirus while maintaining high efficiency. This technology utilizes the tea geometrid moth (… Ectropis grisescens The *Warren* moth was used as the propagation host, and the highly efficient strain QF4 of the tea geometrid moth nucleopolyhedrovirus was used as the inoculation source (see reference: Tang Meijun, Guo Huawei, Ge Chaomei, Yin Kunshan, Xiao Qiang, 2017). Eo Pathogenicity of NPV against the tea geometrid moth and screening of highly efficient strains. Zhejiang Journal of Agricultural Sciences, 29(10):1686-1691.), a specific concentration of virus suspension was inoculated into the tea geometrid moth at specific instars, and then a large amount of highly efficient nucleopolyhedrovirus stock solution of the tea geometrid moth was obtained through a specific purification method. The main steps are as follows: 1) Mass rearing of healthy larvae of the tea geometrid moth: Adult tea geometrid moths are reared in groups using insect rearing cages, with white paper attached to the outside of the cage mesh to allow them to lay eggs. On the second day, the eggs are brushed off with a brush and placed in 500 mL wide-mouthed glass bottles for rearing. After the eggs hatch, they are reared in the bottles with tender tea leaves. After 1 to 2 days of rearing, the insects and tea leaves are transferred to hydroponically grown tea tree branches for rearing at a temperature of 22 to 24℃ and under natural light.

[0019] 2) Propagation of highly effective strains of tea geometrid moth virus: When the larvae of the tea geometrid moth reach the middle of the 3rd instar, use 1×10 7 Polyhedrons (PIB) / mL ~5×10 7 Spray the insect-infested branches with PIB / mL EoNPV-QF4 virus solution, and feed them with the virus for 5-7 days. Then add virus-free tea branches and continue feeding. The feeding temperature should be 22-24℃ and the light should be natural.

[0020] 3) Collection of dead insects: Start collecting dead insects 8 days after feeding the poison, collect them 1-2 times a day, and stop 13-15 days after feeding the poison. Collect the dead insects in a wide-mouthed bottle and store at room temperature; if not to be purified immediately, store at 4℃.

[0021] 4) Virus purification: Soak the insect carcasses in a small amount of water for 1-2 days, then grind and filter. Place the filtrate in a 2000 mL graduated cylinder for natural sedimentation. After separation, immediately aspirate the upper layer, add 0.1% detergent, and then centrifuge the upper layer at 3000-4000 r / min for 30 min. After centrifugation twice, wash the polyhedral precipitate with purified water to obtain the virus stock solution. After microscopic counting, store at 4℃ for later use.

[0022] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0023] Example 1 First, large-scale rearing of the larvae of the tea geometrid moth is conducted. Adult larvae are reared in groups using rearing cages (25 cm long × 25 cm wide × 45 cm high, made of plastic mesh) (800-1000 pupae per cage). White paper is attached to the outside of the cage mesh to allow them to lay eggs. The next day, the eggs are brushed off with a brush and placed in 500 mL wide-mouthed glass bottles, sealed with white paper and rubber bands. After hatching, the eggs are initially reared in the bottles using tender tea leaves. After 1-2 days, the larvae, along with the tea leaves, are transferred to hydroponically grown tea branches. Tea branches are cut from the field, bundled, and placed in 500 mL wide-mouthed bottles filled with water. Three to four bottles of tea branches are placed close together in a 60 cm diameter plastic basin. The first-instar larvae reared in the bottles are then poured out onto the tea foliage, allowing them to spread and feed. Tea branches are added or replaced as needed based on the feeding activity. When the larvae reach the third instar, most are moved to the virus room for virus reproduction, while a small portion is used for breeding and continued rearing. Mature larvae will fall into the container to pupate. Rearing conditions include a constant temperature of 22-24℃ and natural light.

[0024] Take 3rd instar larvae of the tea geometrid moth and feed them with poison. Use a small sprayer to apply 1×10 7 Spray a PIB / mL solution of EoNPV-QF4 virus evenly onto fresh tea cuttings grown hydroponically. After the virus solution dries, knock healthy 3rd instar larvae of the tea geometrid moth onto the tea cuttings, allowing them to feed on the virus-infected leaves. After feeding on the virus for 5-7 days, transfer the larvae that have consumed the virus to a rearing box and continue rearing them with virus-free leaves. Each box contains 200-500 larvae, and the lid is covered with a glass cover to maintain humidity. A total of 8 boxes are used.

[0025] Eight days after feeding the insects with the poison, collect the dead insects from each box until day 15, collecting 1-2 times a day. Place the dead insects in a 500 mL wide-mouth bottle. Randomly select one box, mark it, and record the number of dead insects collected each day. Place the dead insects in a separate 500 mL wide-mouth bottle; place the pupae in a separate bottle.

[0026] Soak the insect carcasses or pupae in a small amount of water for 1-2 days, then grind and filter. Place the filtrate in a 2000 mL graduated cylinder for natural sedimentation. After separation, immediately aspirate the upper layer, add 0.1% detergent, and then centrifuge the upper layer at 3000-4000 r / min for 30 min. After centrifugation twice, wash the polyhedral precipitate with purified water, count the precipitate under a microscope, and calculate the virus yield.

[0027] During the production of the above-mentioned viruses, the rearing temperature was controlled at around 22-24℃, the humidity was controlled at 75%-80%, and natural light was used.

[0028] In this embodiment, the yield of the highly effective strain of the tea geometrid moth nucleopolyhedrovirus is shown in Table 1, and the mortality dynamics of the grey tea geometrid moths after being fed the highly effective strain of the tea geometrid moth virus are shown in Table 1. Figure 1 As shown.

[0029] Table 1. Sampling and yield measurement of high-efficiency strains of tea geometrid moth nucleopolyhedrovirus.

[0030] Example 2 First, a large number of larvae of the tea geometrid moth were reared. The rearing method was the same as in Example 1.

[0031] Poisoning should be carried out when the larvae are reared to the middle of the 3rd instar. Use a small sprayer to apply 5×10 7 Spray the infected tea branches directly with a PIB / mL solution of EoNPV-QF4 virus. When most of the leaves have been consumed, add new branches infected with the virus. After feeding on the virus for 5-7 days, transfer the infected tea geometrid moth larvae to rearing boxes and continue rearing them with virus-free leaves. Each box contains 200-500 larvae, and the lid is covered with a glass cover to maintain humidity. A total of 6 boxes are used.

[0032] Eight days after feeding the insects with the poison, the dead insect carcasses were collected from each box until day 15, 1-2 times a day. The carcasses were placed in 500 mL wide-mouth bottles. One box was randomly selected, marked, and the number of insect carcasses collected each day was recorded. The carcasses were placed in a separate 500 mL wide-mouth bottle; the pupae were placed in a separate bottle.

[0033] Soak the insect carcasses or pupae in a small amount of water for 1-2 days, then grind and filter. Place the filtrate in a 2000 mL graduated cylinder for natural sedimentation. After separation, immediately aspirate the upper layer, add 0.1% detergent, and then centrifuge the upper layer at 3000-4000 r / min for 30 min. After centrifugation twice, wash the polyhedral precipitate with purified water, count the precipitate under a microscope, and calculate the virus yield.

[0034] During the production of the above-mentioned viruses, the rearing temperature was controlled at around 22-24℃, the humidity was controlled at 75%-80%, and natural light was used.

[0035] In this embodiment, the yield of the tea geometrid moth nucleopolyhedrovirus strain is shown in Table 2. The morphology of the viral polyhedrosis is as follows: Figure 2 As shown.

[0036] Table 2. Sampling and yield measurement of high-efficiency strains of tea geometrid moth nucleopolyhedrovirus.

[0037] Example 3 First, a large number of larvae of the tea geometrid moth were reared. The rearing method was the same as in Example 1.

[0038] When the larvae reach the 3rd instar, they are fed poison. The method is the same as in Example 1.

[0039] Eight days after feeding the insects with poison, the dead insect bodies were collected one box at a time until day 15, with collection occurring 1-2 times a day. The dead insect bodies and pupae were placed in 500 mL wide-mouth bottles respectively.

[0040] Soak the insect carcasses or pupae in a small amount of water for 1-2 days, then grind and filter. Place the filtrate in a 2000 mL graduated cylinder for natural sedimentation. After separation, immediately aspirate the upper layer, add 0.1% detergent, and then centrifuge the upper layer at 3000-4000 r / min for 30 min. After centrifugation twice, wash the polyhedral precipitate with purified water, count the precipitate under a microscope, and calculate the virus yield.

[0041] During the production of the above-mentioned viruses, the rearing temperature was controlled at around 22-24℃, the humidity was controlled at 75%-80%, and natural light was used.

[0042] To determine whether the highly effective EoNPV strain retains its high efficiency after large-scale propagation, the bioactivity of purified samples of the highly effective EoNPV-QF4 strain obtained above was determined. The purified virus stock solution was diluted with purified water to 1.25 × 10⁻⁶. 6 PIB / mL, with a common strain (the original strain in Tang Meijun et al., 2017) of 1.25 × 10⁻⁶. 6 Using PIB / mL as a control, indoor bioassays were conducted using a feeding method on second-instar gray tea geometrid moths. The results showed that the pathogenicity of the highly effective strain EoNPV-QF4 obtained in this embodiment was significantly higher than that of the control strain, and the corrected mortality rate during the larval stage was 64.1% higher than that of the control strain. Figure 3 This indicates that the high-efficiency strain still maintains its high efficiency after being propagated using this method.

[0043] Comparative Example 1 This comparative example uses existing technology as a comparative or control example to illustrate the technical problems solved by the technical solution of the present invention and the beneficial effects obtained.

[0044] (1) Effects of insect age and feed virus concentration on the yield of highly efficient strains of tea geometrid moth virus Using the brown geometrid moth as a host, three concentrations of EoNPV-QF4 virus suspension were fed to the second and third instar larvae during their mid-stages. After collecting and purifying the larvae, the virus content was determined. The rearing conditions and purification methods were as described previously. Based on the number of tested insects, the virus yield (10T) was calculated. 8 (PIB / head), the experimental results showed that the virus yield of 3-year-old feeder was significantly higher than that of 2-year-old feeder. Among them, in the 3-year-old gray tea geometrid moth, the virus yield was higher at a feeder concentration of 0.1×10⁻⁶. 8 The highest viral yield was observed at a PIB / mL concentration, followed by the 3-year-old stage and a feed concentration of 0.5 × 10⁻⁶. 8 PIB / mL, viral yield was 6×10 8 PIB / head or higher. This experiment clarified that the optimal conditions for EoNPV-QF4 propagation and production were feeding the toxicant to the third-instar tarantula at a concentration of 0.1 × 10⁻⁶. 8 ~ 0.5×10 8 PIB / mL.

[0045] Table 3. Effects of insect age and feed virus concentration on the yield of highly effective strains of tea geometrid moth virus.

[0046] Note: Virus yield is the average of three replicates.

[0047] (2) Effect of different treatments of insect carcass homogenate on virus purification loss rate Using conventional virus purification methods (insect carcass homogenate without detergent treatment) as a control, insect carcass homogenate was treated with 0.1% detergent before centrifugation. The results showed that after adding 0.1% detergent, the number of viral polyhedra in the supernatant after centrifugation was significantly reduced. The virus loss rate caused by centrifugation treatment decreased from 1.7% in the conventional method to 0.3%, which is 1.4% less than the conventional treatment (Table 4).

[0048] Table 4. Effects of different treatments of insect carcass homogenate on virus purification loss rate.

[0049] Note: The loss rate is the average of two repetitions.

[0050] (3) Comparison of yield and efficacy of highly effective strains of tea geometrid moth virus under two production methods The highly effective strain of tea geometrid moth virus, EoNPV-QF4, was propagated using both the traditional method (Yin Kunshan 2008) and the method of this invention. The virus yield and virulence of the samples obtained were compared between the two methods. The traditional method uses the tea geometrid moth (… Ectropis obliqua Prout was used as the propagation host, and the feeding conditions were 0.01 × 10⁻⁶ to 3rd instar worms.8 PIB / mL; The method of this invention uses the gray tea geometrid moth (PIB / mL); Ectropis grisescens Warren was used as the host, and the feeding conditions were 0.1 × 10⁻⁶ larvae fed with the poison at 3rd instar. 8 PIB / mL; other feeding conditions and purification methods were the same as before. The results of the virus yield test showed that the virus yield of the highly efficient strain of tea geometrid moth virus produced by the method of this invention was significantly higher than that of the traditional method, with a virus yield increase of 122% (Table 5).

[0051] Table 5 Two production methods E O NPV-QF4 production comparison

[0052] Note: Virus yield is the average of three replicates.

[0053] The two E values ​​obtained after production using different methods in the above experiment O Bioassays were performed on NPV-QF4 samples of 2-year-old tea geometrid moths using E... O The original NPV strain served as a control, and the viral fluid concentration was adjusted to 1×10⁻⁶. 6 The results showed that the toxicity of the highly effective strain QF4 obtained by the method of the present invention was significantly higher than that of the sample obtained by the traditional method and the ordinary strain. The toxicity of the highly effective strain QF4 obtained by the method of the present invention against the gray tea geometrid moth was increased by 75.5% compared with the ordinary strain, and the high efficiency of the highly effective strain was maintained. In contrast, the highly effective strain QF4 obtained by the traditional method was only 15.8% higher than the ordinary strain, and lost the high efficiency of the highly effective strain (Table 6).

[0054] Table 6. Comparison of the toxic effects of EoNPV-QF4 produced by the two methods on the tea geometrid moth.

[0055] Note: The corrected mortality rate of larvae is the average of three replicates.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for producing a highly efficient strain of tea geometrid moth nucleopolyhedrovirus, characterized in that, Includes the following steps: With the gray tea inchworm ( Ectropis grisescens The larvae of the tea geometrid moth (Warren) were used as the host; the highly effective strain QF4 of the tea geometrid moth nucleopolyhedrovirus was used as the inoculum; when the larvae of the tea geometrid moth reached the 3rd instar, a concentration of 1×10⁻⁶ was used. 7 PIB / mL ~5×10 7 Inoculation with a PIB / mL EoNPV-QF4 virus suspension was performed; finally, dead insect carcasses were collected and treated with detergent for virus purification.

2. The production method according to claim 1, characterized in that, The larvae of the gray tea geometrid moth were obtained through the following steps: 1) Collect adult gray tea geometrid moths to obtain their eggs; 2) Place the insect eggs in a container to hatch, and feed the newly hatched larvae with tender tea leaves; 3) After raising them for 1-2 days, transfer the larvae to tea tree branches for continued rearing.

3. The production method according to claim 1, characterized in that, After inoculation, the larvae were kept in the rearing program for 5 to 7 days, and then supplemented with virus-free tea tree feed.

4. The production method according to claim 1, characterized in that, The feeding temperature in the production method is 22℃~24℃, and natural light conditions are used.

5. The production method according to claim 1, characterized in that, The dead insect bodies were collected starting 7 to 9 days after inoculation and continued until 13 to 15 days after inoculation.

6. The production method according to claim 1, characterized in that, The virus purification process includes the following steps: 1) Soak the insect carcasses in water for 1-2 days, then grind and filter them to obtain the filtrate; 2) Allow the filtrate to settle naturally into layers, and collect the upper layer. 3) Add 0.05-0.15% detergent to the upper layer and centrifuge. 4) Wash away the polyhedral precipitate to obtain the stock solution of the highly efficient strain of the tea geometrid moth nucleopolyhedrovirus.

7. The production method according to claim 6, characterized in that, The centrifugation conditions are: centrifugation at 3000-4000 r / min for 25-35 minutes, and repeated 1-3 times.

8. A high-efficiency strain of tea geometrid moth nucleopolyhedrovirus stock solution produced using the production method described in any one of claims 1 to 7.

9. A product resistant to inchworm pests, characterized in that, It contains the high-efficiency strain of the tea geometrid moth nucleopolyhedrovirus as described in claim 8.

10. The application of the highly effective strain of tea geometrid moth nucleopolyhedrovirus stock solution as described in claim 8 or the anti-geometrid moth product as described in claim 9 in the control of geometrid moth pests.

Images