Prodenia litura nuclear polyhedrosis virus SpltMNPV-c5 and application thereof

Abstract

The invention discloses a prodenia litura nuclear polyhedrosis virus SpltMNPV-c5 and an application of the prodenia litura nuclear polyhedrosis virus SpltMNPV-c5. Relates to the technical field of biological control. According to the invention, SpltMNPV-c 5 is processed into a preparation for preventing and treating lepidoptera pests such as prodenia litura, and the preparation has important significance; according to the invention, mother powder prepared by SpltMNPV-c 5 is used as an effective component, an optimal formula is determined by measuring wetting time, suspension rate and the like of a carrier, an auxiliary agent and an ultraviolet protective agent, the carrier, the auxiliary agent and the ultraviolet protective agent are mixed according to a ratio to prepare the wettable powder, and various performance indexes are measured; the virulence to prodenia litura is measured by a leaf soaking method and an insect soaking method, so that a theoretical basis is provided for developing SpltMNPV-c5 into a preparation for preventing and treating prodenia litura.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and more specifically to a Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 and an application thereof. Background Art

[0002] 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 muhieapsid 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.

[0003] However, the insecticidal effect of the Spodoptera litura nuclear polyhedrosis virus preparations in the city is unstable during use in the prevention and control of local noctuid pests. The SpltMNPV-c5 virus provided by the present invention comes from the tobacco-growing area of ​​Hunan, has stronger toxicity, has good development and application prospects, and has application value in the preparation of insecticides and the prevention and control of flat moths.

[0004] Therefore, whether it is possible to provide a Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 and its application to overcome the above-mentioned technical deficiencies is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 and applications thereof.

[0006] Collection information:

[0007] Spodoptera litura multinucleocapsidnucleopolyhedroviruse SpltMNPV-c5 is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; deposit date: March 27, 2024; deposit number: CCTCC NO: V 202439.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5, with a deposit number of CCTCC NO: V 202439.

[0010] The present invention also provides the application of the Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 in fermentation, preparation and agricultural production.

[0011] The present invention also provides a biological control preparation for lepidopteran pests prepared by utilizing the Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5.

[0012] Preferably, the dosage form is a wettable powder, comprising the following raw materials: SpltMNPV-c5 mother powder, a carrier, an adjuvant, and an ultraviolet protective agent.

[0013] Preferably, the mass ratio of SpltMNPV-c5 master powder, carrier, auxiliary agent and ultraviolet protective agent is 3-5:75-85:10-15:2-5.

[0014] Preferably, the mass ratio of SpltMNPV-c5 masterbatch, carrier, auxiliary agent and ultraviolet protective agent is 3.3:81.7:12:3.

[0015] Preferred carrier: any one of kaolin, vermiculite powder, diatomaceous earth, white carbon black, light calcium carbonate, sodium bentonite or attapulgite;

[0016] Additives: any one of NNO, calcium alkylbenzene sulfonate, polyethylene glycol 8000, saponin powder, laquer powder, sodium dodecyl sulfonate, sodium carboxymethyl cellulose and sodium dodecyl sulfate;

[0017] UV protectant: any one of titanium dioxide, BHT, and VBL.

[0018] Preferred: Carrier: kaolin; auxiliary agent: NNO; UV protectant: VBL.

[0019] The present invention also provides a method for preparing any of the above-mentioned biocontrol agents, which comprises adding SpltMNPV-c5 mother powder, carrier, auxiliary agent and ultraviolet protective agent into a super micro pulverizer for pulverization and mixing to obtain the biocontrol agent.

[0020] Preferred: Preparation method of SpltMNPV-c5 mother powder:

[0021] 11) Select healthy adults of Spodoptera litura, mate and lay eggs, and disinfect;

[0022] 12) Rearing the larvae after hatching;

[0023] 13) When the larvae develop to the fourth instar, the virus is propagated;

[0024] 14) Raising infected larvae and collecting dead larvae after virus feeding;

[0025] 15) Add distilled water to the collected dead insects for grinding and filtering, add distilled water to the filtered virus stock solution for centrifugation and impurity removal, collect the precipitate, and finally freeze-dry and grind into powder to obtain SpltMNPV-c5 mother powder.

[0026] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 and its application, and the technical effects achieved are:

[0027] It is of great significance to process SpltMNPV-c5 into preparations for the control of lepidopteran pests such as Spodoptera litura.

[0028] The mother powder prepared by SpltMNPV-c5 was used as the effective ingredient. The best formula was determined by measuring the wetting time and suspension rate of the carrier, auxiliary agent and UV protective agent. The wettable powder was prepared by mixing according to the proportion, and various performance indicators were measured.

[0029] The toxicity to Spodoptera litura was determined by leaf dip method and insect dip method, aiming to develop SpltMNPV-c5 into a preparation for the control of Spodoptera litura and provide a theoretical basis. DETAILED DESCRIPTION

[0030] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The embodiment of the invention discloses a Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 and application thereof.

[0032] In the examples, the sources of raw materials not mentioned are all commercially available, for example: carrier kaolin (Tianjin Komiou Chemical Reagent Co., Ltd.), auxiliary agent NNO (Linyi Lusen Chemical Co., Ltd.), ultraviolet protective agent (VBL) (Qingshan Chemical), and their sources are not limited as long as they meet the product requirements. The processes not mentioned are all conventional processes and will not be repeated here.

[0033] Example 1

[0034] A Spodoptera litura nuclear polyhedrosis virus was isolated from the dead larvae of Spodoptera litura in the tobacco fields of Chenzhou, Hunan, and named SpltMNPV-c5. It was also deposited under patent procedures.

[0035] Example 2

[0036] Preparation of SpltMNPV-c5 master powder

[0037] Healthy adults of Spodoptera litura were selected and placed in an egg-laying cage for mating and egg-laying. The eggs were laid on a carrier made of non-woven tape. The eggs were collected, disinfected with sodium hypochlorite, and placed in an insect box for hatching. The artificial feed consisted of 100 parts of distilled water, 15 parts of soybean powder, 15 parts of wheat germ powder, 1 part of casein, 1 part of yeast powder, 1 part of agar powder, 0.5 parts of complex vitamin B, and 0.8 parts of ascorbic acid. After the larvae hatched, they were raised in an environment with a light intensity of L14:D10, a temperature of (27±1)℃, and a relative humidity of (70±5)%. When the larvae developed to the fourth instar, 5% were used for seed preservation and 95% were used for virus propagation. The infected larvae were raised at 24-27℃, and dead larvae were collected after 5-6 days of virus feeding. The collected dead insects were ground by adding 10 to 15 ml of distilled water per gram, and filtered with 100 mesh and 120 mesh gauze respectively. The filtered virus stock solution was added with 20 to 30 times the volume of distilled water and centrifuged at 300 r / min for 5 minutes. After removing the precipitate, it was centrifuged at 800 r / min for 5 minutes. The supernatant was taken and centrifuged at 4000 r / min for 20 minutes to collect the precipitate, and finally freeze-dried and ground into powder, which is the SpltMNPV-c5 mother powder (about 30 billion PIB / g).

[0038] Example 3

[0039] A wettable powder containing SpltMNPV-c5 comprises the following raw materials by weight: 3.3 parts of SpltMNPV-c5 mother powder, 81.7 parts of a carrier (kaolin), 12 parts of an auxiliary agent (NNO), and 3 parts of an ultraviolet protective agent (VBL).

[0040] Preparation method:

[0041] Add SpltMNPV-c5 mother powder, kaolin, NNO and VBL according to the above ratio into a super micro grinder for grinding and mixing to obtain a wettable powder.

[0042] Example 4

[0043] A wettable powder containing SpltMNPV-c5 comprises the following raw materials in parts by weight: 3 parts of SpltMNPV-c5 mother powder, 75 parts of a carrier (kaolin), 10 parts of an auxiliary agent (NNO), and 2 parts of an ultraviolet protective agent (VBL).

[0044] The preparation method is the same as Example 3.

[0045] Example 5

[0046] A wettable powder containing SpltMNPV-c5 comprises the following raw materials by weight: 5 parts of SpltMNPV-c5 mother powder, 85 parts of carrier (kaolin), 15 parts of auxiliary agent (NNO), and 5 parts of ultraviolet protective agent (VBL). The preparation method is the same as that of Example 3.

[0047] Technical effect verification

[0048] Quality indicators:

[0049] The performance indicators of SpltMNPV-c5 wettable powder were determined according to relevant national standards. SpltNPV content: determined by the blood cell counting plate method; suspension rate: determined by referring to the national standard GB / T14825-2023 pesticide wettable powder suspension rate determination method; wetting time: determined by referring to the national standard GB / T 5451-2001 pesticide wettable powder wettability determination method; fineness: determined by referring to the national standard GB / T 16150-1995 pesticide powder, wettable powder fineness determination method; drying loss: determined by referring to the national standard GB / T 30361-2013 pesticide drying loss determination method; pH value: determined by referring to the national standard GB / T 1601-2023 pesticide pH value determination method; persistent foaming: determined by referring to the national standard GB / T 28137-2011 pesticide persistent foaming determination method.

[0050] The results are shown in Table 1. The appearance of the SpltMNPV-c5 wettable powder prepared in Example 3 is a white powder with a fineness of 98.58%, a wetting time of 76.6 s, a suspension rate of 85.13%, a drying loss of 2.12%, a pH value of 6.5, and a number of viral inclusion bodies of 1.14×10 10 PIB / g and persistent foaming were 0 mL.

[0051] Since there is no national standard for nuclear polyhedrosis virus wettable powder, the national quality indicators of some fungal and bacterial microbial pesticide wettable powders were used to evaluate SpltMNPV-c5 wettable powder. The test results showed that the main performance indicators of SpltMNPV-c5 wettable powder met the national standards of most microbial pesticide wettable powders, indicating that the agent is qualified and can be used for pest control.

[0052] Table 1 Test results of main indicators of SpltMNPV-c5 wettable powder

[0053]

[0054]

[0055] Toxicity of SpltMNPV-c5 wettable powder to larvae of Spodoptera litura

[0056] Indoor bioassay

[0057] In the leaf immersion method, the collected fresh tobacco leaves were washed, immersed in different dilutions of the drug solution for 10s, taken out and air-dried, and then placed in a petri dish. The 2nd or 3rd instar larvae of Spodoptera litura were placed in a square petri dish (d = 125mm, moistened with wet cotton), 30 heads for each treatment, repeated 3 times, with tobacco leaves soaked in water as the control, placed at (26 ± 1) ° C, and the number of larval deaths was counted every day, and the total number of larval deaths was counted for 7 days, and the larval mortality rate was calculated. Insect immersion method bioassay: The agent was diluted with sterile water to different multiples, and the 2nd or 3rd instar larvae of Spodoptera litura were immersed in different concentrations of drug solution for 10s, taken out and air-dried, and then placed in a square petri dish, fed with fresh tobacco leaves, 30 heads for each treatment, repeated 3 times, with water as the control, placed at (26 ± 1) ° C, and the number of larval deaths was counted every day, and the total number of larval deaths was counted for 7 days, and the larval mortality rate was calculated.

[0058] The results of the toxicity test of SpltMNPV-c5 wettable powder to the 2nd instar larvae of Spodoptera litura are shown in Table 2. The results showed that the mortality of the 2nd instar larvae treated by the leaf dipping method was higher than that of the insect dipping method at the same time. The concentrations of the agent treatments that exceeded 90% on the seventh day of the 2nd instar larvae in the leaf dipping method were 200, 400 and 800 times the liquid, while the concentration of the agent treatments that exceeded 90% on the seventh day of the 2nd instar larvae in the insect dipping method was 200 times the liquid.

[0059] The results of the toxicity test of SpltMNPV-c5 wettable powder to the 3rd instar larvae of Spodoptera litura are shown in Table 3. The results showed that the mortality of the 3rd instar larvae treated by the leaf dipping method was higher than that of the insect dipping method at the same time, which was the same as that of the 2nd instar larvae. The concentrations of the agent that caused the mortality of the 3rd instar larvae on the seventh day to exceed 90% in the leaf dipping method were also 200, 400 and 800 times the liquid, while there was no concentration of the agent that caused the mortality of the 3rd instar larvae on the seventh day to exceed 90% in the insect dipping method.

[0060] Table 2 Toxicity of SpltMNPV-c5 wettable powder to the 2nd instar larvae of Spodoptera litura

[0061]

[0062]

[0063] Table 3 Toxicity of SpltMNPV-c5 wettable powder to the third instar larvae of Spodoptera litura

[0064]

[0065] Comparative test

[0066] Vector screening

[0067] The mother powder of SpltMNPV-c5 was mixed with the carrier to be screened in a certain proportion to prepare a wettable powder of 1 billion / g. The wetting time and suspension rate of the preparation were measured. The best carrier was selected by comprehensively considering the wetting time, suspension rate and cost.

[0068] The carriers were screened with wetting time and suspension rate as indicators, and the screening results are shown in Table 4. The wetting time of the seven carriers was significantly different. White carbon black and light calcium carbonate had the longest wetting time, both exceeding 900s, followed by bentonite and attapulgite, with wetting times of 155.90 and 96.05s, respectively, and diatomaceous earth had the shortest wetting time of 4.10s; white carbon black had the highest suspension rate of 67.67%, followed by bentonite and kaolin, with suspension rates of 56.28 and 48.40%, respectively, and attapulgite had the lowest suspension rate of 18.28%. Kaolin was selected as the carrier of SpltMNPV-c5 wettable powder based on the comprehensive wetting time and suspension rate.

[0069] Table 4 Screening of vectors

[0070]

[0071] Additive screening

[0072] Based on the carrier screening, the additive to be screened was mixed with SpltMNPV-c5 master powder and the optimal carrier at a ratio of 10% to form a 1 billion / g wettable powder, and the wetting time and suspension rate of the preparation were measured; the optimal additive was screened out based on the measurement results. The optimal additive was mixed with SpltMNPV-c5 master powder and the optimal carrier at an addition rate of 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16% and 18% to form a 1 billion / g wettable powder, and the wetting time and suspension rate of the preparation were measured. Taking the wetting time, suspension rate and cost into consideration, the optimal dosage of the optimal additive was screened out.

[0073] The results of the adjuvant screening are shown in Table 5. Taking the wetting time as the index, the wetting time of sodium carboxymethyl cellulose is the longest, which is more than 900s, and the wetting time of sodium dodecyl sulfate is the shortest, which is 7.65s. Taking the suspension rate as the index, the suspension rate of NNO is the highest, which is 83.36%, and the suspension rate of pull open powder is the lowest, which is 39.87%. Therefore, NNO is selected as the adjuvant of SpltMNPV-c5 wettable powder.

[0074] Table 5 Screening of additives

[0075]

[0076]

[0077] Additive dosage screening

[0078] As shown in Table 6, when the adjuvant dosage is 14%, the suspension rate is the highest, which is 85.01%, followed by the suspension rates of 84.73% and 84.31% when the adjuvant dosage is 16% and 12%, respectively. There is no significant difference between the suspension rate at the dosage of 12% and the suspension rate at the dosage of 14%, but the wetting time of the former is significantly lower than that of the latter, so the adjuvant dosage of 12% is selected as the adjuvant dosage of SpltMNPV-c5 wettable powder.

[0079] Table 6 Screening of additive dosage

[0080]

[0081] UV protection agent screening

[0082] On the basis of the screening of auxiliary agents, the UV protective agent to be screened was mixed with SpltMNPV-c5 mother powder and the optimal carrier at a ratio of 3% to prepare a 1 billion / g wettable powder, which was placed together with the Splt MNPV-c5 wettable powder without adding UV protective agent at a distance of 40 cm from the UV lamp for 1 hour. The SpltMNPV-c5 wettable powder after UV irradiation was prepared into 5 gradient suspensions (1×10 4 PIB / mL, 1×10 5 PIB / mL, 1×10 6 PIB / mL, 1×10 7 PIB / mL, 1×10 8 PIB / mL) for later use, pick out healthy 3rd instar larvae and put them into a 12-well cell culture plate, starve them for 6 hours, use a pipette to draw 20 μL of virus suspension and drop it into the artificial feed, air-dry it for feeding. After feeding for 24 hours, replace it with fresh non-toxic feed, and record the number of larval deaths from the 2nd day for a total of 10 days. SpltMNPV-c5 wettable powder without adding UV protectant and irradiating ultraviolet rays was used as a control, and clean water was set as a blank control.

[0083] The screening results of UV protective agents are shown in Table 7. After 1 h of UV irradiation, LC treated with VBL, BHT, and TiO2 50 They are 6.9735×10 4 , 1.0057×10 5 and 1.4418×10 5 PIB / mL, all lower than that without protective agent, UV irradiation, and LC 50 (1.1247×10 6 PIB / mL), and LC without protective agent and UV irradiation 50 (3.9405×10 4 PIB / mL) compared with VBL-treated LC 50The increase was the smallest and the UV protection effect was the strongest. Therefore, VBL was selected as the UV protectant for SpltMNPV-c5 wettable powder.

[0084] Table 7 Screening of UV protective agents

[0085]

[0086] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0087] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5, characterized in that The deposit number is CCTCC NO: V202439.

2. Application of the Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 according to claim 1 in fermentation, preparation and agricultural production.

3. A biological control preparation for lepidopteran pests prepared using the Spodoptera litura nuclear polyhedrosis virus SpltMNPV-c5 described in claim 1.

4. The biocontrol agent according to claim 3, characterized in that The dosage form is a wettable powder, including the following raw materials: SpltMNPV-c5 mother powder, carrier, adjuvant, and ultraviolet protective agent.

5. The biocontrol agent according to claim 4, characterized in that The mass ratio of the SpltMNPV-c5 master powder, the carrier, the auxiliary agent and the ultraviolet protective agent is 3-5:75-85:10-15:2-5.

6. The biocontrol agent according to claim 5, characterized in that The mass ratio of the SpltMNPV-c5 masterbatch, the carrier, the auxiliary agent and the ultraviolet protective agent is 3.3:81.7:12:

3.

7. The biocontrol agent according to claim 6, characterized in that The carrier is any one of kaolin, vermiculite powder, diatomaceous earth, white carbon black, light calcium carbonate, sodium bentonite or attapulgite; The auxiliary agent is any one of NNO, calcium alkylbenzene sulfonate, polyethylene glycol 8000, saponin powder, pull-open powder, sodium dodecyl sulfonate, sodium carboxymethyl cellulose and sodium dodecyl sulfate; The ultraviolet protective agent is any one of titanium dioxide, BHT and VBL.

8. The biocontrol agent according to claim 7, characterized in that The carrier is kaolin; the auxiliary agent is NNO; and the ultraviolet protective agent is VBL.

9. The method for preparing the biocontrol agent according to any one of claims 4 to 8, characterized in that: The method comprises the following steps: adding SpltMNPV-c5 mother powder, a carrier, an auxiliary agent and an ultraviolet protective agent into an ultra-micro grinder for grinding and mixing to obtain a biocontrol agent.

10. The preparation method according to claim 9, characterized in that: Preparation method of the SpltMNPV-c5 mother powder: 11) Select healthy adults of Spodoptera litura, mate and lay eggs, and disinfect; 12) Rearing the larvae after they hatch; 13) When the larvae develop to the fourth instar, the virus is propagated; 14) Raising infected larvae and collecting dead larvae after virus feeding; 15) Add distilled water to the collected dead insects for grinding and filtering, add distilled water to the filtered virus stock solution for centrifugation and impurity removal, collect the precipitate, and finally freeze-dry and grind into powder to obtain SpltMNPV-c5 mother powder.

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