CN111727980B

CN111727980B - Pesticide for preventing and treating cotton bollworm by using nuclear polyhedrosis virus and preparation method and application thereof

Info

Publication number: CN111727980B
Application number: CN202010535866.8A
Authority: CN
Inventors: 柴连琴; 荆玉谱; 张林杰; 刘鹏飞; 孟景会; 张海燕; 王喆
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2021-08-20
Anticipated expiration: 2040-06-12
Also published as: CN111727980A

Abstract

The present invention belongs to the field of biological control of bollwormIn the field of operation, the effect of preventing and treating the aged cotton bollworm can be greatly improved by adding the extract of the ecdysone-containing dewy grass into the cotton bollworm nuclear polyhedrosis virus, and the effect of preventing and treating the cotton bollworm with low age is better than that of the traditional method. 5mg of Cyanotis Hypoglauca extract (30% ecdysone) and 2.0 × 10 per 6-year-old cotton bollworm⁶The usage amount of the PIB nuclear polyhedrosis virus can obviously enhance the effect of the nuclear polyhedrosis virus on preventing and treating the aged cotton bollworm. The pesticide provided by the invention has the mortality rate of 100% in 48 hours for larvae below 3 years old; the composition has the advantages of quick response, high efficiency and reduced egg laying amount for preventing and controlling the aged cotton bollworms, has obvious effect on preventing and controlling the next-year cotton bollworm outbreak, and fills the blank that the cotton bollworms are not effectively killed at present.

Description

Pesticide for preventing and treating cotton bollworm by using nuclear polyhedrosis virus and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological prevention and control of cotton bollworms, and particularly relates to a pesticide for preventing and controlling cotton bollworms by using nuclear polyhedrosis virus, and a preparation method and application thereof.

Background

The cotton bollworm is a parasitic pest with serious harm, and the 5-6-year old aged cotton bollworm larvae enter a binge eating period and have the greatest harm to crops. The cotton bollworm is the species with the largest egg laying amount in insects, the single female moth can lay more than 3000 eggs at most, if the prevention and control are not in place, one overwintering female pupa can propagate millions of larvae in the next year by 1 year on average, and more than one hundred million larvae are grown, so that a small amount of old cotton bollworms with net leakage completely have destructive effect. However, the effect of chemical pesticide or biological pesticide on preventing and controlling the aged cotton bollworm is poor at present. With the wide planting of transgenic Bt cotton, although the damage to cotton fields is reduced to a certain extent, cotton bollworms damage various crops such as corn, wheat and the like and vegetables such as tomato, pepper and the like, and meanwhile, resistant cotton bollworms appear in fields, so that the cotton bollworms still pose great threat to national food safety and are still key points in pest prevention and control. Although the chemical pesticide has fast effect on preventing and treating larvae of 2-3 years old, the chemical pesticide has great risk on biological safety and ecological safety, so that the development of the biological pesticide for preventing and treating the aged cotton bollworms is urgently needed. The nucleopolyhedrosis virus is the earliest commercialized virus biological pesticide, is an important pathogenic microorganism of lepidoptera insects, can infect other larvae with disease insect excrement and dead insects, can also be transmitted to offspring through eggs, is suitable for low-age larvae below 3 ages, and has almost no control effect on aged cotton bollworms above 3 ages, especially at 6 ages.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a pesticide for preventing and treating cotton bollworm by using nuclear polyhedrosis virus, and a preparation method and application thereof. Effectively kill the cotton bollworm and prevent and control the outbreak of the cotton bollworm.

The technical scheme adopted by the invention is as follows:

a pesticide containing nucleopolyhedrosis virus for biologically preventing and treating cotton bollworm contains effective components of nucleopolyhedrosis virus and extract of Erysiphe Divaricata containing ecdysone. The effective amount of the pesticide is that the content ratio of the nuclear polyhedrosis virus to the ecdysone is 2.0 x 10⁶-2.5×10⁶PIB：1.5-1.8mg。

Preferably, the effective components of the pesticide for biologically controlling cotton bollworm by using nuclear polyhedrosis virus are nuclear polyhedrosis virus and an extract of Erysiphe aquatica containing ecdysone. The effective amount of the pesticide is that the content ratio of the nuclear polyhedrosis virus to the ecdysone is 2.0 x 10⁶PIB：1.5mg。

Preferably, the pesticide for biologically controlling cotton bollworm by using nuclear polyhedrosis virus comprises 30 mass percent of ecdysone extracted from the common dew plant containing ecdysone.

A preparation method of a pesticide for biologically preventing and controlling cotton bollworms by using nuclear polyhedrosis virus specifically comprises the following steps:

(1) dissolving the extract of the dewy grass containing the effective amount of the ecdysone with a certain amount of water, and uniformly mixing to obtain a solution of the extract of the dewy grass;

(2) and mixing the dew grass extract solution with the solution containing the nuclear polyhedrosis virus with the effective amount to obtain the pesticide.

Preferably, the preparation method of the pesticide for biologically controlling the cotton bollworm by the nuclear polyhedrosis virus specifically comprises the following steps:

(1) dissolving herba Eragrostidis extract containing 1.6-2.0Kg ecdysone with water, and mixing well to obtain herba Eragrostidis extract solution;

(2) the extract solution of herba Eragrostidis and the extract solution containing 2.8 × 10¹²PIB-3.5×10¹²And mixing the solutions of the PIB nuclear polyhedrosis virus to obtain the pesticide.

An application of the nuclear polyhedrosis virus as agricultural chemical in preventing and eliminating cotton bollworm is disclosed.

Preferably, the application of the nuclear polyhedrosis virus pesticide in biological control of cotton bollworms comprises the following application method per mu: containing 1.6-2.0Kg of ecdysone and 2.8X 10¹²PIB-3.5×10¹²The pesticide of the PIB nuclear polyhedrosis virus is diluted into a certain volume and is uniformly mixed and sprayed.

The invention has the beneficial effects that:

1. the nuclear polyhedrosis virus pesticide provided by the invention is characterized in that 5mg of dew grass extract (30% ecdysone) and 2.0X 106 PIB nuclear polyhedrosis virus are fed to 6-year-old cotton bollworms, the prevention and treatment effect is optimal, and the cotton bollworms can be effectively killed.

2. The pesticide for preventing and controlling the cotton bollworm by the nuclear polyhedrosis virus provided by the invention has quick insecticidal effect, and the death rate of the 6-year cotton bollworm on day 3 is obvious; the insecticidal efficiency is improved, and the survival rate at the 4 th day is reduced by 28.4%; the survival rate at day 5 was 52.5%, and the survival rate at day 7 was only 34.7%. Usually, the nuclear polyhedrosis virus is used for preventing and treating 3-year-old larvae, the prevention and treatment effect is poor after 3 years old, and if the method is used for preventing and treating 3-year-old larvae, the death rate reaches 100% within 48 hours. Therefore, the method has obvious effects on reducing the harm of the aged cotton bollworms to the crops in the current year, reducing the egg laying amount and preventing and controlling the outbreak of the cotton bollworms in the next year. Meanwhile, the killing effect on the cotton bollworms before 3 years old is better than that of the traditional pesticide.

Drawings

FIG. 1 feeding 5mg of Cyanotis japonica extract (30% ecdysone) and 2.0X 10⁶Survival Rate of 3-instar Cotton bollworm at PIB Nuclear polyhedrosis Virus

Figure 2 feeding ecdysone pure product 20E and 2.5X 10⁶Survival rate of 6-instar cotton bollworm of PIB nuclear polyhedrosis virus

FIG. 3 viral infection induced upregulation of VHDLR expression

FIG. 4 silencing VHDLR leads to a reduction in the amount of viral infection

Blocking of VHDLR by the antibody of figure 5 reduced viral infection.

Detailed Description

Example 1:

a pesticide containing nucleopolyhedrosis virus for preventing and treating cotton bollworm contains effective components of nucleopolyhedrosis virus and extract of Erythrophloe with ecdysone. Mixing the two according to a certain proportion.

To identify the effect of different proportions and concentrations of nucleopolyhedrosis virus and extract of Cyanotis plants on 6-instar Helicoverpa L, experiments were performed according to Table 1 to determine the survival rate. Divided into 12 groups of 50 heads each. The results are shown in Table 1. The experimental method of the pesticide with the concentration of one of the preferred embodiments provided by the invention is as follows, and other groups of experimental methods are similar and can be deduced according to the technical proposal of the person skilled in the art.

Dissolving 21 g of herba Eragrostidis extract containing 30% ecdysone in 5 ml of 60-degree water, adding 25-degree water to 100 ml, and respectively sucking 25 μ l of the herba Eragrostis extract and 25 μ l of the extract containing 8.5 × 10⁷Mixing with PBS solution of nuclear polyhedrosis virus PIB/mL, and spraying onto 6.15 cm²The normal feed is used for feeding 6-year-old single-head cotton bollworms, and after the food sprayed with the medicine is eaten, the normal feed is added for conventional feeding. The preparation process of the normal feed comprises the following steps:

1. dissolving 3 g of sorbic acid in 600 ml of water, and uniformly stirring with 80 g of soybean meal, 150 g of wheat bran and 30 g of yeast powder;

2. adding 20 g of agar into 800 ml of water, heating to melt, adding 40 g of casein, uniformly stirring, standing and cooling to 40 ℃;

3. adding 3 g of ascorbic acid, 1.715 g of compound vitamin, 8 g of glucose and 20 g of cane sugar into 100 ml of water, stirring uniformly,

4. mixing the

components

1, 2 and 3 together, adding 2 ml of formaldehyde and 4 ml of acetic acid, stirring uniformly, and standing at 4 ℃ for later use.

Table 1: survival rates of 6-instar cotton bollworms when fed with nuclear polyhedrosis virus and extract of Cyanotis aquatica (30% ecdysone) at different concentrations

As can be seen from the statistical results in Table 1, the extract of Cyanotis japonica (30% ecdysone) and 2.0X 10 were added in an amount of 5mg per 6 th instar bollworm⁶The usage amount of the PIB nuclear polyhedrosis virus can achieve better prevention and treatment effect. Particularly, in the case of the mixture ratio, the survival rate of the cotton bollworm fed with the nuclear polyhedrosis virus HaNPV only is reduced to 71.66% on the 4 th day, and the death rate of the cotton bollworm is increased by 14.45% compared with the survival rate of the cotton bollworm fed with the nuclear polyhedrosis virus HaNPV only to 86.11% on the 4 th day; the survival rate decreased to 34.67% by day 7, while the survival rate of Heliothis armigera fed with HaNPV alone increased 32% by day 4 compared to 66.67%. The survival rate of the cotton bollworm is not obviously affected by adding the extract of the dewy grass (30 percent of the ecdysone) alone, and the nuclear polyhedrosis virus HaNPV added with the extract of the dewy grass (30 percent of the ecdysone) can shorten the death time of the cotton bollworm of 6 years old, accelerate the effect, and greatly improve the insecticidal efficiency, thereby not only reducing the harm of the aged cotton bollworm to the crops of the same year, but also reducing the pupation rate, and having important effect on preventing and controlling the outbreak of the cotton bollworm of the next year.

The pesticide provided by the invention is used for preventing and treating 3-instar pre-larvae, and is divided into three groups, wherein each group comprises 50, one group is fed with 5 mg/dew grass extract (30% ecdysone), and the other group is fed with nuclear polyhedrosis virus HaNPV2.0 x 10⁶PIB, one group was fed with 5mg of Cyanotis variegata extract (30% ecdysone) and 2.0 × 10⁶PIB nuclear polyhedrosis virus, the experimental procedure is as described above. As shown in figure 1, the 48-hour survival rate of the 3-year-old cotton bollworms fed with the extract of the dewy grass is 96.3 percent, and the survival rate of the cotton bollworms is slightly influenced; survival rate of feeding HaNPV only for 12 hours97.3 percent, and the survival rate in 48 hours is 71 percent; however, when the two are added simultaneously, the survival rate of the 3-year-old cotton bollworms in 12 hours is reduced to 71 percent, and the survival rate in 48 hours is 0. The extract of the dewy grass (30 percent of ecdysone) obviously improves the efficiency of killing 3-year-old cotton bollworms by the HaNPV, shortens the death time of the cotton bollworms caused by the HaNPV, accelerates the effect, and greatly reduces the harm to crops.

In order to determine the active ingredient of the agricultural chemical provided by the present invention, experiments were conducted using an ecdysone pure product (purchased from Sigma-Aldrich, Cat. No.: H5142) and nuclear polyhedrosis virus. Divided into two groups of 50. As can be seen in fig. 2, a significant difference in survival occurred at day 7. It is shown that very small amount of pure 20E can accelerate virus-induced bollworm death, and the extract of Cyanotis is effective in containing ecdysone. The principle that the pesticide provided by the invention can play a role is that ecdysone can induce the up-regulated expression of a virus receptor VHDLR. Our studies found that VHDLR is an on-membrane receptor for infection by this virus, and that the virus was unable to infect host cells when the gene was silenced or blocked with antibodies.

The experimental method comprises the following steps: taking 40 larvae of 6-year-old at feeding stage, starving for 12 hr, and feeding with feed containing 1 × 10⁸The feed of the nuclear polyhedrosis virus HaNPV of PIB is eaten after 12 hours. Then, 5 larvae inducing for 0h, 3h, 6h, 12h, 18h, 24h, 36h and 48h are taken according to the induction time respectively, hemolymph is collected, RNA and protein are extracted, and the expression change of VHDLR induced by the HaNPV at different times is detected by real-time quantitative PCR and Western blot respectively. Relative expression was quantified in real time using the general 2- Δ Ct algorithm and then divided by the relative fold of the control.

As can be seen from the experimental results of fig. 3: the nuclear polyhedrosis virus HaNPV infection can induce the up-regulation expression of mRNA and protein of VHDLR.

The gene silencing method comprises the following steps: taking 20 larvae at the late stage of 6 th instar and 10 larvae per group by using VHDLR double-stranded RNA synthesized in vitro, injecting VHDLR double-stranded RNA at 2.5ug/5ul, and injecting dsGFP in a control group; 24h after injection, double stranded RNA was injected at 2.5ug/5 ul. After 48h of 2 times of interference, 5 larvae in the experimental group and the control group are respectively taken to extract hemolymph RNA. Injecting 5ul HaNPV budding virus into the remaining 5 larvae of each group, performing BV induction for 12h, taking hemolymph, centrifuging for 3min at 200rcf, discarding supernatant, adding 1 XPBS for cleaning for 3 times, extracting blood cell RNA, performing reverse transcription, and detecting by qPCR and Western blot. Relative expression was quantified in real time using the general 2- Δ Ct algorithm and then divided by the relative fold of the control.

As can be seen from the experimental results of fig. 4: when VHDLR gene is silent (FIG. 4A), the F gene of the virus is detected to be reduced by real-time quantitative PCR (FIG. 4B), the VHDLR protein is detected to be reduced by Western blot (FIG. 4C), the F protein of the virus is also reduced, and the result is consistent with the mRNA, which indicates that the infection of the virus depends on the expression of the VHDLR.

The antibody blocking method comprises the following steps: VHDLR rabbit anti-IgG protein concentrations were measured and diluted with PBS into 4 groups of different concentrations: 0, 5, 10, 50 (μ g/ml); taking 50 mul of each group of antibodies, adding equivalent BV, and incubating for 10min at 4 ℃; then 4 groups of 6-instar 12h larvae of the cotton bollworm are taken, and 10 larvae are taken for each group. Respectively injecting 10 mul of antibody-virus mixture, and sampling after raising in a constant temperature incubator at 28 ℃ for 6 hours for subsequent detection. The viral infection rate is the fluorescence of the BV-F protein observed under a microscope, and the number of infected cells with fluorescence divided by the percentage of the total number in the field. FIG. 5C shows the total protein in blood cells after equal amounts of virus infection after blocking with different antibodies to VHDLR, and then the amount of VHDLR and the amount of viral marker protein BV-F were measured.

As can be seen from the experimental results of fig. 5: a and B, antibodies block VHDLR, viral infection decreases with increasing antibody concentration; C. western blot protein levels with increasing VHDLR antibody concentrations, the amount of virus F protein in the cells decreased.

The amount of virus F detected by immunohistochemistry decreased with increasing antibody concentration (fig. 5A); the virus infection rate and the blocking concentration of the VHDLR antibody are inversely proportional (FIG. 5B), although the VHDLR protein exists, the binding sites are blocked by the antibody detected by Western blot, and therefore the virus F protein infection amount in the cells is reduced (FIG. 5C), which indicates that the VHDLR is used as a receptor to mediate the infection of the host cells by the HaNPV.

VHDLR is only expressed in 6-year-old fat body and midgut tissue when ecdysone 20E is highest in the cotton bollworm, and VHDLR is not expressed at all when 20E is at a lower level in other development stages; ecdysone, which is a typical steroid hormone, is capable of upregulating the expression of VHDLR, the 20E receptor is formed by the nuclear receptors EcR and USP, 20E promotes transcription of VHDLR by activating EcR-USP heterodimers; when silencing EcRB1 or USP1, respectively, with RNA interference technology, 20E is not able to upregulate expression of VHDLR; 20E is also capable of promoting phosphorylation promotion of VHDLR and ligand binding.

20E at age 6 is high in titer, and the VHDLR receptor is up-regulated by 20E, so that the expression is high, but the virus carries ecdysteroid UDP-glucopyranosyl transferase itself, and 20E can be inactivated. Therefore, the virus can promote self-propagation without causing the host to die immediately, and the balance which is most beneficial to the survival of the virus is achieved. Thus, by increasing the expression of the receptor by 20E, breaking this balance, would lead to accelerated death by viral infection beyond the host's tolerance due to over-expression of the receptor.

Example 2:

a preparation method of a pesticide for preventing and controlling cotton bollworm by using nuclear polyhedrosis virus comprises the following steps:

An application of the nuclear polyhedrosis virus as agricultural chemical in preventing and eliminating cotton bollworm is disclosed. The method for applying the pesticide per mu comprises the following steps: diluting the prepared nuclear polyhedrosis virus pesticide to obtain a solution containing 1.6-2.0Kg of ecdysone and 2.8 × 10 Kg of ecdysone¹²PIB-3.5×10¹²The pesticide of the PIB nuclear polyhedrosis virus is prepared into 500-1000mL, diluted to a certain volume such as 20-30Kg, mixed and sprayed.

Claims

1. A pesticide for biologically preventing and controlling cotton bollworm by using cotton bollworm nuclear polyhedrosis virus is characterized in that: effective components of nuclear polyhedrosis virus and extract of Cyanotis aquatica containing ecdysone, and the effective amount of the pesticide isThe content ratio of nuclear polyhedrosis virus to ecdysone is 2.0 × 10⁶-2.5×10⁶PIB：1.5-1.8mg。

2. The pesticide of claim 1, wherein the nuclear polyhedrosis virus biological control of cotton bollworm comprises: the effective amount of the pesticide is that the content ratio of the nuclear polyhedrosis virus to the ecdysone is 2.0 x 10⁶PIB：1.5mg。

3. The pesticide of claim 1, wherein the nuclear polyhedrosis virus biological control of cotton bollworm comprises: the mass fraction of the ecdysone in the extract of the common dew containing the ecdysone is 30 percent.

4. A method for preparing a pesticide for biologically controlling Heliothis armigera by using the nuclear polyhedrosis virus as claimed in claim 1, which comprises the following steps:

5. A preparation method of a pesticide for biologically preventing and controlling cotton bollworm by using cotton bollworm nuclear polyhedrosis virus is characterized by comprising the following steps:

6. Use of a nucleopolyhedrosis virus pesticide, as claimed in any one of claims 1-3, for the biological control of cotton bollworms.

7.An application of cotton bollworm nuclear polyhedrosis virus pesticide in biological prevention and control of cotton bollworms is characterized in that the pesticide application method per mu is as follows: containing 1.6-2.0Kg of ecdysone and 2.8X 10¹²PIB-3.5×10¹²The pesticide of the PIB nuclear polyhedrosis virus is diluted into a certain volume and is uniformly mixed and sprayed.