CN114794120A

CN114794120A - Method for reducing toxic amount of TSWV in thrips adults

Info

Publication number: CN114794120A
Application number: CN202210577058.7A
Authority: CN
Inventors: 杨金广; 袁莲莲; 李莹; 葛明; 焦裕冰; 宋丽云; 申莉莉; 王凤龙; 褚贵德; 薛新田
Original assignee: Heilongjiang Dadifeng Agriculture Technology Development Co ltd; Tobacco Research Institute of CAAS
Current assignee: Heilongjiang Dadifeng Agriculture Technology Development Co ltd; Tobacco Research Institute of CAAS
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2022-07-29
Anticipated expiration: 2042-05-25
Also published as: CN114794120B

Abstract

The invention discloses a method for reducing the toxic content of TSWV in a thrips adult, belonging to the technical field of virus prevention and control. The invention prepares the prodigiosin into a slow-release preparation, and selectively irrigates soil at the pupal stage of the thrips so as to utilize the characteristic of immobility at the pupal stage of the thrips to make the thrips compelled to receive the effect of the prodigiosin slow-release preparation and reduce the toxic carrying amount in the pupal body of the thrips, thereby finally reducing the toxic carrying amount in the body of the adults of the thrips and greatly reducing the risk of the adult thrips spreading the tomato spotted wilt virus to other plants.

Description

Method for reducing toxic amount of TSWV in thrips adults

Technical Field

The invention belongs to the technical field of virus prevention and treatment, and particularly relates to a method for reducing toxic dose of TSWV in a thrips adult.

Background

Tomato Spotted Wilt Virus (TSWV) is an important virus which endangers agricultural production and is mainly infected through sap contact, and can invade as long as a host has a wound. The tomato spotted wilt virus has wide host plants, and can cause serious harm and even stop production after infecting various crops such as grains, tobacco, vegetables, flowers and the like. In recent years, the tomato spotted wilt virus disease becomes more and more prevalent with the change of global climate, and is one of the 10 most harmful plant viruses in the world.

Tomato spotted wilt virus spreads the virus mainly through the mediator thrips to feed on the plant, once the thrips is poisoned, the virus can be carried out for the lifetime. Thrips occur all the year round, spring, summer and autumn mainly occur in open field, winter mainly occurs in greenhouse, generations are overlapped, and propagation is carried out all the year round. The peak period generally occurs in autumn or in 11-12 months of winter, and the second peak period is 3-5 months. The life history of thrips pests is composed of eggs, nymphs (1-year and 2-year nymphs), pupae (fake pupae) and adults respectively. The thrips nymphs stop feeding from the back of the leaves to the late age, fall into the surface soil and pupate, generally do not feed in the pupate stage, emerge from the post-eclosion as adults breaking the soil, have a wide range of activity, can cause mutual harm between new species and new strains by feeding new plants, and have obviously increased destructive power. Therefore, how to reduce the risk of the tomato spotted wilt virus spread from the thrips to the overground plants has important significance. Chemical pesticide is irrigated to underground soil to kill the thrips before the thrips are grown, so that the tomato spotted wilt virus can be effectively confined in the soil, and the method is a main measure for preventing and treating the tomato spotted wilt virus at present. However, long-term use of chemical pesticides can cause pollution of soil environment, and finally harm human health.

Disclosure of Invention

In addition to chemical pesticides, there is currently no safe and efficient way to kill adult thrips in soil before they are killed, and for this reason, the present invention attempts to reduce the toxic load in the adult thrips to control the widespread spread of adult thrips to ground plants by tomato spotted wilt virus.

Prodigiosin compounds (PGs) are natural secondary metabolites of microorganisms, have Prodigiosin (PG) polypyrrole frameworks, have wide biological activity, have an inhibiting effect on tomato spotted wilt virus and are safe to the environment. The control effect of effectively reducing the toxic amount in the thrips adults cannot be achieved by directly irrigating prodigiosin underground, and the two main reasons are as follows: firstly, prodigiosin is easy to degrade in natural environment and has short drug effect; second, thrips nymphs can be kept away from prodigiosin, resulting in reduced efficacy.

In order to overcome the technical problems, prodigiosin is prepared into a slow-release preparation, and soil is selectively irrigated in the pupal stage of the thrips, so that the effect of the prodigiosin slow-release preparation is forced to be accepted by the prodigiosin in the pupal stage, and the toxic carrying amount in the pupal body of the thrips is reduced, thereby finally reducing the toxic carrying amount in the adult body of the thrips and greatly reducing the risk of the adult thrips spreading tomato spotted wilt virus to other plants.

The technical scheme of the invention is as follows:

a method for reducing TSWV toxic amount in a thrips adult comprises the following steps: and (3) irrigating a prodigiosin sustained-release preparation to the land in the pupal stage of the thrips to inhibit the toxic carrying amount in the pupal bodies of the thrips, thereby finally reducing the toxic carrying amount in the adult bodies of the thrips.

The concentration of the prodigiosin sustained-release preparation is 200-400 mug/mL.

The irrigation method of the prodigiosin sustained-release preparation comprises the following steps: and (3) irrigating roots of the plants, and irrigating 100-200 mL of prodigiosin sustained-release preparation for each plant.

The coating material for preparing the prodigiosin into the sustained-release preparation has the following characteristics: the coating has good performances in the aspects of biodegradability, compatibility, adhesion and the like, and can play roles in preventing the coating from releasing suddenly and controlling the release speed of the coating. Preferably, sodium alginate is used.

The invention provides a prodigiosin sustained-release preparation which is composed of prodigiosin, salicylic acid and sodium alginate. Preferably, the mass ratio of the sodium alginate to the prodigiosin to the salicylic acid is 1:2: 1. In the process of preparing the prodigiosin sustained-release preparation, an emulsifier can be selected, and preferably, the emulsifier is tween-20.

The structural formula of prodigiosin in the prodigiosin sustained-release preparation is shown as the following formula I, and the preparation method is disclosed in the patent CN 103387529A.

The invention provides a preparation method of a prodigiosin sustained-release preparation, which comprises the following steps:

sequentially adding isopropanol and glutaraldehyde into the sodium alginate solution, and uniformly stirring; then adding tween-20, the prodigiosin solution and the salicylic acid solution drop by drop, and stirring and mixing uniformly; centrifuging, removing supernatant, and washing to obtain prodigiosin nanocapsule, i.e. prodigiosin sustained-release preparation.

The method for reducing the toxic quantity of TSWV in the thrips imagoes can be applied to preventing and controlling the wide spread of tomato spotted wilt viruses from the thrips imagoes to ground plants. Especially preventing and controlling the propagation of tomato spotted wilt virus from thrips to disease-free plant.

The invention has the beneficial effects that:

the prodigiosin sustained-release preparation can slowly and continuously release prodigiosin and maintain stable effective drug concentration for a long time. The prodigiosin sustained-release preparation is applied to the soil in the pupal stage of the thrips, so that the characteristics of inactiveness in the pupal stage of the thrips can be effectively utilized, the prodigiosin sustained-release preparation is forced to take the effect, and the toxic quantity in the thrips pupae is reduced, so that the toxic quantity in the thrips imagoes is finally reduced, and the risk of the propagation of tomato spotted wilt viruses to other plants by the thrips imagoes is greatly reduced. Meanwhile, the prodigiosin belongs to a microbial secondary metabolite, and is safe and pollution-free to the environment. In addition, salicylic acid is used as a plant endogenous hormone, can induce plants to generate systemic resistance by activating plant anaphylaxis and regulating the expression of a plant disease course related protein gene, and resists the invasion of pathogens to crops.

Drawings

FIG. 1 is an electron microscope image of a prodigiosin sustained-release preparation;

FIG. 2 is a graph showing the average particle size distribution (left) and Zeta potential distribution (right) of a prodigiosin sustained-release preparation;

FIG. 3 is a graph showing the release profiles of prodigiosin at different temperatures, wherein the upper curve is at 35 deg.C, the middle curve is at 25 deg.C, and the lower curve is at 15 deg.C;

FIG. 4 is a graph showing the release of salicylic acid at different temperatures, wherein the upper curve is 35 deg.C, the middle curve is 25 deg.C, and the lower curve is 15 deg.C;

FIG. 5 is a representation of symptoms at day seven after inoculation of TSWV with Nicotiana benthamiana, wherein the left panel is a control and the right panel is an inoculation of TSWV;

figure 6 is the relative TSWV content of benschniakia tobacco leaves in different treatment groups.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1

The preparation method of the prodigiosin sustained-release preparation comprises the following steps:

adding 5mL of sodium alginate solution (0.1g/mL) into a 50mL triangular flask, sequentially adding 4mL of isopropanol and 2mL of glutaraldehyde under the condition of magnetic stirring, uniformly stirring, and standing for 30 min; then 0.5mL of tween-20, 1mL of prodigiosin solution (1g/mL) and 0.5mL of salicylic acid solution (1g/mL) are added dropwise, stirred for 1h and mixed evenly; centrifuging at 4 deg.C and 11000 Xg for 10min, removing supernatant, washing with deionized water for three times to obtain prodigiosin nanocapsule, i.e. prodigiosin sustained release preparation, and storing in dark for use.

(1) Analysis of physical Properties

The results of detecting the particle size, Zeta potential, PDI and other data of the prodigiosin sustained-release preparation are shown in figure 1 and figure 2, the average particle size of the prodigiosin sustained-release preparation is 100-200 nm, the Zeta potential +31.5mV, and the polydispersity index (PDI) is 0.542. The results show that the prodigiosin sustained-release preparation has the characteristics of excellent nano medicaments.

(2) Analysis of sustained Release characteristics

5mg of the prodigiosin sustained-release preparation was placed in a 3.5KD dialysis bag and immersed in 100mL of PBS buffer. Shaking at 60rpm at 15 deg.C, 25 deg.C and 35 deg.C, respectively, periodically and equally taking out 5mL of each sample for analysis, and replacing with 5mL of fresh PBS buffer. The drug release amounts of prodigiosin and salicylic acid were then calculated by measuring the absorbance of the PBS-releasing buffer at 535nm and 298nm, respectively.

The drug release curves are shown in fig. 3 and fig. 4, the prodigiosin sustained-release preparation has good sustained-release characteristics, and the release speed of prodigiosin and salicylic acid is gradually increased along with the increase of temperature, but the cumulative release amount is basically the same. The results show that the prodigiosin sustained-release preparation has the characteristic of slowly and continuously releasing the medicines, and can maintain stable effective medicine concentration for a long time, thereby being beneficial to improving the field application effect.

Example 2

The method for testing the toxicity-obtaining capability of frankliniella occidentalis comprises the following steps:

healthy and nontoxic Benzena was cultivated in a flowerpot and used for the test when 5 to 6 true leaves were grown. 1g of the N.benthamiana leaf with TSWV was put into a mortar sterilized in advance, 10mL of 1 XPBS buffer was added thereto, and the mixture was ground to prepare a seed stock solution. Taking a healthy Nicotiana benthamiana plant to be inoculated, scattering a small amount of 500-mesh carborundum powder on 2-3 true leaves on the top of the healthy Nicotiana benthamiana plant, dipping a proper amount of inoculation liquid with a cotton swab, gently rubbing the inoculation liquid at a vein position for inoculation, slightly washing the leaves with clear water after 30min, placing the inoculated leaves in a dark environment for 24h, and transferring the inoculated leaves to a normal environment for continuous culture. After inoculation for about 7-10 days, typical symptoms such as malformation, chlorosis, yellowing, mottle and the like appear, as shown in fig. 5, namely the plants with the virus are used for subsequent experiments.

Putting five healthy fresh green beans into an adult feeding glass tank, taking out the green beans after the adults lay eggs on the green beans for 24 hours, putting the green beans into another new glass tank, and transferring nymphs to TSWV-infected detached Bunshi tobacco leaves after the nymphs hatch one year after three days. The leaf was placed with the back side up in a glass petri dish containing 5mm thick 2% agar and a layer of filter paper, sealed and then placed in an incubator at 27 + -1 deg.C under illumination with L: D of 16:8h for 120 h.

The experiment then set two treatments, one: transferring about 200 second-instar aged nymphs (standard that body color is changed into orange red) to a flat-bottomed glass tank with fresh green beans, flatly paving and sieving the bottom of the tank, sterilizing surface soil at high temperature, diluting the prodigiosin sustained-release preparation to 200 mu g/mL, and then irrigating the soil to keep the humidity at 70%, thus obtaining a test group. And (5) processing: transferring about 200 aged second-instar nymphs to a flat-bottomed glass jar containing fresh green beans, spreading and sieving at the bottom of the jar, sterilizing surface soil at high temperature, and irrigating and wetting the soil to keep the humidity at 70%, which is a control group. Each treatment was housed in an incubator at 27 ± 1 ℃ under illumination with L: D ═ 16:8h until eclosion of adult insects occurred. When the adults are 5 days old, 20 female worms and 20 male worms are respectively taken and placed in a 1.5mL RNA-Free PE tube for each treatment, the treatment is repeated for 3 times, the eggs are quickly frozen by liquid nitrogen, then RNA is extracted, and the content of TSWV in the adults treated by different methods is quantitatively detected by utilizing the RT-qPCR technology.

The results are shown in Table 1, and in both treatment trials, a small number of thrips were not infected with TSWV, and individuals infected with TSWV exceeded 90% of the total. The virus content in single Frankliniella occidentalis is classified into 1(0< R <1), 2(1< R <2), 3(2< R <3), 4(3< R <4) and 5(R >4), compared with the contrast, the content of virus in the Frankliniella occidentalis adults treated by the prodigiosin sustained-release preparation is relatively low, the proportion of virus-carrying adults with low virus content (grades 1 and 2) is 82.78%, wherein the virus content is 60.35% of 1 and is obviously higher than 31.80% of the contrast group. The results show that the brevibacillin sustained-release preparation can reduce the virus multiplication capacity, thereby reducing the toxic carrying amount of the frankliniella occidentalis adults.

TABLE 1 ratio of TSWV concentration in different treated Frankliniella occidentalis

Example 3

The method for testing the virus-transmitting capacity of Frankliniella occidentalis comprises the following steps:

adult insects fed in the two treatment tests in the example 2 are taken as test insects, after emergence for 5 days, the toxin transmission capacity of the adult insects is detected by using live Benzenbaci, and the specific method comprises the following steps: transplanting the Nicotiana benthamiana seedlings into a plastic bucket (the thickness of a plant culture medium at the bottom of the bucket is about 10cm) with the height of 35cm and the diameter of 20cm, and covering the plastic bucket with a 200-mesh insect-proof gauze. When 4-5 true leaves are cultured in the Nicotiana benthamiana, the experiment is divided into three treatments, namely a treatment: each barrel was inoculated with one head of a female or male adult treated as described in example 2 above; and (b) processing: one end of each female adult or male adult treated in the second breeding in the above example 2 is inoculated into each barrel; and c, processing: test insects were not inoculated as Control (CK); each treatment requires 10 barrels, 5 barrels are accessed with female worms, 5 barrels are accessed with male worms, and 3 times of repetition is set, namely, each treatment is accessed with 30 heads. After the test insects are inoculated, the cover is tightly covered, the test insects are placed in an incubator to be cultured for two weeks, and the disease incidence condition of each treated tobacco strain is investigated according to the virus disease grading standard in GB/T23222-2008.

The test results are shown in table 2, and the control tobacco plants are asymptomatic; the severity of disease development was lower for the tobacco plants treated a than for b. The copy number of TSWV was checked by qRT-PCR, as shown in figure 6, the copy number of TSWV in treatment a was significantly lower than treatment b. The results show that the prodigiosin sustained-release preparation is irrigated into the soil in the pupal stage of Frankliniella occidentalis, so that the toxic carrying amount of Frankliniella occidentalis can be obviously reduced, the toxic transmission amount is reduced, and the disease degree of crops is reduced.

TABLE 2 different treatment of tobacco plant disease

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims

1. A method for reducing the toxic amount of TSWV in the body of the adult thrips is characterized in that a prodigiosin sustained-release preparation is irrigated to the land in the pupal stage of the thrips to inhibit the toxic amount in the pupal of the thrips, thereby finally reducing the toxic amount in the body of the adult thrips.

2. The method according to claim 1, wherein the concentration of the prodigiosin sustained-release preparation is 200 to 400 μ g/mL.

3. The method according to claim 2, wherein the prodigiosin sustained-release preparation is applied by irrigation as follows: and (3) irrigating roots of the plants, and irrigating 100-200 mL of prodigiosin sustained-release preparation for each plant.

4. The method according to any one of claims 1 to 3, wherein the prodigiosin sustained-release preparation consists of prodigiosin, salicylic acid and sodium alginate in a mass ratio of 1:2: 1.

5. The method of claim 4, wherein the prodigiosin has the formula I:

6. the method according to claim 4, wherein the prodigiosin sustained-release preparation is prepared by:

7. The use of the method as claimed in any one of claims 1 to 6 for the prevention and control of the widespread transmission of tomato spotted wilt virus from adult thrips towards ground plants.

8. Use according to claim 7, wherein the plant is a plant not infected with tomato spotted wilt virus.