CN114794120B

CN114794120B - Method for reducing TSWV (total suspended solid Virus) toxin carrying amount in adult thrips

Info

Publication number: CN114794120B
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: 2023-08-11
Anticipated expiration: 2042-05-25
Also published as: CN114794120A

Abstract

The invention discloses a method for reducing TSWV (total suspended solid Virus) carrying amount in adult thrips, belonging to the technical field of virus prevention and control. According to the invention, the prodigiosin is prepared into a slow-release preparation, and soil is irrigated in the thrips pupa period, so that the prodigiosin is forced to accept the action of the prodigiosin slow-release preparation by utilizing the inactive characteristic of the thrips pupa period, and the toxin carrying amount in the thrips pupa is reduced, so that the toxin carrying amount in the thrips adult is finally reduced, and the risk of transmitting tomato spotted wilt viruses to other plants by the thrips adult is greatly reduced.

Description

Method for reducing TSWV (total suspended solid Virus) toxin carrying amount in adult thrips

Technical Field

The invention belongs to the technical field of virus prevention and control, and particularly relates to a method for reducing TSWV (total suspended solid Virus) carrying amount in adult thrips.

Background

Tomato Spotted Wilt Virus (TSWV) is an important virus that jeopardizes agricultural production, and is primarily transmitted by sap contact, and invades only if the host has a wound. The tomato spotted wilt virus has very wide host plants, and can produce serious harm and even absolute production after being infected with various crops such as grains, tobacco, vegetables, flowers and the like. In recent years, tomato spotted wilt virus diseases are becoming more and more popular with global climate change, and are becoming one of the 10 most harmful plant viruses worldwide.

Tomato spotted wilt virus is transmitted by feeding plants mainly through mediator thrips, and once the thrips acquire the virus, the thrips can carry the virus for life. Thrips occur all the year round, mainly occur in open field in spring, summer and autumn, mainly occur in greenhouse in winter, overlap generation and reproduce all the year round. The peak period generally occurs in autumn or 11-12 months in winter, and the second peak period is 3-5 months. The life history of thrips pests consists of four stages, namely eggs, nymphs (1-and 2-year-old nymphs), pupae (pseudopupae) and adults, respectively. Thrips nymphs stop feeding at the end of the advanced age after feeding on the leaf backs, fall into the surface soil to pupate, are usually not fed and do not move in the pupae stage, then emerge as adults are broken into soil, the moving range is widened, the damage between new species and new strains can be caused by feeding new plants, and the destructive power is obviously increased. Therefore, the method has important significance on how to reduce the risk of transmitting tomato spotted wilt virus to overground plants by thrips adults. The chemical pesticide is applied to underground soil to kill thrips before adult thrips, so that tomato spotted wilt virus can be effectively confined in the soil, and the method is a main measure for preventing and treating tomato spotted wilt virus at present. However, long-term use of chemical pesticides can cause pollution to the soil environment, ultimately endangering human health.

Disclosure of Invention

Besides chemical pesticides, no other safe and efficient mode is available at present for killing the thrips adults in soil before the thrips adults, and for this purpose, the invention tries to control the thrips adults to widely spread tomato spotted wilt viruses to ground plants by reducing the amount of the toxic substances in the thrips adults.

Prodigiosin (PGs) is a natural secondary metabolite of microorganisms, has a skeleton of Prodigiosin (PG) polypyrrole, has wide biological activity, has an inhibitory effect on tomato spotted wilt virus, and is environmentally safe. Directly irrigate Shi Ling bilirubin underground, can not reach the prevention and cure effect of effectively reducing the in vivo amount of toxicity of adult thrips, mainly have the following two reasons: firstly, the prodigiosin is easy to degrade in natural environment and has short drug effect; second, the thrips nymphs may be far away from prodigiosin, resulting in reduced efficacy.

In order to overcome the technical problems, the prodigiosin is prepared into a slow-release preparation, and soil is irrigated in the thrips pupa period to make use of the inactive characteristic of the thrips pupa period, so that the prodigiosin slow-release preparation is forced to accept the action of the prodigiosin slow-release preparation, the toxin carrying amount in the thrips pupa is reduced, the toxin carrying amount in the thrips adult is finally reduced, and the risk that the thrips adult transmits tomato spotted wilt viruses to other plants is greatly reduced.

The technical scheme of the invention is as follows:

a method for reducing TSWV (total suspended solids) toxin carrying amount in adult thrips comprises the following steps: and (3) filling the Shi Ling bilirubin slow-release preparation into the land in the thrips pupa period to inhibit the toxin carrying amount in the thrips pupa, thereby finally reducing the toxin carrying amount in the thrips adult.

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

The method for applying the prodigiosin slow-release preparation comprises the following steps: and (3) root irrigation treatment is carried out on plants, and 100-200 mL of prodigiosin slow release preparation is irrigated to each plant.

The coating material used for preparing the prodigiosin into the sustained-release preparation has the following characteristics: the coating has good performance in terms of biodegradability, compatibility, adhesiveness and the like, and can play a role in preventing the coating from being suddenly released and controlling the release speed of the coating. Preferably sodium alginate is used.

The invention provides a prodigiosin slow-release preparation, which consists of prodigiosin, salicylic acid and sodium alginate. Preferably, the mass ratio of sodium alginate, prodigiosin and salicylic acid is 1:2:1. In the preparation of the prodigiosin sustained release preparation, an emulsifier may be optionally used, preferably, the emulsifier is tween-20.

The structural formula of the prodigiosin in the prodigiosin sustained-release preparation is shown in the following formula I, and the preparation method is disclosed in patent CN103387529A.

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, prodigiosin solution and salicylic acid solution dropwise, and stirring and mixing uniformly; centrifuging, removing supernatant, and washing to obtain prodigiosin nanocapsule, namely prodigiosin sustained release preparation.

The method for reducing the TSWV virus carrying amount in the adult thrips can be applied to preventing and controlling the adult thrips from widely transmitting tomato spotted wilt virus to ground plants. In particular to prevention and control of the spread of tomato spotted wilt virus by thrips adults to uninfected plants.

The beneficial effects of the invention are as follows:

the prodigiosin slow release preparation can slowly and continuously release prodigiosin and maintain stable and effective drug concentration for a long time. The prodigiosin slow-release preparation is applied to soil in the thrips pupa period, so that the characteristic of inactivity in the thrips pupa period can be effectively utilized, the prodigiosin slow-release preparation is forced to be accepted, the toxin carrying amount in the thrips pupa is reduced, the toxin carrying amount in the thrips adult is finally reduced, and the risk of spreading tomato spotted wilt viruses to other plants by the thrips adult is greatly reduced. Meanwhile, the prodigiosin belongs to a microorganism secondary metabolite, and is safe and pollution-free to the environment. In addition, salicylic acid is used as plant endogenous hormone, and can induce plants to generate systemic resistance by activating plant allergic reaction and regulating plant disease course related protein gene expression, so as to resist the invasion of pathogens to crops.

Drawings

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

FIG. 2 shows 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 profile of prodigiosin at various temperatures, wherein the upper curve is at 35℃and the middle curve is at 25℃and the lower curve is at 15 ℃;

FIG. 4 is a graph showing the release profile of salicylic acid at various temperatures, wherein the upper curve is 35℃and the middle curve is 25℃and the lower curve is 15 ℃.

FIG. 5 is a graph showing symptoms on the seventh day after inoculation of TSWV with Nicotiana benthamiana, wherein the left panel is a control and the right panel is an inoculated TSWV;

fig. 6 is a graph showing the relative TSWV content of burley tobacco leaves in different treatment groups.

Detailed Description

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated. The invention will be described in further detail below in connection with specific embodiments and with reference to the data. 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 (0.1 g/mL) into a 50mL triangular flask, sequentially adding 4mL of isopropyl alcohol and 2mL of glutaraldehyde under the magnetic stirring condition, and standing for 30min after stirring uniformly; then adding 0.5mL Tween-20, 1mL prodigiosin solution (1 g/mL) and 0.5mL salicylic acid solution (1 g/mL) dropwise, and stirring for 1h to mix uniformly; centrifuging at 4deg.C in 11000 Xg low temperature centrifuge 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 place for use.

(1) Analysis of physical Properties

The particle size, zeta potential, PDI and other data of the prodigiosin sustained release preparation were measured, and as shown in FIG. 1 and FIG. 2, the average particle size of the prodigiosin sustained release preparation was 100 to 200nm, zeta potential +31.5mV, and polydispersity index (PDI) was 0.542. The results show that the prodigiosin slow-release preparation has the characteristic of excellent nano medicament.

(2) Analysis of sustained release Properties

5mg of the prodigiosin slow release formulation was placed in a 3.5KD dialysis bag and immersed in 100mL PBS buffer. 5mL samples were taken periodically in equal amounts for analysis and replaced with 5mL fresh PBS buffer, with shaking at 60rpm at 15, 25 and 35℃respectively. The absorbance of the release PBS buffer was then measured at 535nm and 298nm, respectively, to calculate the drug release amounts of prodigiosin and salicylic acid.

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

Example 2

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

healthy and nontoxic benthamia tinctoria is cultivated in a flowerpot and used for test when growing to 5-6 true leaves. 1g of the leaf of Benshi tobacco with TSWV is put into a mortar which is sterilized in advance, and 10mL of 1 XPBS buffer solution is added and then ground to be used as an inoculation liquid. And (3) taking healthy benthamiana plants to be inoculated, scattering a small amount of 500-mesh silicon carbide powder on 2-3 pieces of true leaves at the top of the healthy benthamiana plants, dipping a proper amount of inoculation liquid with a cotton swab, lightly rubbing and inoculating at the vein position, slightly washing the leaves with clear water after 30min, and transferring to a normal environment for continuous culture after being placed in a dark environment for 24 h. Typical symptoms of deformity, chlorosis, yellowing, mottle and the like appear after about 7-10 days of inoculation, and are shown in fig. 5, namely, plants with toxicity are used for subsequent experiments.

Five healthy fresh beans are placed in an adult feeding glass tank, after the adults lay eggs on the beans for 24 hours, the beans are taken out and placed in another new glass tank, after three days, the nymphs of one age are hatched, and the nymphs are transferred to TSWV infected in vitro Nicotiana benthamiana leaves. The leaf blade was placed with its back face facing upwards in a glass petri dish containing 5mm thick 2% agar and a layer of filter paper, sealed and placed in an incubator with illumination at 27.+ -. 1 ℃ and with illumination at L: d=16:8 h for 120h.

Then, two treatments were set up for the test, treatment one: transferring about 200 second-age aged nymphs (taking body color as orange red as standard) to a flat-bottom glass tank containing fresh green beans, spreading and sieving high-temperature sterilized earth surface soil at the bottom of the tank, diluting the prodigiosin slow-release preparation to 200 mug/mL, and then pouring soil to keep the humidity at 70%, thus obtaining the test group. And (2) treatment II: transferring about 200 mature two-year-old nymphs to a flat-bottom glass tank with fresh beans, spreading and sieving high-temperature sterilized ground surface soil at the bottom of the tank, and watering to wet the soil to keep the humidity at 70%, wherein the humidity is the control group. The treatments were placed in an incubator at 27±1 ℃ with light at L: d=16:8 h until the adults emerged. When adults are 5 days old, respectively placing 20 female worms and 20 male worms in 1.5mL RNA-Free PE pipes, repeating each treatment for 3 times, quick freezing with liquid nitrogen, extracting RNA, and quantitatively detecting TSWV content in the adults in different treatments by using RT-qPCR technology.

The results of the test are shown in Table 1, with a small number of thrips not infected with TSWV in both treatment trials, and with TSWV infected individuals 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), 5 (R > 4), compared with the control, the virus content in the thrips adult treated by the prodigiosin slow-release preparation is relatively low, the proportion of the virus-carrying adults with low virus content (grades 1 and 2) is 82.78%, wherein the virus content is classified into 60.35% of 1, which is obviously higher than that of the control group. The result shows that the slow-release preparation of the Shi Ling bilirubin can reduce the virus proliferation capacity, thereby reducing the toxin carrying amount of the frankliniella occidentalis adults.

TABLE 1 ratio of TSWV concentrations in different treatments of Frankliniella occidentalis

Example 3

The method for testing the toxin-transmitting capability of frankliniella occidentalis comprises the following steps:

taking the adults fed by the two treatment tests in the example 2 as test insects, and detecting the toxin transmission capacity of the test insects by adopting living Benshi cigarettes after the test insects emerge for 5 days, wherein the specific method comprises the following steps: the young Nicotiana benthamiana is transplanted into a plastic bucket (the thickness of plant culture medium at the bottom of the bucket is about 10 cm) with the height of 35cm and the diameter of 20cm, and an insect screen with 200 meshes is covered on the plastic bucket. When 4-5 true leaves are cultivated in Benshi tobacco, the test is divided into three treatments, treatment a: each barrel was inoculated with one end of a female or male adult raised in example 2 above; treatment b: each barrel was inoculated with one end of the female or male adult treated for the second feeding in example 2 above; treatment c: no test insects were inoculated as a Control (CK); 10 barrels are needed for each treatment, 5 barrels are accessed to female worms, 5 barrels are accessed to male worms, and 3 times of repetition are set, namely 30 heads are accessed for each treatment. After the test insects are inoculated, the cover is covered tightly, and the test insects are placed in an incubator for two weeks to be cultured, and the disease condition of each treatment 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 group tobacco plants are asymptomatic; treatment a had a lower severity of disease than treatment b. The copy number of TSWV was detected by qRT-PCR, as shown in fig. 6, with the copy number of TSWV in treatment a being significantly lower than treatment b. The result shows that the Shi Ling bilirubin slow-release preparation is filled into soil in the pupal stage of frankliniella occidentalis, so that the toxin carrying amount of the frankliniella occidentalis can be obviously reduced, the toxin transmission amount of the frankliniella occidentalis is reduced, and the disease degree of crops is reduced.

TABLE 2 treatment of tobacco plant morbidity

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for reducing TSWV (total suspended solids) toxin carrying amount in adult thrips is characterized in that Shi Ling bilirubin slow-release preparation is irrigated to the land in the pupa period of the thrips to inhibit toxin carrying amount in the adult thrips, so that the toxin carrying amount in the adult thrips is reduced finally;

the structural formula of the prodigiosin is shown in the following formula I:

2. the method of claim 1, wherein the concentration of the prodigiosin slow release formulation is 200-400 μg/mL.

3. The method according to claim 2, wherein the slow release formulation of prodigiosin is applied by the following method: and (3) root irrigation treatment is carried out on plants, and 100-200 mL of prodigiosin slow release preparation is irrigated to each plant.

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

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

6. Use of the method according to any one of claims 1 to 5 for controlling the broad spread of tomato spotted wilt virus by adults to ground plants.

7. The use according to claim 6, wherein the plant is a plant not infected with tomato spotted wilt virus.