CN111543437B

CN111543437B - Parasitic nematode preparation, preparation method and application

Info

Publication number: CN111543437B
Application number: CN202010327257.3A
Authority: CN
Inventors: 李星月; 张鸿; 杨武云; 符慧娟; 陈鹏; 李其勇; 易军; 陆文壹
Original assignee: Institute of Plant Protection Sichuan Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection Sichuan Academy of Agricultural Sciences
Priority date: 2020-04-23
Filing date: 2020-04-23
Publication date: 2021-08-17
Anticipated expiration: 2040-04-23
Also published as: CN111543437A

Abstract

The invention discloses a parasitic rhabditis elegans preparation, a preparation method and application, belongs to the technical field of biological pesticides, and solves the problems that in the prior art, the biological control is low in quick-acting performance, and Spodoptera frugiperda cannot be efficiently and quickly killed. The parasitic nematode formulation of the present invention comprises: parasitic nematodes, RNA fragments that interfere with the expression of the polyphenol oxidase PO gene of spodoptera frugiperda; also comprises a pest phagostimulant matrix, a nematode humectant and nematode symbiotic bacteria nutrients. The invention creatively starts from the aspects of induction, infection, saprophytic, inhibition of the immune response of pests by adding dsRNA and the like, and effectively improves the killing effect of the nematodes on spodoptera frugiperda.

Description

Parasitic nematode preparation, preparation method and application

Technical Field

The invention belongs to the technical field of biological pesticides, and particularly relates to a parasitic nematode preparation as well as a preparation method and application thereof.

Background

Spodoptera frugiperda (j.e. smith) is a moth agricultural pest of the genus spodoptera of the family noctuidae, native to tropical and subtropical americans. The Spodoptera frugiperda larva can gnaw a large number of gramineae crops such as oryza sativa, sugarcane and corn, and various crops such as Compositae and Cruciferae, the development speed of the Spodoptera frugiperda larva becomes fast along with the rise of the air temperature, the Spodoptera frugiperda larva can be propagated for several generations in one year, and more than 1000 eggs can be produced by one female moth. After 1 month in 2016, the spread to 44 countries in south of sahara soon occurred. The invasion of Spodoptera frugiperda in 5 months in 2018, migrated from Burma into China in 12 months and 11 months in 2018, and diffused to 26 provinces (districts and cities) in China in 10 months in 2019. The fall armyworm quickly enters a serious occurrence stage after the fall armyworm invades, which has great influence on the production of crops such as corns in many countries of Africa and Asia, and forms a long-term threat to the national food production safety.

In the prior art, the work of preventing and controlling Spodoptera frugiperda is divided into two modes of killing by adopting chemical agents and biological agents. At present, the problem of emergency prevention and control is mainly solved by implementing a comprehensive prevention and control strategy which mainly adopts chemical prevention and control, and the problem of grain safety caused by seriously injuring crops such as corn, wheat and the like is prevented. However, such a technical route has high investment cost and has certain food safety risks and higher environmental safety risks. Since the invaded and colonized spodoptera frugiperda has already generated high resistance to organophosphorus, pyrethroid, carbamate and nicotine chemical insecticides, the generation of drug resistance can be quickly caused by the large-area use of few high-efficiency insecticides such as amides and the like. Once Spodoptera frugiperda has resistance to several types of pesticides with high control effect at present, a passive situation of lacking effective control measures appears in production, and the serious consequence of the uncontrolled cotton bollworm in the past 90 years of the century is likely to be reproduced. In order to meet the increasing requirements of human beings on the aspects of environment, food safety and the like, the development of new pest control measures is not slow, and the vigorous development and application of biopesticides can not only protect natural enemies, but also relieve the selection pressure of pesticides.

The insect parasitic nematodes have the double characteristics of natural enemy insects and pathogenic microorganisms, are important biological pest control factors (high efficiency on pests, and safe on non-target organisms and environment), and have great application potential in sustainable pest control as microbial pesticides, like entomopathogenic nematodes. However, the adoption of insect parasitic nematodes for spodoptera frugiperda has the problem of low biocontrol and quick-acting performance, and the spodoptera frugiperda cannot be efficiently and quickly killed. Therefore, by means of biological reasonable design, aiming at the expression gene of polyphenol oxidase PO of Spodoptera frugiperda, RNA interference technology is utilized to construct and interfere the gene dsRNA which is used together with insect parasitic nematodes, so that the PO-mediated humoral immunity in the insect body can be artificially inhibited, the pathogenic process of the Spodoptera frugiperda infected by the insect parasitic nematodes is accelerated, and the insect biocontrol efficiency is improved. The preparation can utilize the insect parasitic nematodes to kill Spodoptera frugiperda quickly, efficiently and sustainably, and becomes a problem to be solved by the technical personnel in the field.

Disclosure of Invention

One of the purposes of the invention is to provide a parasitic nematode preparation, which can lure spodoptera frugiperda to take the preparation, inhibit the immune response of spodoptera frugiperda, accelerate the infection of the nematodes with lepidoptera larvae such as spodoptera frugiperda and the like, and rapidly propagate on the corpses of the larvae, thereby efficiently and rapidly killing the spodoptera frugiperda.

The second object of the present invention is to provide a process for producing the parasitic nematode agent.

The invention also aims to provide application of the parasitic nematode preparation.

The technical scheme adopted by the invention is as follows:

the invention relates to a parasitic nematode preparation, which comprises the following components: parasitic nematodes, RNA fragments that interfere with the expression of the polyphenol oxidase PO gene of Spodoptera frugiperda.

In the technical scheme, the parasitic corynebacteria is Oscheius sp., is an insect parasitic corynebacteria separated and purified from pupal spodoptera frugiperda which naturally dies in soil, can quickly infect lepidoptera larvae such as spodoptera frugiperda and can be massively and quickly propagated on the corpses of the lepidoptera larvae.

The specific separation and purification operations of the parasitic corynebacterium family nematodes are as follows: cleaning pupa of Spodoptera frugiperda which dies naturally in soil with clear water, disinfecting the surface of the pupa with 75% alcohol, collecting the pupa into distilled water by adopting a White trap method, picking single-strip egg female nematodes, propagating the single-strip egg female nematodes in vitro by using large wax moth body fluid, and purifying to obtain a single variety.

In the technical scheme of the invention, the RNA segment is dsRNA, and the nucleotide sequence of the dsRNA is selected from any one of the following three groups of sequences:

group A: forward sequence (5 '-3'): CCAUGGAGCUGCCCUAUAA, respectively; reverse sequence (5 '-3'): UUAUAGGGCAGCUCCAUGG, respectively;

group B: forward sequence (5 '-3'): GGAAACGUUGAUAGGAGAU, respectively; reverse sequence (5 '-3'): AUCUCCUAUCAACGUUUCC, respectively;

group C: forward sequence (5 '-3'): CCUUCCUUCUGCCAUAUAU, respectively; reverse sequence (5 '-3'): AUAUAUGGCAGAAGGAAGG are provided.

The invention creatively designs a small RNA sequence (dsRNA) segment for interfering the expression of the gene according to the polyphenol oxidase gene sequence of Spodoptera frugiperda. The dsRNA enters the spodoptera frugiperda midbody, silences polyphenol oxidase PO gene of the Spodoptera frugiperda midbody, inhibits the expression of the Spodoptera frugiperda midbody, and specifically inhibits the humoral immunity mediated by the polyphenol oxidase in the Spodoptera frugiperda midbody, so that the rate of killing the Spodoptera frugiperda by nematode parasitism and the control effect are improved.

As some examples of the invention, the number of parasitic nematodes per 1L of formulation is: 8,000,000 IJs/L-12,000,000 IJs/L. IJs is English abbreviation of infested larva infested juveniles.

Or/and the dosage of the RNA fragment is as follows: 80 to 160 mu g.

Preferably, the amount of the RNA fragment is: 100 mu g-120 mu g; more preferably, it is 120. mu.g.

Preferably, in some embodiments of the present invention, the formulation further comprises 500ml to 800ml of the pest phagostimulant substrate per 1L of the formulation.

The pest phagostimulant matrix comprises 20-40 g of soybean meal, 2-5 g of yeast powder, 40-60 g of corn flour, 0.2-0.5 g of ascorbic acid and 1-3 g of soybean oil per 1L.

Preferably, in some embodiments of the present invention, the formulation further comprises a humectant for maintaining the vitality of nematodes, wherein the humectant comprises 2g to 5g of agar powder, 2g to 6g of α, α -trehalose, and 1g to 1.5g of an addition polymer (polyether) of polypropylene glycol and ethylene oxide per 1L of the formulation.

Preferably, in some embodiments of the present invention, the formulation further comprises symbiotic nutrients, wherein the symbiotic nutrients comprise peptone 2.5g to 5g and beef extract 2.5g to 6g per 1L of the formulation.

The preparation method of the parasitic nematode preparation comprises the following steps: preparing the components according to the proportion and mixing uniformly to obtain the product.

Preferably, the parasitic nematodes are used after being infected and propagated by 5-6 instar larvae of spodoptera frugiperda; more preferably, the parasitic rhabditis elegans after infection propagation is collected into distilled water by a White trap method and is used after being stored for a period of time at low temperature;

preferably, weighing the pest phagostimulant matrix, or/and the humectant, or/and the symbiotic bacteria nutrient according to the proportion, adding distilled water to a specified amount, sealing, and sterilizing for later use;

preferably, the RNA fragment is artificially synthesized. The synthesis method of the RNA fragment is the prior art.

The RNA fragment of the invention is from a RiboMAXExpressRNAiSystems kit of a promega company.

The invention relates to application of a parasitic pratylenchus xylophilus preparation in control of spodoptera frugiperda.

Compared with the prior art, the invention has the following beneficial effects:

the food pest phagostimulant substrate adopted by the invention can induce pests to actively approach the preparation and actively take target dsRNA; by adopting parasitic rhabditis, lepidoptera larvae such as spodoptera frugiperda and the like can be quickly infected and killed, and a large amount of lepidoptera larvae can be quickly propagated on the bodies of the lepidoptera larvae, so that the killing effect is further enhanced; the polyphenol oxidase PO plays the most important role in resisting (immunoreaction) when the nematode parasitizes insects, and the dsRNA interfering the expression of Spodoptera frugiperda PO gene is adopted in the invention, thereby inhibiting the humoral immune response. The invention creatively starts from various aspects of inducing, adding dsRNA to inhibit immune response, infection, saprophytic and the like, and effectively improves the killing effect of the nematodes on spodoptera frugiperda.

The preparation method is simple, simple and convenient to operate and easy to popularize and apply.

Detailed Description

The present invention is further illustrated by the following examples, which include, but are not limited to, the following examples.

Example 1

This example provides ingredient content tables and methods for making the parasitic nematode formulation of the present invention, as shown in table 1.

TABLE 1

The preparation method of the parasitic nematode preparation of the invention using the raw materials in table 1 is as follows:

step 1, propagation culture of parasitic rhabditis elegans:

infecting and propagating the parasitic nematodes by 5-6 instar larvae of Spodoptera frugiperda; collecting the bred third instar larvae (IJs) of parasitic nematodes in distilled water by White trap method, storing in refrigerator at 4 deg.C for 2 weeks, and using;

step 2, weighing the raw material components in proportion:

and (3) concentrating the parasitic rhabditis elegans suspension collected in the step (1) by using a static precipitation method, adding a dsRNA sample, adding distilled water to supplement the dsRNA to 1000mL, uniformly mixing at room temperature, and storing in a refrigerator at 4 ℃ for later use.

The specific sequence of the dsRNA in this example is:

group A: the forward sequence (5'-3') is shown in seq. id 1: CCAUGGAGCUGCCCUAUAA, respectively; the reverse sequence (5'-3') is shown in SEQ. ID2: UUAUAGGGCAGCUCCAUGG, respectively;

group B: the forward sequence (5'-3') is shown in seq. id 3: GGAAACGUUGAUAGGAGAU, respectively; the reverse sequence (5'-3') is shown in SEQ. ID4: AUCUCCUAUCAACGUUUCC, respectively;

group C: the forward sequence (5'-3') is shown in seq. id 5: CCUUCCUUCUGCCAUAUAU, respectively; the reverse sequence (5'-3') is shown in SEQ. ID6: AUAUAUGGCAGAAGGAAGG are provided.

Example 2

This example provides the ingredient content scale for the parasitic nematode formulation of the present invention, as shown in table 2.

TABLE 2

The preparation method of the parasitic nematode preparation of the invention using the raw materials in table 2 is as follows:

step 1, propagation culture of parasitic rhabditis elegans:

infecting and propagating the parasitic nematodes by 5-6 instar larvae of Spodoptera frugiperda; collecting parasitic third instar larva of caenorhabditis elegans (IJs) in distilled water by White trap method, storing in refrigerator at 4 deg.C for 2 weeks;

step 2, weighing the pest phagostimulant matrix, the humectant and the symbiotic bacteria nutrients according to the proportion into the same container, adding distilled water to supplement the distilled water to 900mL, uniformly mixing, then filling into a triangular flask, covering a sealing film, then putting into an autoclave, sterilizing (melting) for 20-40 min under the conditions that the pressure is 1.0MPa and the temperature is 121 ℃, and taking out the obtained product for later use after cooling;

and 3, concentrating the parasitic rhabditis elegans suspension collected in the step 1 to 100mL (the number of the included nematodes is unchanged) by using a static precipitation method, adding the dsRNA sample and the product obtained in the step 2, uniformly mixing at room temperature, and storing in a refrigerator at 4 ℃ for later use.

Example 3

This example provides the use of the formulations of the invention to control spodoptera frugiperda.

The concrete application is as follows:

1. indoor biological activity determination:

the area is about 10cm²Sterilizing the surface of the corn leaf with 75% alcohol, naturally drying, and culturing in a culture dish (diameter 9cm) filled with 2 layers of filter paper. Each dish was inoculated with 10 spodoptera frugiperda 5 instar larvae. Diluting different rhabditis elegans preparations by 10 times, adding onto filter paper of culture dish, adding 0.25ml rhabditis elegans preparation (combined with diluted preparation 2.5 ml/dish) into each culture dish, repeating for 3 times, and treating with clear water as blank control. And observing and counting the number of dead insects treated after 8h, 16h, 24h and 32h of spodoptera exigua larvae respectively.

Survival rate (survival number/number of test insects) × 100%; corrected mortality ═ treatment mortality-control mortality)/(1-control mortality) ] × 100%;

experimental treatment 1 group: parasitic caenorhabditis elegans preparation a, prepared according to the procedure described in example 1, was prepared using the raw material ratios listed in table 1, numbered 3, in example 1.

Experimental treatment 2 groups: parasitic caenorhabditis elegans preparation B, prepared according to the procedure described in example 1, using the raw material ratios listed in table 1, numbered 7, in example 1.

Experimental treatment 3 groups: parasitic caenorhabditis elegans preparation C prepared according to the procedure described in example 1, using the raw material ratios listed in Table 1, numbered 11 in example 1

Experimental treatment 4 groups: parasitic caenorhabditis elegans preparation D, prepared according to the procedure described in example 1, using the raw material ratios listed in table 2, numbered 3, in example 2.

Experimental treatment 5 groups: parasitic caenorhabditis elegans preparation E, prepared according to the procedure described in example 1, using the raw material ratios listed in table 2, numbered 13, in example 2.

Experimental treatment 6 groups: parasitic caenorhabditis elegans preparation F, prepared according to the procedure described in example 1, was prepared using the raw material ratios listed in table 2, number 14, in example 2.

Example 7 group: parasitic caenorhabditis elegans preparation G, prepared according to the procedure described in example 1, was prepared using the raw material ratios listed in table 2, number 15, in example 2.

Experimental treatment 8 groups: only parasitic caenorhabditis elegans, no RNA fragments were administered. Compared to the parasitic caenorhabditis elegans preparation a of experimental treatment 1, no dsRNA was present, and the rest was identical.

Blank control group: and (5) treating with clear water.

The results are shown in Table 1.

TABLE 1

2. And (3) field control effect determination:

the field control test is carried out in Zea mays test base of Yongtaizhen county of Yangjiang county in Sichuan for 8 treatments, each treatment is set to be 3 times, 24 cells are counted, and each cell is 50m²(10m by 5m) cells are distributed in random blocks.In the small trumpet period of the corn, the parasitic rhabditis elegans preparation is diluted by 10 times, is applied to soil with the diameter of 20 cm-30 cm and the depth of 5 cm-10 cm in the rhizosphere range of the corn in a circle mode by using an injector (without a needle head) with the capacity of 30 ml-100 ml, is covered by surface soil, and is poured into proper water for moisture preservation. Thus, the larval pupation characteristic of spodoptera frugiperda is mainly utilized: the larva pupates in the depth of the soil, and the depth is 2 cm-8 cm. Wherein the depth is influenced by the texture, temperature and humidity of the soil, and the pupal stage is 7-37 days. The higher the humidity, the slower the pupation and the aging time of the larvae are, and the larvae are convenient for the parasitic larvae of the nematodes. The dosage of the parasitic nematode preparation is 6L/mu to 10L/mu (12 to 20m10 times of diluent L/hole corn), 50L to 80L (10 to 16 ml/hole corn) is watered per mu of corn, and the humidity is kept. The survey used a 5-point sampling method. Each test plot investigates 15 maize plants. The population base number of insects is investigated before application, the age of insects is mainly 3 years, and part of insects is 4-6 years. The degree of new leaf damage of corn was investigated and classified on the 10 th day after the administration. Grading standard: grade 0, new leaves are not harmed by the insect; grade 1, the new leaves have smaller lesion spots; grade 3, connecting the harmful spots into pieces and forming transparent films; grade 5, which is a defect that causes new leaves to be notched; grade 7, the damage causes the new leaves to have larger and more gaps; grade 9, causing new leaf rupture. And simultaneously, the number of the Spodoptera frugiperda living insects on the corn samples is stripped and recorded. And calculating the damage index, the leaf protection effect and the insect population prevention effect. The effect of each agent treatment on crops and on non-target organisms was observed during the experiment.

The damage index [ Σ (number of damaged plants at each stage × relative damaged-stage representative value)/(total investigated plants × highest-stage value) ] × 100

Leaf retention (%) [ (control zone injury index-treatment zone injury index)/control zone injury index ] × 100

Insect control effect (%) [1- (number of live insects after drug treatment x number of live insects before drug control region)/(number of live insects before drug treatment x number of live insects after drug control region) ] × 100

Blank control group: and (5) treating with clear water.

The results are shown in Table 2.

TABLE 2

Note: in the table, the letters a, b, c, d, e, f, g and h are different and represent significant differences, and P is less than 0.05.

The result shows that the preparation has good control effect on Spodoptera frugiperda.

The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.

SEQUENCE LISTING

<110> institute of plant protection of academy of agricultural sciences of Sichuan province

<120> a parasitic nematode preparation, preparation method and application

<130> 20200320

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<170> PatentIn version 3.5

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Claims

1. A parasitic nematode formulation comprising: parasitic nematodes, RNA fragments that interfere with the expression of the polyphenol oxidase PO gene of spodoptera frugiperda;

wherein the parasitic nematodes are of the genus Oscheius sp, and are isolated and purified from Spodoptera frugiperda in pupal stage, which naturally dies in soil;

wherein the RNA fragment is dsRNA, and the nucleotide sequence of the dsRNA is selected from any one of the following three groups of sequences:

group A: forward sequence: CCAUGGAGCUGCCCUAUAA, respectively; reverse sequence: UUAUAGGGCAGCUCCAUGG, respectively;

group B: forward sequence: GGAAACGUUGAUAGGAGAU, respectively; reverse sequence: AUCUCCUAUCAACGUUUCC, respectively;

group C: forward sequence: CCUUCCUUCUGCCAUAUAU, respectively; reverse sequence: AUAUAUGGCAGAAGGAAGG, respectively;

wherein, in each 1L of the preparation, the number of the parasitic nematodes is as follows: 8,000,000 IJs/L-12,000,000 IJs/L, wherein the dosage of the RNA fragment is as follows: 80 to 160 mu g.

2. The parasitic nematode formulation of claim 1 further comprising 600ml to 900ml of pest phagostimulant base per 1L of said formulation.

3. The parasitic pratylenchus praecox preparation according to claim 2, wherein the pest phagostimulant substrate comprises 20 g-40 g of soybean meal, 2 g-5 g of yeast powder, 40 g-60 g of corn meal, 0.2 g-0.5 g of ascorbic acid and 1 g-3 g of soybean oil per 1L.

4. The parasitic nematode formulation of claim 1, further comprising a humectant for maintaining nematode viability, wherein said humectant comprises agar powder 2-5 g, alpha-trehalose 2-6 g, and an addition polymer of polypropylene glycol and ethylene oxide 1-1.5 g per 1L of the formulation.

5. The parasitic nematodes according to claim 1, wherein said preparation further comprises symbiotic nutrients, and each 1L of said preparation comprises peptone 2.5-5 g and beef extract 2.5-6 g.

6. A process for the preparation of a parasitic nematodes formulation according to any one of claims 1 to 5, comprising the steps of: preparing the components according to the proportion and mixing uniformly to obtain the product.

7. The method for producing a parasitic nematodes according to claim 6, wherein said parasitic nematodes are used after being infected and propagated by 5-6 instar larvae of Spodoptera frugiperda.

8. The method of claim 7, wherein the parasitic nematodes after being infested and propagated are collected in distilled water by White trap method and stored at low temperature for a certain period of time.

9. The method for preparing a parasitic nematodes according to claim 6, wherein the pest phagostimulant matrix, or/and the humectant, or/and the symbiotic bacteria nutrient are weighed in proportion, distilled water is added to a specified amount, and the mixture is sealed and sterilized for later use.

10. The method of any one of claims 6-9, wherein said RNA fragment is artificially synthesized.

11. Use of a parasitic gracilis formulation according to any of claims 1 to 5 for the control of spodoptera frugiperda.