阅读说明：本技术 一种寄生性小杆线虫制剂及制备方法和应用 (Parasitic nematode preparation, preparation method and application ) 是由 李星月 张鸿 杨武云 符慧娟 陈鹏 李其勇 易军 陆文壹 于 2020-04-23 设计创作，主要内容包括：本发明公开了一种寄生性小杆线虫制剂及制备方法和应用,属于生物农药技术领域,解决了现有技术中生防速效性不高的问题,不能高效、快速地杀灭草地贪夜蛾的问题。本发明的寄生性小杆线虫制剂包括：寄生性小杆线虫,干扰草地贪夜蛾的多酚氧化酶PO基因表达的RNA片段；还包括害虫诱食剂基质、线虫保湿剂、线虫共生菌养分。本发明创造性地从引诱、侵染、腐生、加dsRNA抑制害虫免疫反应等多方面入手,共同作用,有效地提高了线虫对草地贪夜蛾的杀灭效果。(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.)

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

2. The parasitic gracilis formulation as claimed in claim 1, wherein said parasitic gracilis is of the genus Oscheius sp, an insect parasitic gracilis isolated and purified from natural dead soil Spodoptera frugiperda.

3. A parasitic nematode formulation as claimed in claim 1 or claim 2 wherein said RNA fragments are dsRNA having a nucleotide sequence 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 are provided.

4. A parasitic gracilis formulation according to claim 1, wherein the number of parasitic gracilis per 1L of formulation is: 8,000,000IJs/L to 12,000,000IJs/L,

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

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

6. The parasitic pratylenchus praecox preparation according to claim 5, 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.

7. 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.

8. 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.

9. A process for the preparation of a parasitic nematode formulation as claimed in any one of claims 1 to 8, comprising the steps of: 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.

10. Use of a parasitic gracilis formulation according to any of claims 1 to 9 for the control of spodoptera frugiperda.

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.

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.