阅读说明：本技术 蛔甙在制备促进H.indica LN2感染期线虫恢复发育制剂中的应用 (Application of ascaroside in preparation of preparation for promoting recovery and development of nematodes in H.indica LN2 infection stage ) 是由 谷星慧 王杰 屈玲 崔永和 曹莉 韩日畴 黄智华 李江舟 周文兵 杨晓俊 于 2021-08-19 设计创作，主要内容包括：本发明公开了蛔甙在制备促进H.indica LN2感染期线虫恢复发育制剂中的应用。H.indica LN2线虫在韭菜迟眼覃蚊Bradysia odoriphaga(简称韭蛆)绿色防控中发挥重要作用。本发明发现,相比于作为对照的共生菌菌液上清液,蛔甙能够显著地促进H.indica LN2感染期线虫恢复发育,可以使用蛔甙诱导H.indica LN2感染期线虫恢复发育。因此,在线虫大量培养过程中,将蛔甙添加到业已加入含相应共生细菌的H.indica LN2感染期线虫的培养基中,诱导感染期线虫恢复发育和生长。本发明为商品化生产具有成本优势的H.indica LN2线虫提供了核心技术。(The invention discloses application of ascaroside in preparation of a preparation for promoting recovery and development of nematodes in an H.indica LN2 infection stage. The indica LN2 nematode plays an important role in the green control of the bradycardia mosquito odoriphaga (shortly called leek maggot). The ascaroside can obviously promote the recovery and development of the nematodes in the H.indica LN2 infection stage and can induce the recovery and development of the nematodes in the H.indica LN2 infection stage by using the ascaroside compared with the supernatant of the symbiotic bacteria liquid serving as a control. Therefore, during the mass culture of nematodes, ascaroside is added to the culture medium of H.indica LN2 infected phase nematodes, which have been added with the corresponding commensal bacteria, to induce the restoration of development and growth of the infected phase nematodes. The invention provides a core technology for commercial production of the H.indica LN2 nematode with cost advantage.)

1. The application of ascaroside in preparing a preparation for promoting the recovery and development of nematodes in the Heterorhabditis indica LN2 infection stage, wherein the ascaroside has a structural formula shown in any one of ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9, ascr #10 and ascr #11ascr #12 in the following formula:

2. the use of claim 1 wherein said ascaroside is ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9 or ascr #10 of said formula.

3. A method for inducing restoration of development of nematodes in the stage of H.indica LN2 infection, which comprises adding ascaroside to a culture medium containing H.indica LN2 of commensal bacteria to induce restoration of development and growth of nematodes in the stage of infection;

the structural formula of the ascaroside is shown as any one of the following formulas;

4. the method of claim 3 wherein said ascaroside is present in the formula ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9 or ascr # 10.

5. The method of claim 3 or 4, wherein the concentration of ascaroside in the culture medium is from 0.04pM to 0.04. mu.M.

Technical Field

The invention belongs to the field of biological control, and particularly relates to application of ascaroside in preparation of a preparation for promoting recovery and development of nematodes in a Heterorhabditis indica LN2 infection stage.

Background

The entomopathogenic Steinernema and Heterorhabditis nematodes actively search host insects in the 3-year infection period, are safe to non-target organisms and environment, can be produced in large scale, are natural enemy biological preparations with huge application potential, and are widely used for controlling pests in the fields of agriculture and forestry, pasture, flowers, sanitation and the like. Entomopathogenic nematodes enter the insect body in the infected state with the host food or from the natural opening (e.g. anus, stoma), internode membranes of the insect, and subsequently release symbiotic bacteria of the genus Xenorhabdus (symbiotic with Steinernema, a. Steinernema) or Photorhabdus (symbiotic with Heterorhabditis, a. Heterorhabditis) carried in the intestinal lumen. The entomopathogenic nematodes act with symbiotic bacteria carried in the host to kill host insects, and then use the nutrients in the host to breed offspring. The indica LN2 nematode plays an important role in the green control of the bradycardia mosquito odoriphaga (shortly called leek maggot).

When the nematode in the infection phase infects a suitable host insect or when suitable commensal bacteria are added to the nematode in vitro culture system, the nematode in the infection phase will begin to enter a feeding state and resume Development, a process called Development recovery. The signal substance contained in the insect haemolymph has the function of inducing the recovery of the nematode in the infection period. Symbiotic bacteria cultured in artificial media can also produce such information substances to induce recovery of infected stage larvae. This signal substance that induces recovery of nematodes in the infected phase is termed the food signal. In the process of isolated culture, the main steps are to insert symbiotic bacteria into a nematode culture medium and then add pollution-free infection-stage nematode seeds. The recovery ratio of the larvae in the infection stage influences the process flow and the cost of nematode culture. Therefore, how to improve the recovery ratio of the nematodes in the inoculation infection phase is the core technology of commercial culture of the entomopathogenic nematodes.

One symbiont failed to recover all non-specific symbiotic infective stage nematodes, indicating that different species of symbiotic bacteria produce different food signals. Isopropylstilbene is known as a signal substance for inducing the development of nematodes in the infection phase from Photorhabdus bacteria. However, no report is found about other compounds inducing the restoration of the development of nematodes in the entomopathogenic infection stage. Ascarosides information substances commonly existing in linear animals play an important role in regulating behaviors of aggregation, avoidance, mating, formation of a Duolur (Dauer), diffusion and propagation of nematodes and the like. However, whether the ascarosides can induce the recovery and development of nematodes in the entomopathogenic infection stage is not reported yet.

Disclosure of Invention

The first purpose of the invention is to provide application of ascaroside in preparation of a preparation for promoting recovery and development of nematodes in an H.indica LN2 infection stage.

Experiments show that ascaroside can induce nematodes in the H.indica LN2 infection stage to recover development. Therefore, the invention provides application of ascaroside in preparing a preparation for promoting recovery and development of nematodes in the H.indica LN2 infection stage, wherein the structural formula of the ascaroside is any one of ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9, ascr #10 and ascr #11ascr #12 in formula 1.

Preferably, the ascaroside is ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9 or ascr #10 of formula 1.

The second purpose of the invention is to provide a method for inducing the recovery development of the nematodes in the H.indica LN2 infection stage, which is to add ascaroside to a culture medium of the nematodes in the H.indica LN2 infection stage, which contains corresponding symbiotic bacteria, to induce the recovery development and growth of the nematodes in the infection stage.

The ascaroside has a structural formula shown in formula 1, wherein the structural formula is any one of ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9, ascr #10 and ascr #11as cr # 12.

Preferably, the ascaroside is ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9 or ascr #10 of formula 1.

Preferably, the concentration of ascaroside in the culture medium is 0.04pM to 0.04. mu.M.

Compared with the supernatant of symbiotic bacteria liquid serving as a control, ascaroside can remarkably induce the nematodes in the H.indica LN2 infection stage to recover and develop, and the nematodes in the infection stage have good recovery rate. Therefore, the ascaroside is used for inducing the recovery and development of the nematodes in the H.indica LN2 infection stage, so that a core technology is provided for commercial production of the H.indica LN2 nematodes with cost advantages and a supporting function is exerted.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

First, experimental material

1. The ascarosides comprise 11 kinds of artificially synthesized ascarosides with the code numbers of ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7, ascr #8, ascr #9, ascr #10, ascr #11 and ascr #12 respectively, and the structural formulas are listed as follows:

respectively dissolving 11 ascarosides in the above formula to 0.04mM with sterile PBS, diluting to a certain concentration, adding into symbiotic bacteria supernatant or culture medium at a certain amount to make the concentration in symbiotic bacteria supernatant or culture medium respectively 0.04 μ M, 0.04nM, 0.04 pM.

2. Symbiotic bacteria monoculture h.indica LN 2: after washing nematodes cultured in a conventional solid medium, adding a streptomycin sulfate solution (action concentration: 100ppm) to the solution at 25 ℃ overnight, washing the solution three times with sterile PBS buffer, preparing the precipitate into 10. mu.l of a solution containing 50-stage nematodes (IJs) (5000IJs/mL) in sterile PBS, placing the solution in a 250mL triangular flask (containing 50mL of nematode suspension), and placing the flask at 9-13 ℃. Preferably within 7 days.

3. Preparation of Photorhabdus luminescens LN2 symbiotic bacteria: culturing symbiotic bacteria of H.indica LN2 nematodes (P.luminescens LN2) in sterile nutrient broth (sterilized at 121 ℃ for 30min) in a shaker at 25 ℃ and 120rpm for 3 days; centrifuging at 12000rpm at 4 deg.C for 10 min; the obtained supernatant was filtered with a bacterial filter (0.22 μm) on a clean bench for IJ recovery (IJ recovery) assay.

4. Streptomycin sulfate: 100 mg/mL.

5. Sterile 1 × PBS buffer.

6. Sterile ultrapure water.

7. 0.2 μm filter.

Second, Experimental methods

Respectively adding 0.1ml of supernatant of P.luminescens LN2 symbiotic bacteria into a disposable 48-hole cell culture plate; mu.l streptomycin sulfate (stock solution is 100mg/ml, diluted 10 times for use) is added into each hole; about 50 nematodes in the infection phase were added in an amount of 10. mu.l; then adding 10 μ l ascaroside with different concentrations to make its concentration in symbiotic bacteria supernatant or culture medium 0.04 μ M, 0.04nM, 0.04pM respectively. As a control, the supernatant to which streptomycin sulfate was added at an equal volume of 10mg/ml, sterile PBS to which streptomycin sulfate was added at an equal volume of 10mg/ml, and DMSO at a mass fraction of 0.1% were added. Each treatment was set up with 4 well replicates.

Sealing the pore plate with sealing film, placing into a sterile plastic box with cover, and culturing at 25 deg.C in dark. The number of recovered nematodes in the infection phase (i.e., the number of nematodes recovered to develop) in each well was counted by observation with a dissecting mirror on day 6, and the recovery rate was calculated as recovery number/total number × 100%.

The results are shown in Table 1.

Table 1: effect of ascaroside on nematode recovery in the stage of H.indica LN2 infection (day 6)

Note: PBS represents sterile PBS and streptomycin sulfate is added; PBS (none) represents sterile PBS without streptomycin sulfate added. The capital letters in the table indicate significant differences between different concentrations under the same treatment; lower case letters indicate significant differences between different treatments at the same concentration.

As can be seen from table 1, all of the 11 ascarosides have good effects of promoting recovery and development of h.indica LN2 infected nematodes, and particularly, ascr #1, ascr #2, ascr #3, ascr #5, ascr #6, ascr #7 and ascr #8 among them have very good effects of promoting recovery of infected nematodes.