Use of ascaridole in the preparation of a preparation for promoting the recovery development of S. carpocapsae All infected nematodes

Abstract

The application discloses application of ascaridole in preparation of a preparation for promoting recovery development of Steinernema carpocapsae All infection period nematodes. The application finds that, compared with symbiotic bacteria liquid supernatant as a control, ascaridole can significantly promote recovery development of S. carpocapsae All infection period nematodes. Therefore, in the process of mass culture of nematodes, ascaridole can be used to induce recovery development of S. carpocapsae All infection period nematodes, and core technology is provided for commercialized production of S. carpocapsae All nematodes with cost advantages.

Description

Technical fields:

[0001] This invention belongs to the field of biological control, specifically relating to the application of ascaroside in the preparation of a formulation that promotes the recovery and development of nematodes during the infection period of S. carpocapsae All. Background technology:

[0002] Insect pathogenic nematodes of the families Steinernematidae and Heterorhabditidae actively search for host insects in their third instar infective stage. They are safe for non-target organisms and the environment, can be mass-produced, and are promising natural enemies already widely used to control pests in agriculture, forestry, pasture, floriculture, and sanitation. Insect pathogenic nematodes, together with their symbiotic bacteria, kill host insects. They enter the insect in their infective stage with food or through natural openings (such as the anus, spiracles), or intersegmental membranes, subsequently releasing symbiotic bacteria of the genera *Xenorhabdus* (symbiotic with *Steinernema*) or *Photorhabdus* (symbiotic with *Heterorhabditis*) carried in their intestinal lumen. The toxins (toxic agents) secreted by the nematodes and symbiotic bacteria cause the insect's death, and the nematodes then use the host's nutrients to reproduce. *S. carpocapsae* All nematodes have shown good control effects against a variety of pests.

[0003] When infective nematodes infect a suitable insect host or when suitable symbiotic bacteria are introduced into an in vitro nematode culture system, the infective nematodes will begin to feed and resume growth and development; this process is called development recovery. Signaling substances contained in insect hemolymph can induce this recovery in infective nematodes. Symbiotic bacteria cultured in artificial media can also produce such signaling substances to induce recovery in infective larvae. Some symbiotic strains of the genus *Photorhabdus* can induce the development of *S. carpocapsae* nematodes across species boundaries, but signaling substances produced by bacteria of the genus *Xenorhabdus* cannot induce the development of *Heterobacter nematodes*. This signaling substance that induces recovery in infective nematodes is named the food signal.

[0004] The inability of a single symbiotic bacterium to induce recovery in all non-specific symbiotic nematodes during their infective stage suggests that different species of symbiotic bacteria produce different food signals. Isopropylstilbene is considered a signaling molecule that induces development in infective nematodes from *Photorhabdus* bacteria. However, no other compounds have been reported to induce recovery in nematodes infected with entomopathogenic pathogens. Ascarosides, a ubiquitous nematode signaling molecule, play an important role in regulating nematode aggregation, avoidance, mating, Dauer formation, and dispersal behaviors. However, whether these ascarosides can induce recovery in nematodes infected with entomopathogenic pathogens remains unreported. Summary of the Invention:

[0005] The first objective of this invention is to provide the use of ascaroside in the preparation of formulations that promote the recovery and development of nematodes infected with S. carpocapsae All.

[0006] This invention has experimentally demonstrated that ascaridin can induce the recovery and development of *S. carpocapsae All* nematodes during their infective stage. Therefore, this invention provides the application of ascaridin in the preparation of formulations that promote the recovery and development of *S. carpocapsae All* nematodes during their infective stage, wherein the ascaridin has the structural formula shown in any one of Formula 1:

[0007]

[0008]

[0009] Preferably, the ascaroside is ascr#1, ascr#2, ascr#3, ascr#5, ascr#7, ascr#8, ascr#10, ascr#11 or ascr#12.

[0010] The second objective of this invention is to provide a method for inducing the recovery and development of S. carpocapsae All infected nematodes, which involves adding ascaroside to a culture medium containing S. carpocapsae All infected nematodes to induce the recovery and development of infected nematodes.

[0011] The aforementioned ascaroside has the structural formula shown in any of Formula 1.

[0012] Preferably, the ascaroside is ascr#1, ascr#2, ascr#3, ascr#5, ascr#7, ascr#8, ascr#10, ascr#11 or ascr#12.

[0013] Preferably, when ascaroside is ASICS#1, its concentration in the culture medium is 0.04 pM-0.04 nM; or when ascaroside is ASICS#2, its concentration in the culture medium is 0.04 nM; or when ascaroside is ASICS#3, its concentration in the culture medium is 0.04 nM; or when ascaroside is ASICS#5, its concentration in the culture medium is 0.04 pM-0.04 nM; or when ascaroside is ASICS#6, its concentration in the culture medium is 0.04 nM; or when ascaroside... When it is ascr#7, its concentration in the culture medium is 0.04 pM-0.04 nM; or when ascaroside is ascr#8, its concentration in the culture medium is 0.04 nM; or when ascaroside is ascr#10, its concentration in the culture medium is 0.04 pM-0.04 μM; or when ascaroside is ascr#11, its concentration in the culture medium is 0.04 pM-0.04 nM; or when ascaroside is ascr#12, its concentration in the culture medium is 0.04 pM.

[0014] This invention reveals that, compared to the supernatant of the symbiotic bacterial suspension used as a control, ascaroside significantly induces the recovery and development of infected S. carpocapsae All nematodes, with the infected nematodes exhibiting a good recovery rate. Therefore, using ascaroside to induce the recovery and development of infected S. carpocapsae All nematodes will provide a core technology for the commercial production of cost-effective S. carpocapsae All nematodes. Detailed implementation method:

[0015] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0016] Example 1:

[0017] I. Experimental Materials

[0018] 1. Ascaroside includes 11 synthetically produced ascarosides, designated as ASCII#1, ASCII#2, ASCII#3, ASCII#5, ASCII#6, ASCII#7, ASCII#8, ASCII#9, ASCII#10, ASCII#11, and ASCII#12, with their structural formulas listed below:

[0019]

[0020] Eleven ascarosides were dissolved in sterile PBS to a concentration of 0.5 mM, then diluted to a certain concentration and added to the supernatant or culture medium of symbiotic bacteria in a certain amount, so that their concentrations in the supernatant or culture medium of symbiotic bacteria were 0.04 μM, 0.04 nM and 0.04 pM, respectively.

[0021] 2. Single-strain culture of S. carpocapsae All: After washing S. carpocapsae All nematodes cultured on conventional solid medium, add streptomycin sulfate solution (concentration: 100 ppm) and incubate overnight at 25°C. Wash three times with sterile PBS buffer, and reconstitute the precipitate with sterile PBS to a concentration of 50 infectious stage nematodes (IJs) per 10 μl (5000 IJs / mL). Pour this into a 250 ml Erlenmeyer flask (containing 50 ml of nematode suspension) and incubate at 9-13°C. It is best used within 7 days.

[0022] 3. Preparation of symbiotic bacteria of Xenorhabdus nematophila All: The symbiotic bacteria of S. carpocapsae All (X. nematophila All) were cultured in sterile nutrient broth (sterilized at 121℃ for 30 min) at 25℃ and 120 rpm in a shaker for 3 days; centrifuged at 12000 rpm and 4℃ for 10 min; the supernatant obtained was filtered through a bacterial filter (0.22 μm) on a clean bench and used for IJ recovery assay.

[0023] 4. Streptomycin sulfate: 100 mg / mL.

[0024] 5. Sterile 1×PBS buffer.

[0025] 6. Sterile ultrapure water.

[0026] 7. 0.2μm filter.

[0027] II. Experimental Methods

[0028] In disposable 48-well cell culture plates, 0.1 ml of supernatant from *X. nematophila* All symbiotic bacteria was added to each well; 10 μl of streptomycin sulfate (100 mg / ml stock solution, diluted 10 times) was added to each well; approximately 50 IJs were added in 10 μl increments; then 10 μl of different concentrations of ascaridin were added to each well, resulting in concentrations of 0.04 μM, 0.04 nM, and 0.04 pM in the symbiotic bacterial supernatant or culture medium, respectively. Controls included equal volumes of supernatant containing 10 mg / ml streptomycin sulfate, sterile PBS, sterile PBS containing 100 mg / ml streptomycin sulfate, and 0.1% DMSO. Each treatment was replicated in 4 wells.

[0029] After sealing the well plates with sealing film, they were placed in sterile, covered plastic boxes and incubated in the dark at 25°C. On the 6th day, the number of recovered nematodes in each well was observed and counted using a dissecting microscope in a clean bench. The recovery rate was calculated as follows: recovery rate = number of recovered nematodes / total number of nematodes × 100%.

[0030] The results are shown in Table 1:

[0031] Table 1: Effect of ascaroside on recovery rate of S. carpocapsae All during infection (6 days)

[0032]

[0033] Note: PBS represents sterile PBS with added streptomycin sulfate; PBS (without) represents sterile PBS without added streptomycin sulfate. Uppercase letters in the table indicate significant differences between different concentrations under the same treatment; lowercase letters indicate significant differences between different treatments at the same concentration.

[0034] As shown in Table 1, all 11 ascarosides have a good effect on promoting the recovery and development of nematodes in the infecting stage of S. carpocapsae All, especially ascr#7, which has a very good effect on promoting the recovery of nematodes in the infecting stage.

Claims

1. A method for inducing the recovery and development of *S. carpocapsae All* nematodes during their infective stage, characterized in that, Ascaroside was added to the culture medium containing the corresponding symbiotic bacteria of S. carpocapsae All infected nematodes, and the S. carpocapsae All infected nematodes were cultured to induce their recovery and development. The aforementioned ascaroside has the structural formula shown in any of Formula 1; ascr#1 ascr#5 ascr#7 ascr#10 ascr#11 Formula 1.

2. The method according to claim 1, characterized in that, When ascaroside is ASSR#1, its concentration in the culture medium is 0.04 pM-0.04 nM; or when ascaroside is ASSR#5, its concentration in the culture medium is 0.04 pM-0.04 nM; or when ascaroside is ASSR#7, its concentration in the culture medium is 0.04 pM-0.04 nM; or when ascaroside is ASSR#10, its concentration in the culture medium is 0.04 pM-0.04 μM; or when ascaroside is ASSR#11, its concentration in the culture medium is 0.04 pM-0.04 nM.

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