Efficient method for mass rearing of entomopathogenic nematodes

Abstract

The application belongs to the field of biological agents, and particularly relates to a high-efficiency trapping method of insect pathogenic nematodes, which adopts Galleria mellonella treated by immune barrier weakening and cocooning inhibition in sequence for trapping and collecting; and specifically comprises the following steps: S1, collecting fresh soil samples from the wild; S2, inoculating biocontrol bacteria into the soil samples, spraying pheromones and moisturizers, and mixing; S3, placing the Galleria mellonella treated in the above step into a trapping agent ball, and filling the soil samples treated in the step S2 into the trapping agent ball and compacting; S4, burying the trapping agent ball in the remaining soil samples, and after completion, placing the soil samples in a sample bag in a thermostat with sufficient oxygen for trapping, the application can improve the success rate of nematode infection, is beneficial to further propagation, collection and application of insect pathogenic nematode resources, and has the advantages of simple method and simple operation.

Description

Technical Field

[0001] This invention belongs to the field of biological agents, specifically relating to a highly efficient method for attracting entomopathogenic nematodes. Background Technology

[0002] Entomopathogenic nematodes (EPNs) are microbial insecticides that possess the dual characteristics of being both natural enemy insects and pathogenic microorganisms. Entomopathogenic nematodes have advantages such as a wide host range, safety for humans, animals, and the environment, ease of large-scale cultivation, convenient use, and recyclability in the environment. They are commonly used for the control of pests in fruit trees, vegetables, and lawns.

[0003] Currently, the collection of entomopathogenic nematodes mainly adopts traditional methods, which involve collecting soil samples from the field and bringing them back indoors, then using the large wax moth (Galleria mellonella) trapping method to separate and collect the nematodes.

[0004] The entomopathogenic nematodes Steinernema spp. and Heterorhabditis spp. in fresh soil carry highly pathogenic symbiotic bacteria and can actively seek out insect hosts (great wax moths). Therefore, the infected and dead great wax moths can be picked out and collected separately for propagation to obtain entomopathogenic nematode resources.

[0005] While conventional methods are effective and can collect some entomopathogenic nematode strains highly pathogenic to the wax moth, the significant differences in pathogenicity among different entomopathogenic nematode species or strains in fresh soil mean that many nematode resources with weak pathogenicity and small populations fail to be collected, leading to resource waste. Furthermore, because fresh soil is a complex micro-ecosystem, the successful infection of the wax moth host by entomopathogenic nematodes is also related to other soil microorganisms. For example, an excessive population of fungi (entomopathogenic fungi) and bacteria (entomopathogenic bacteria) that infect the wax moth can interfere with and reduce the infection probability of entomopathogenic nematodes. The natural enemies of entomopathogenic nematodes in fresh soil are mainly fungi that capture nematodes; an excess of these fungi will preferentially infect the insect host, leading to nematode trapping failure.

[0006] In addition, the indoor trapping process also affects the success of entomopathogenic nematode attractants. Currently, the conventional setup involves burying a stainless steel porous spherical mesh in fresh soil samples, then spraying water to maintain moisture. Often, to retain moisture, the soil sample bags are sealed and placed in a dark environment for 7-20 days. This creates a stuffy, poorly ventilated environment, leading to the growth and decay of the large wax moth due to the presence of corrosive bacteria, pathogenic fungi, and other microorganisms. Furthermore, the sealed bags result in low oxygen levels, reducing nematode activity or causing death. Therefore, the trapping effect is ultimately poor, with a very low nematode detection rate (less than 10%), resulting in unsatisfactory collection of entomopathogenic nematode resources. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a highly efficient method for attracting entomopathogenic nematodes, which can improve the success rate of nematode infection compared with the prior art.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A highly efficient method for attracting entomopathogenic nematodes involves using large wax moths that have undergone sequential treatments to weaken their immune barrier and inhibit cocooning for trapping and collection.

[0010] Specifically, the following steps are included:

[0011] S1. Collect fresh soil samples from the field;

[0012] S2. Inoculate the soil sample with biocontrol bacteria, and simultaneously spray with pheromone and moisturizer, and mix well;

[0013] S3. Place the treated large wax moth into the attractant ball, then fill the attractant ball with the soil sample treated in step S2, and press and compact it;

[0014] S4. Bury the attractant balls in the remaining soil samples. After completion, place the soil samples in a sample bag and put them in a constant temperature chamber with sufficient oxygen for attraction.

[0015] Furthermore, the immune barrier weakening treatment involves feeding immunosuppressants to large wax moths that have been normally cultured to the 5th age.

[0016] Furthermore, the immunosuppressant includes resveratrol oxidase (C... 14 H 12 O4), immunosuppressants are added to the feed at a rate of 10-15 mg / kg for 3-5 days.

[0017] Furthermore, the cocoon-inhibiting treatment involves placing the large wax moth at 8-12℃ for 24 hours.

[0018] Furthermore, in step S2, the biocontrol bacteria include Bacillus subtilis and Pseudomonas pulveratum, with the inoculation amount of Bacillus subtilis being 10-15 g / kg of fresh soil and the inoculation amount of Pseudomonas pulveratum being 5-8 g / kg of fresh soil.

[0019] And / or, the information hormone is ascaroside, and the spraying dosage is 0.020 nmol-0.025 nmol / kg fresh soil;

[0020] And / or, the moisturizer is a hyaluronic acid aqueous solution with a concentration of 0.2%-0.5%, and the spraying amount is 20-30 mL / kg fresh soil.

[0021] Furthermore, in step S3, the attractant ball is a porous mesh ball with a diameter of 6-8 cm and a mesh size of 0.10-0.15 cm.

[0022] Furthermore, in step S3, the number of large wax moths placed is 1-2 per bait ball.

[0023] Furthermore, in step S4, the density of the attractant balls is 3-5 per 1.0-1.5 kg of fresh soil sample.

[0024] Furthermore, in step S4, the temperature of the constant temperature chamber is 20-25°C, and the oxygen concentration is 25%-30%.

[0025] Furthermore, in step S4, the induction period is 15-20 days.

[0026] This invention feeds oxidized resveratrol, an immunosuppressant, to 5-instar larvae that have been normally cultured. This compound has an inhibitory effect on the humoral immunity of larvae, which can weaken the immune barrier of larvae against entomopathogenic nematodes, but does not affect the normal development of larvae. This can effectively solve the problem of low numbers, insufficient attack, and unsuccessful infection of some nematode strains with weak pathogenicity in soil samples.

[0027] Meanwhile, since the host's cocooning also makes it difficult for it to be infected by nematodes, storing the large wax moth at 8-12℃ for 24 hours before trapping can inhibit its cocooning during the trapping process.

[0028] This invention involves placing the large wax moth in a bait ball for trapping, securing it with cotton thread to ensure the moth is positioned as centrally as possible within the bait ball. The bait ball can be made of stainless steel or silicone.

[0029] Inoculating fresh soil in attractant balls with Bacillus subtilis and Pseudomonas erythropoietinus can inhibit some other fungi and bacteria, reducing the chance of insects being infected by harmful bacteria and increasing the chance of nematode infection.

[0030] When placing the attractant balls, the balls are connected to each other by stainless steel or silicone chains.

[0031] Spraying the pheromone ascaridin into fresh soil can mobilize the swarm attack of entomopathogenic nematodes, shorten the recovery time of larvae during the infection period, enable them to recover quickly, strengthen the nematode swarm's ability to spread, enhance the nematode swarm's aggressiveness, achieve the ability to quickly find, infect, and kill hosts, and improve the nematode induction and aggregation success rate.

[0032] Adding an appropriate amount of hyaluronic acid aqueous solution to fresh soil as a humectant is beneficial. Hyaluronic acid molecules contain numerous carboxyl and hydroxyl groups, forming intramolecular and intermolecular hydrogen bonds in the aqueous solution. This gives it a powerful water-retention capacity, capable of binding more than 400 times its own weight in water, preventing moisture loss from the fresh soil. It provides excellent humectant properties without making the soil sticky like other gel-like humectants. This allows the sample bags containing the fresh soil to be left open rather than sealed, ensuring sufficient oxygen levels in the soil, promoting better survival for nematodes, and reducing the suffocation and mold growth of large wax moths in the soil.

[0033] After placing the fresh soil sample in the insulated box, the present invention needs to ensure that the oxygen content in the insulated box is sufficient, which can be achieved by setting up a conventional oxygenation device.

[0034] During the trapping process, the condition of the wax moths is checked every other day, and any dead wax moths are removed promptly.

[0035] The beneficial effects of this invention are: it can effectively improve the success rate of infection of the original nematode, which is conducive to further propagation, collection and application, and the method is simple and easy to operate. Attached Figure Description

[0036] Figure 1 For the large wax moth after the trapping process is over;

[0037] Figure 2 These are attractant balls after being filled with soil. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0039] The fresh soil samples in the following examples and comparative examples were all collected from the field, including forest humus soil, weed soil, uncultivated wasteland soil, and garden soil (the soil samples were collected from the top 20-40cm depth after removing surface debris such as dead branches and gravel, and each sample weighed about 30kg). Each soil sample was mixed evenly and then divided into 3 equal parts for 3 replicates of the induction experiment. Each treatment in each replicate consisted of 3 replicates.

[0040] The attractant balls were all porous mesh balls with a diameter of 7cm and a mesh size of 0.10-0.15cm, made of stainless steel; the immunosuppressants, Bacillus subtilis, Bacillus erythropoietin, and ascaridin were all purchased from the market.

[0041] Example 1:

[0042] Before proceeding with this embodiment, the host plant, the large wax moth, was subjected to immune barrier weakening treatment and cocoon inhibition treatment. Specifically, the large wax moths were cultured normally until around the 5th instar. 15 mg of oxidized resveratrol was added to every 1 kg of feed given to the large wax moths. After culturing for another 4 days, the large wax moths were stored at 10°C for 24 hours and then taken out for use.

[0043] A highly efficient method for attracting entomopathogenic nematodes includes the following steps:

[0044] S1. Inoculate the collected fresh soil with Bacillus subtilis and Pseudomonas erythrosporum at inoculation rates of 15 g / kg and 8 g / kg of fresh soil, respectively. At the same time, spray with ascaroside ASCRI#3 and hyaluronic acid aqueous solution at inoculation rates of 0.025 nmol / kg and 30 mL / kg of fresh soil, respectively, and mix well. The concentration of the hyaluronic acid aqueous solution is 0.4%.

[0045] S2. Fix the treated large wax moth host in the attractant ball with cotton thread, place 2 large wax moths in each attractant ball, and then fill the attractant ball with the soil sample treated in step S1, and press and compact it.

[0046] S3. Bury the attractant balls in the remaining soil samples. Place 5 balls in 1.5 kg of fresh soil sample. After completion, put the soil sample in a sample bag and place it in a constant temperature chamber with an oxygen content of 30% and a temperature of 25℃ for 20 days. During the collection process, the sample bag opening is kept open and the oxygen in the constant temperature chamber is replenished at any time through an oxygen pump.

[0047] Three replicate experiments were conducted, with 10 large wax moths in each group.

[0048] Example 2

[0049] Before proceeding with this embodiment, the host plant, the large wax moth, was subjected to immune barrier weakening treatment and cocoon inhibition treatment. Specifically, the large wax moth was cultured normally until around the 5th instar. 10 mg of oxidized resveratrol was added to every 1 kg of feed given to the large wax moth. After culturing for another 4 days, the large wax moth was stored at 10°C for 24 hours and then taken out for use.

[0050] A highly efficient method for attracting entomopathogenic nematodes includes the following steps:

[0051] S1. Inoculate the collected fresh soil with Bacillus subtilis and Pseudomonas erythrosporum at inoculation rates of 12 g / kg and 5 g / kg of fresh soil, respectively. At the same time, spray with ascaroside ASCRI#3 and hyaluronic acid aqueous solution at inoculation rates of 0.02 nmol / kg and 20 mL / kg of fresh soil, respectively, and mix well. The concentration of the hyaluronic acid aqueous solution is 0.4%.

[0052] S2. Fix the treated large wax moth host in the attractant ball with cotton thread, place one large wax moth in each attractant ball, and then fill the attractant ball with the soil sample treated in step S1, and press and compact it.

[0053] S3. Bury the attractant balls in the remaining soil samples, placing 3 balls in 1 kg of fresh soil sample. After completion, place the soil samples in a constant temperature chamber with an oxygen content of 25% and a temperature of 20℃ for 15 days. During the collection process, keep the sample bag open and replenish the oxygen in the constant temperature chamber at any time through an oxygen pump.

[0054] Three replicate experiments were conducted, with 10 large wax moths in each group.

[0055] Compare with Example 1

[0056] Traditional methods were used to attract entomopathogenic nematodes, namely, no immunosuppressants were added to the feed for the large wax moth, and the fresh soil samples were not inoculated with Bacillus subtilis and Bacillus erythropoietin. No ascaridin was sprayed, and the samples were kept moist by spraying water. The sample bags containing the fresh soil samples were sealed to prevent moisture evaporation, and the samples were attracted in a dark environment at 25°C for 20 days.

[0057] Compare with Example 2

[0058] The difference between this comparative example and Example 1 is that the large wax moth in step S2 is a normally cultured and matured healthy large wax moth, that is, no oxidized resveratrol is added to the feed. The other steps are the same.

[0059] Compare with Example 3

[0060] The difference between this comparative example and Example 1 is that in step S3, during the induction process, the sample bag opening was not opened, and oxygen was directly replenished into the sample bag through an oxygen pump.

[0061] Compare with Example 4

[0062] The difference between this comparative example and Example 1 is that, in step S1, Bacillus subtilis and Pseudomonas pulveratum were not inoculated into the fresh soil; the other steps are the same.

[0063] Compare with Example 5

[0064] The difference between this comparative example and Example 1 is that, in step S1, the pheromone ascaridin was not sprayed onto the fresh soil sample; the other steps are the same.

[0065] Compare with Example 6

[0066] The difference between this comparative example and Example 1 is that, in step S1, instead of adding an aqueous solution of hyaluronic acid to the fresh soil sample, an equal amount of water was added; the other steps are the same.

[0067] While observing the host plant, the large wax moth, in the above embodiments and comparative examples, nematode detection was performed. The detection method was as follows: the dead large wax moth was dissected and the presence of nematodes was observed. If nematodes were found, the attractant infection was considered successful.

[0068] The results are shown in Table 1:

[0069] Table 1

[0070]

[0071] Note: In this table, different letters a, b, c, d, and e indicate significant differences (P < 0.05).

[0072] As can be seen from Table 1, the solution of the present invention can significantly reduce the infection and death of the large wax moth by other insect pathogenic microorganisms in the soil compared with the prior art, improve the detection rate of insect pathogenic nematodes, and facilitate the resource collection of insect pathogenic nematodes.

[0073] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A highly efficient method for attracting entomopathogenic nematodes, characterized in that, The large wax moth, which had undergone sequential treatments to weaken its immune barrier and inhibit cocooning, was used for trapping and collection. Specifically, the following steps are included: S1. Collect fresh soil samples from the field; S2. Inoculate the soil sample with biocontrol bacteria, and simultaneously spray with pheromone and moisturizer, and mix well; S3. Place the treated large wax moth into the attractant ball, then fill the attractant ball with the soil sample treated in step S2, and press and compact it; S4. Bury the attractant balls in the remaining soil sample. After completion, put the soil sample in a sample bag and place it in a constant temperature chamber with sufficient oxygen for attraction. The immune barrier weakening treatment involves feeding 5-year-old wax moths with immunosuppressants. The immunosuppressants include oxidized resveratrol, which is added to the feed at a rate of 10-15 mg / kg for 3-5 days.

2. The highly efficient method for attracting entomopathogenic nematodes according to claim 1, characterized in that, The treatment to inhibit cocooning involves placing the large wax moth at 8-12℃ for 24 hours.

3. The highly efficient method for attracting entomopathogenic nematodes according to claim 1, characterized in that, In step S2, the biocontrol bacteria include Bacillus subtilis and Pseudomonas pulvinata. The inoculation amount of Bacillus subtilis is 10-15 g / kg fresh soil, and the inoculation amount of Pseudomonas pulvinata is 5-8 g / kg fresh soil. And / or, the information hormone is ascaridin, and the spraying dosage is 0.020 nmol-0.025 nmol / kg fresh soil: And / or, the moisturizer is a hyaluronic acid aqueous solution with a concentration of 0.2% to 0.5%, and the spraying amount is 20-30 mL / kg fresh soil.

4. The highly efficient method for attracting entomopathogenic nematodes according to claim 1, characterized in that, In step S3, the attractant ball is a porous mesh ball with a diameter of 6-8 cm and a mesh size of 0.10-0.15 cm.

5. The highly efficient method for attracting entomopathogenic nematodes according to claim 1, characterized in that, In step S3, the number of large wax moths placed is 1 to 2 per bait ball.

6. The highly efficient method for attracting entomopathogenic nematodes according to claim 1, characterized in that, In step S4, the density of the attractant balls is 3 to 5 per 1.0-1.5 kg of fresh soil sample.

7. The highly efficient method for attracting entomopathogenic nematodes according to claim 1, characterized in that, In step S4, the temperature of the constant temperature chamber is 20-25℃, and the oxygen concentration is 25%-30%.

8. The highly efficient method for attracting entomopathogenic nematodes according to claim 1, characterized in that, In step S4, the induction period is 15 to 20 days.

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