Screening method of entomopathogenic nematodes for preventing and controlling plagiospermum erythrorhizon larvae in pine soil

Abstract

The invention provides a screening method of entomopathogenic nematodes for preventing and controlling plagiospermus giganteus larvae in pine soil, and relates to the technical field of biology. The method comprises the following steps: collecting a plurality of entomopathogenic nematodes, and digging the larvae and peripheral soil of the platystigma obtusifolium as test materials; screening entomopathogenic nematode types by calculating the death rate and the corrected death rate of the larvae of the platystigma oblasioides; screening out two high-virulence pathogenic nematode strains, and testing the lethal effect of different doses of the high-virulence pathogenic nematodes on the larvae of the plagiospermus giganteus; testing the lethal effect of the screened high-toxicity pathogenic nematodes on the larvae of the plagiospermus giganteus in different soil layer thicknesses; the test data is analyzed, a conclusion is given, and an important basis is provided for preventing and controlling the larvae of the flat leaf bees by utilizing the entomopathogenic nematodes.

Description

Technical field:

[0001] The invention provides a method for screening insect pathogenic nematodes for preventing and controlling larvae of pine sawfly in loose soil, and relates to the field of biotechnology. Background technology:

[0002] Acantholyda posticalis, also known as pine flat sawfly, belongs to the genus Icantholyda of the family Pamphiliidae of the order Hymenoptera (Xiao Gangrou and Li Zhenyu, 2020). It is a highly explosive and harmful leaf-eating pest that mainly harms Pinus tabulaeformis, P. koraiensis and P. sylvestris. Acantholyda posticalis has one generation per year. The larvae pupate underground in late April, the adults emerge in early May, the larvae damage pine needles in early to mid-June, and the mature larvae spin silk and descend from the trees in late July to oversummer in soil chambers on the surface of the soil. Currently, the prevention and control of pine flat sawflies is still mainly chemical, which has led to problems such as increased pest resistance, environmental pollution and impacts on non-target organisms.

[0003] Since the larvae of the pine sawfly live underground for up to 9 months, it provides a lot of time to find weak links in its control. As a highly promising biological control method, insect pathogenic nematodes are particularly suitable for underground pests. Steinernema and Heterorhabditis pathogenic nematodes have good control effects on a variety of underground pests. For example, the use of S. longicaudum X-7 and H. bacteriophora H06 to control peanut grubs in the control field can increase the yield of peanuts by 70%; the pathogenicity of H. bacteriophora Cangzhou strain to the larvae of Holotrichia parallela reaches 93.3%; the control effect of insect pathogenic nematodes on the larvae of yellow striped flea beetles in the soil is 73.47% to 83.21%. Insect pathogenic nematodes have certain host specificity. Different species and strains will have different control effects on the same host insect, and the same species or strain will have different control effects on different host insects. Therefore, screening the best nematode species or strains is the key to pest control.

[0004] Therefore, the present invention screens out insect pathogenic nematodes with high pathogenicity by conducting indoor biological assays on the pathogenicity of five species (strains) of insect pathogenic nematodes to the larvae of pine sawflies living underground; and tests the pathogenicity of dominant strains of pathogenic nematodes to the larvae of pine sawflies in different soil thicknesses, in order to provide a reference for the biological control of pine sawflies. Summary of the invention:

[0005] In order to solve the above technical problems, the present invention proposes a method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil, comprising the following steps:

[0006] Step S1, collecting a variety of insect pathogenic nematodes, digging out pine sawfly larvae and surrounding soil as test materials;

[0007] Step S2, screening highly toxic insect pathogenic nematode species by calculating the mortality rate and corrected mortality rate of larvae of P. pineella;

[0008] Step S3, testing the lethal effects of different doses of the screened highly virulent pathogenic nematode strains on larvae of pine sawfly;

[0009] Step S4, testing the lethal effect of the selected highly toxic pathogenic nematodes on larvae of pine sawfly in different soil layer thicknesses;

[0010] Step S5: Analyze the test data and draw conclusions.

[0011] Further, in step S2, 5 kinds of insect pathogenic nematodes are respectively prepared into nematode suspensions with a concentration of 2000 IJs / mL, 10 pine flat sawfly larvae are respectively placed in each disposable culture dish padded with two layers of neutral filter paper, 1 mL of nematode suspension is dripped into the culture dish, and an equal amount of clean water is dripped as a control group; the culture dish is sealed with a sealing film and placed in an incubator to keep the filter paper slightly wet; each treatment and control are repeated 3 times, and each repeat is 10 pine flat sawfly larvae; 24, 36, 48, 60, and 72 hours after the experimental treatment, the mortality rate and corrected mortality rate of the pine flat sawfly larvae are calculated:

[0012]

[0013] Furthermore, in step S3, two highly toxic pathogenic nematode strains were screened, and nematode suspensions with concentrations of 1000 IJs / mL, 2000 IJs / mL and 4000 IJs / mL were prepared respectively. 1.0 mL of the nematode suspension was dripped into a disposable culture dish padded with two layers of neutral filter paper, and an equal amount of clean water was dripped into the control; 10 larvae of pine flat sawfly were placed in the culture dish, and different doses of 100 IJs / worm, 200 IJs / worm, and 400 IJs / worm were treated. The culture dish was sealed with a sealing film and placed in an incubator; each treatment or control was repeated 3 times, with 10 larvae of pine flat sawfly in each repeat; the mortality rate and the corrected mortality rate were calculated 24, 36, 48, 60, and 72 hours after the pathogenic nematode killed the insect.

[0014] Furthermore, in the step S4, the screened highly toxic pathogenic nematodes are prepared into 2000 IJs / mL, the soil samples collected in the forest are mixed, 10 healthy mature larvae of pine flat sawfly are randomly selected and placed at the bottom of the aviation plastic cup, the soil samples collected in the forest are evenly sprinkled in the cup, and the soil layer thickness is set to 2 cm, 4 cm, 6 cm, 8 cm, and 10 cm, respectively. 1 mL of the prepared nematode suspension is aspirated with a pipette, covered with plastic wrap, small holes are pierced in the plastic wrap for ventilation, and placed in an environment of 25°C, and an equal amount of clean water is added in the same way as a control group; it is observed every 12 hours, and the mortality rate and corrected mortality rate are calculated at 24, 36, 48, 60, and 72 hours.

[0015] Furthermore, in step S1, the multiple insect pathogenic nematodes collected are Steinernema longicaudum X-7, Steinernema feltiae SN, Steinernema carpocapsae All, Heterorhabditis bacteriophora H06, and Heterorhabditis indica LN2.

[0016] Furthermore, in step S1, when digging out the larvae of the pine sawfly, the distance between the larvae of the pine sawfly and the ground surface is measured, and used as a basis for setting the thickness of the soil layer indoors.

[0017] Furthermore, step S5 includes: analyzing the lethal effect of pathogenic nematodes on pine sawfly larvae, analyzing and screening the optimal dose of pathogenic nematodes, and analyzing the lethal effect of insect pathogenic nematodes on pine sawfly larvae in different soil layer thicknesses.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The present invention selects 5 kinds of pathogenic nematodes, and screens out the strains with the best control effect on pine flat sawfly larvae through indoor lethality tests and lethality tests under different soil layer thicknesses. The results show that under indoor culture dish conditions, the 5 insect pathogenic nematodes measured are all sensitive to pine flat sawfly larvae, and the pine flat sawfly larvae are more sensitive to the nematodes S. longicaudum X-7 and S. carpocapsae All at high concentrations. At a dosage of 400 IJs / worm, the lethality of S. carpocapsae All and S. longicaudum X-7 to pine flat sawfly larvae can reach 83.33% at 72 hours. The mortality rate of the two nematodes to pine flat sawfly larvae in the soil at a shallow soil layer thickness of 2 cm for 72 hours is 90%, and at a soil layer thickness of 10 cm, the lethality of the two nematodes to pine flat sawfly larvae at 72 hours is 76.67%. The field effect of the soil-hidden insect state of the larvae of the pine sawfly deserves further exploration. The present invention provides an important basis for the use of insect pathogenic nematodes to control the larvae of the pine sawfly. Description of the drawings:

[0020] Figure 1 The bar graph shows the lethal effects of five insect pathogenic nematodes on the larvae of the pine sawfly in a petri dish.

[0021] Figure 2 The curve of the corrected mortality of five insect pathogenic nematodes against pine sawfly larvae changing with treatment time at a concentration of 100 IJs / head;

[0022] Figure 3 The curve of the corrected mortality of five insect pathogenic nematodes against pine sawfly larvae at a concentration of 200 IJs / head versus treatment time;

[0023] Figure 4 The curve of the corrected mortality of five insect pathogenic nematodes against pine sawfly larvae at a concentration of 400 IJs / head versus treatment time;

[0024] Figure 5 This is the time-varying curve of the corrected mortality of the nematode S. carpocapsae All to the larvae of the pine sawfly at different soil thicknesses;

[0025] Figure 6 This is the time-varying curve of the corrected mortality of the nematode S. longicaudum X-7 against the larvae of the pine sawfly in different soil thicknesses. Specific implementation method:

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0027] In the drawings of the specific embodiments of the present invention, in order to better and more clearly describe the working principles of the various components in the system, the connection relationship of the various parts in the device is shown, which only clearly distinguishes the relative position relationship between the various components, and cannot constitute a limitation on the signal transmission direction, connection sequence and structural size, size and shape of each part within the component or structure.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0029] Step S1, collecting various insect pathogenic nematodes, digging out larvae of pine sawfly and surrounding soil as test materials.

[0030] S1.1: Collecting multiple entomopathogenic nematodes

[0031] The various insect pathogenic nematodes include: Steinernema longicaudum X-7, SteinernemafeltiaeSN, Steinernema carpocapsae All, Heterorhabditis bacteriophora H06, and Heterorhabditis indica LN2.

[0032] S1.2: Digging up pine sawfly larvae

[0033] The larvae of the pine flat sawfly were collected in the Taohuayu Pinus tabulaeformis forest of Taishan Forest Farm and dug in the soil under the canopy radiation area. When the insect body was dug, the distance between the insect body and the ground surface was measured and used as the basis for setting the soil layer thickness indoors. The dug larvae of the pine flat sawfly were brought back to the laboratory as spare test insects.

[0034] S1.3: Collect soil around pine sawfly larvae

[0035] When digging out the larvae of the pine sawfly, the soil around the larvae was brought back to the laboratory. After the soil was fully mixed indoors, three 50g soil samples were taken and dried in an oven at 105±5℃ to constant weight. The average moisture content was calculated to reflect the humidity of the soil where the larvae of the pine sawfly were located.

[0036]

[0037] Step S2: Screening highly virulent insect pathogenic nematode species by calculating the mortality rate and corrected mortality rate of larvae of P. pineella.

[0038] Prepare nematode suspensions of 2000 IJs / mL for each of the five insect pathogenic nematodes, put 10 larvae of P. pineella in each disposable culture dish (d=9 cm) lined with two layers of neutral filter paper, add 1 mL of nematode suspension (100 IJs / worm) to the culture dish, and add an equal amount of clean water as a control group.

[0039] The culture dish was sealed with sealing film and placed in an incubator (10°C, relative humidity 75%, dark) to keep the filter paper slightly moist. Each treatment and control were repeated 3 times, with 10 larvae in each repeat. At 24, 36, 48, 60, and 72 hours after the experimental treatment, the number of dead larvae of the pine flat sawfly was counted, and the mortality rate and corrected mortality rate of the larvae of the pine flat sawfly were calculated.

[0040]

[0041] Step S3, testing the lethal effects of different doses of the selected highly virulent pathogenic nematode strains on larvae of pine sawfly.

[0042] The two highly virulent pathogenic nematode strains were screened out and prepared into nematode suspensions with concentrations of 1000 IJs / mL, 2000 IJs / mL and 4000 IJs / mL, respectively. 1.0 mL of nematode suspension was dripped into a disposable culture dish (d=9 cm) padded with two layers of neutral filter paper, and an equal amount of water was dripped into the control. 10 pine flat sawfly larvae were placed in the culture dish, that is, the insect pathogenic nematodes were treated with different doses of 100 IJs / worm, 200 IJs / worm, and 400 IJs / worm on the pine flat sawfly larvae. The culture dish was sealed with a sealing film and placed in an incubator (10°C, relative humidity of 75%, dark). Each treatment or control was repeated 3 times, with 10 test worms in each repeat. The number of deaths of pine flat sawfly larvae was counted at 24, 36, 48, 60, and 72 hours after the pathogenic nematodes killed the larvae, and the mortality rate and corrected mortality rate were calculated.

[0043] Step S4, testing the lethal effect of the selected highly virulent pathogenic nematodes on the larvae of Pine Sawfly in different soil layer thicknesses.

[0044] The highly toxic pathogenic nematodes screened out were prepared into a nematode suspension of 2000 IJs / mL, i.e., 200 IJ / worm, and the soil samples collected in the forest were mixed. Ten healthy mature larvae of pine flat sawfly were randomly selected and placed at the bottom of an aviation plastic cup (3 cm in diameter × 12 cm in height). The soil samples collected in the forest were evenly sprinkled in the cup, and the soil layer thickness was set to 2 cm, 4 cm, 6 cm, 8 cm, and 10 cm, respectively. 1 mL of the prepared nematode suspension was taken with a pipette and dripped evenly onto the soil surface from as many directions as possible, covered with plastic wrap, pierced with small holes in the plastic wrap for ventilation, placed in a 25°C environment, and the same amount of clean water was dripped in the same way as the control group. After the experimental treatment, observations were made every 12 hours, and the number of deaths of pine flat sawfly larvae was counted at 24, 36, 48, 60, and 72 hours, and the mortality rate and corrected mortality rate were calculated.

[0045] Step S5: Analyze the test data and draw conclusions.

[0046] 1: Analysis of the lethal effect of pathogenic nematodes on larvae of pine sawfly:

[0047] There was no significant difference in the corrected mortality of the five entomopathogenic nematodes against larvae of the pine sawfly in culture dishes ( Figure 1 At 72 h of treatment, the corrected mortality of S. carpocapsae All was 83.33%, that of S. longicaudum X-7 was 76.67%, that of S. feltiae SN, H. bacteriophora H06 and H. indica LN2 was 73.33%, 73.33% and 70%, respectively, with no significant difference among the treatments (F=0.679, df=4,10, P=0.622).

[0048] 2: Analyze and screen the optimal dose of pathogenic nematodes:

[0049] At different concentrations, the corrected mortality of the five insect pathogenic nematodes against the larvae of the pine sawfly increased with the increase of treatment time, and there was no significant difference among all the pathogenic nematodes at the same treatment time ( Figure 2 , 3 、4).

[0050] After 24 hours of treatment with a concentration of 100 IJs / head, the mortality rate of the five nematodes to the larvae of pine sawfly was 13.33%-23.33% (F=0.758, df=4,10, P=0.575). After 72 hours of treatment, the corrected mortality rate of S.carpocapsae All was the highest, reaching 83.33%; the corrected mortality rates of several other nematodes were also above 60% (F=2.306, df=4,10, P=0.129).

[0051] After 24 hours of treatment with a concentration of 200 IJs / head, the mortality rate of the five nematodes to the larvae of the pine sawfly was ≤26.67% (F=0.901, df=4,10, P=0.499). After 72 hours of treatment, the corrected mortality rate of S. carpocapsae All was the highest, at 83.33%; the corrected mortality rate of S. longicaudum X-7 was 80.00%, and the corrected mortality rates of the other nematodes were ≥70.00% (F=2.170, df=4,10, P=0.146).

[0052] After 24 hours of treatment with a concentration of 400 IJs / head, the mortality rate of the five nematodes to the larvae of pine sawfly was ≤26.67% (F=1.207, df=4,10, P=0.367). After 60 hours of treatment, the corrected mortality rates of the five nematodes to the larvae of pine sawfly increased to above 66.67% (F=0.692, df=4,10, P=0.614). After 72 hours of treatment, the corrected mortality rates of S.carpocapsae All and S.longicaudum X-7 were the highest, at 83.33%; the corrected mortality rates of S.feltiaeSN, H.bacteriophora H06 and H.indica LN2 were 70.00%, 73.33% and 76.67%, respectively (F=0.716, df=4,10, P=0.600).

[0053] 3: Analysis of the lethal effect of insect pathogenic nematodes on larvae of Pine Sawfly in different soil thicknesses:

[0054] According to measurements and calculations, the average moisture content of the soil where the larvae of the pine sawfly live is 4.6%.

[0055] The corrected mortality of the nematode S. carpocapsae All against the larvae of the pine sawfly in the soil increased with the increase of treatment time. There was no significant difference in the corrected mortality of the larvae of the pine sawfly in different soil thicknesses at the same treatment time ( Figure 5 ). After 24 hours of treatment, the corrected mortality of nematodes S.carpocapsae All against pine sawflies in soil at different soil thicknesses was less than 33.33% (F=2.175, df=4,15, P=0.176); after 72 hours of treatment, the corrected mortality was the highest at 2 cm soil thickness, 90.00%, and at 4 cm soil thickness, 86.67%. When the soil thickness was 6-10 cm, the corrected mortality of nematodes S.carpocapsae All against pine sawflies in soil was the same, 76.67% (F=0.229, df=4,15, P=0.916).

[0056] There was no significant difference in the corrected mortality of pine sawfly larvae treated with the nematode S. longicaudum X-7 at different soil thicknesses at the same time ( Figure 6 ). After 24 hours of treatment, the corrected mortality of the nematode S. longicaudum X-7 to the pine sawfly in the soil was less than 13.33% (F=0.500, df=4,15, P=0.737) at different soil thicknesses. After 60 hours of treatment, the corrected mortality of the nematode S. longicaudum X-7 to the pine sawfly in the soil was as high as 76.67% (F=2.470, df=4,15, P=0.112) at a soil thickness of 2 cm. After 72 hours of treatment, the corrected mortality of the nematode S. longicaudum X-7 to the pine sawfly in the soil was as high as 90.00% at soil thicknesses of 2 cm and 4 cm. When the soil thickness was 6 to 10 cm, the corrected mortality of the nematode S. longicaudum X-7 to the pine sawfly in the soil was the same at 76.67% (F=0.371, df=4,15, P=0.824).

[0057] 4: Conclusion and discussion

[0058] The results of indoor bioassays showed that the five tested insect pathogenic nematodes were all sensitive to the larvae of the pine flat sawfly. After 72 hours of treatment with a concentration of 200 IJs / head, the mortality rate of the larvae of the pine flat sawfly was above 73.33%, showing strong toxicity. Therefore, using insect pathogenic nematodes to control the larvae of the pine flat sawfly is an effective biological control method.

[0059] The application dose is an important factor affecting the control effect. Generally speaking, an increase in the application concentration will increase the lethal effect of nematodes on the host, but it will also increase the cost of use. The results of the pathogenicity of different doses of insect pathogenic nematodes to the larvae of pine flat sawfly in the present invention show that under the same treatment time, the corrected mortality rate of pine flat sawfly larvae increases with the increase of the nematode application dose. In general, the five insect pathogenic nematodes have a high toxicity to the larvae of pine flat sawfly at a concentration of 200 IJs / worm, and the lethal effect can reach more than 70% at 72h. At a dosage of 400 IJs / worm, the mortality rate of S.carpocapsaeAll and S.longicaudum X-7 to the larvae of pine sawfly can reach 83.33% at 72 hours, and the corrected mortality rate of the nematode S.carpocapsae All to the larvae of pine sawfly at a dosage of 100 IJs / worm is also 83.33%. Within a certain concentration range, the lethal effect of the nematode S.carpocapsae All is not greatly affected. The mortality rate of S.feltiae SN, H.bacteriophora H06 and H.indica LN2 is relatively low under the same concentration conditions.

[0060] The present invention preliminarily screened from 5 strains of insect pathogenic nematodes and found that the larvae of pine flat sawfly were more sensitive to the nematodes S. longicaudum X-7 and S. carpocapsae All, with a relatively high mortality rate, and showed good control effects on the larvae of pine flat sawfly at different depths of the soil layer. The mortality rate of the two nematodes to the larvae of pine flat sawfly in 72 hours at a shallow soil layer thickness of 2 cm reached 90%, and at a soil layer thickness of 10 cm, the mortality rate of the two nematodes to the larvae of pine flat sawfly in 72 hours was 76.67%. Therefore, the field effect of applying S. carpocapsae All and S. longicaudum X-7 to control the larvae of pine flat sawfly deserves further exploration.

[0061] In summary, the five insect pathogenic nematodes S. carpocapsae All, S. longicaudum X-7, S. feltiaeSN, H. bacteriophora H06 and H. indica LN2 all have a good lethal effect on the larvae of pine sawfly; S. carpocapsae All and S. longicaudum X-7 have relatively good lethal effects and show high toxicity to larvae in the soil, and have the potential to control the hidden insect stage of pine sawfly in the soil.

[0062] The technical features of the above embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0063] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the present application. It should be noted that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil, characterized in that: The steps include: Step S1, collecting a variety of insect pathogenic nematodes, digging out pine sawfly larvae and surrounding soil as test materials; Step S2, screening highly toxic insect pathogenic nematode species by calculating the mortality rate and corrected mortality rate of larvae of P. pineella; Step S3, testing the lethal effects of different doses of the screened highly virulent pathogenic nematode strains on larvae of pine sawfly; Step S4, testing the lethal effect of the selected highly toxic pathogenic nematodes on larvae of pine sawfly in different soil layer thicknesses; Step S5: Analyze the test data and draw conclusions.

2. The method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil according to claim 1, characterized in that: In the step S2, 5 kinds of insect pathogenic nematodes are respectively prepared into nematode suspensions with a concentration of 2000 IJs / mL, 10 pine flat sawfly larvae are respectively placed in each disposable culture dish padded with two layers of neutral filter paper, 1 mL of nematode suspension is dripped into the culture dish, and an equal amount of clean water is dripped as a control group; the culture dish is sealed with a sealing film and placed in an incubator to keep the filter paper slightly wet; each treatment and control are repeated 3 times, and each repetition is 10 pine flat sawfly larvae; at 24, 36, 48, 60, and 72 hours after the experimental treatment, the mortality rate and the corrected mortality rate of the pine flat sawfly larvae are calculated:

3. The method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil according to claim 2, characterized in that: In the step S3, two highly toxic pathogenic nematode strains are screened, and nematode suspensions with concentrations of 1000 IJs / mL, 2000 IJs / mL and 4000 IJs / mL are prepared respectively. 1.0 mL of the nematode suspension is dripped into a disposable culture dish padded with two layers of neutral filter paper, and an equal amount of clean water is dripped into the control; 10 larvae of pine flat sawfly are placed in the culture dish, and different doses of 100 IJs / worm, 200 IJs / worm, and 400 IJs / worm are treated, and the culture dish is sealed with a sealing film and placed in an incubator; each treatment or control is repeated 3 times, and each repeat has 10 larvae of pine flat sawfly; 24, 36, 48, 60, and 72 hours after the pathogenic nematode kills, the mortality rate and the corrected mortality rate are calculated.

4. The method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil according to claim 3, characterized in that: In the step S4, the screened highly toxic pathogenic nematodes are prepared into 2000 IJs / mL, the soil samples collected in the forest are mixed, 10 healthy mature larvae of pine flat sawfly are randomly selected and placed at the bottom of an aviation plastic cup, the soil samples collected in the forest are evenly sprinkled in the cup, and the soil layer thickness is set to 2 cm, 4 cm, 6 cm, 8 cm, and 10 cm, respectively. 1 mL of the prepared nematode suspension is drawn with a pipette, and the plastic wrap is covered with small holes in the plastic wrap for ventilation. The plastic wrap is placed in an environment of 25°C, and an equal amount of clean water is added in the same way as a control group; the control group is observed every 12 hours, and the mortality rate and the corrected mortality rate are calculated at 24, 36, 48, 60, and 72 hours.

5. The method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil according to claim 1, characterized in that: In the step S1, the multiple insect pathogenic nematodes collected are Steinernema longicaudum X-7, Steinernema feltiae SN, Steinernema carpocapsae All, Heterorhabditisbacteriophora H06, and Heterorhabditis indica LN2.

6. The method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil according to claim 1, characterized in that: In the step S1, when digging out the larvae of the pine sawfly, the distance between the larvae of the pine sawfly and the ground surface is measured, and used as a basis for setting the thickness of the soil layer indoors.

7. The method for screening insect pathogenic nematodes for controlling larvae of pine sawfly in loose soil according to claim 1, characterized in that: The step S5 includes: analyzing the lethal effect of pathogenic nematodes on pine sawfly larvae, analyzing and screening the optimal dose of pathogenic nematodes, and analyzing the lethal effect of insect pathogenic nematodes on pine sawfly larvae in different soil layer thicknesses.

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