A method for culturing dispersible third-instar larvae of pine wood nematode and its application

Abstract

This invention relates to a method for culturing third-instar larvae of the pine wood nematode and its application. The culturing method includes the following steps: (1) obtaining second-instar reproductive larvae of the pine wood nematode; (2) inoculating the second-instar reproductive larvae of the pine wood nematode into a culture medium and culturing them; (3) obtaining the third-instar larvae of the pine wood nematode. The culturing method of this invention has the advantages of being simple and quick, and being able to obtain a large number and stable number of third-instar larvae of the pine wood nematode.

Description

Technical Field

[0001] This invention relates to the field of plant pathogen culture technology, and in particular to a method for culturing third-instar larvae of the pine wood nematode and its application. Background Technology

[0002] Pine wood nematode (Bursaphelenchus xylophilus) is a type of nematode widely distributed in pine wood. Taxonomically, it belongs to the phylum Nematoda, class Nematoda, order Nematoda, family Nematodae, and genus Bursaphelenchus. It causes pine wilt disease. Since its occurrence in my country in 1982, pine wilt disease has become one of the most complex and damaging diseases affecting my country's forest ecosystems. With the gradual decrease in temperature, the pine wood nematode transforms from a reproductive second-instar larva to a dispersing third-instar larva. This dispersing third-instar larva can survive adverse environments, allowing the nematode to survive in low-temperature regions and infect more surrounding areas.

[0003] Because the dispersive third-instar larvae of the pine wood nematode are a stage of nematode development resistant to adverse conditions, their formation under natural conditions is closely related to the external environment, especially the temperature of the pine forest. Obtaining a stable population of these dispersive third-instar larvae has always been one of the major problems hindering in-depth research on the pine wood nematode. However, the routine collection and acquisition of these larvae has always relied on the autumn and winter seasons each year, involving felling pine trees infected with pine wood nematode disease in the wild, cutting the logs into small pieces, and collecting the dispersive third-instar larvae from the pine wood. Field collection is limited by the low temperatures of the season, the presence of diseased host pine trees, and numerous uncertainties regarding the identification and collection of pine wood nematodes from the host trees, as well as whether the pine wood nematodes have formed dispersive third-instar larvae.

[0004] Research on pine wood nematodes has been widely carried out in countries around the world. Currently, the reported methods are all for the preservation of third-instar larvae of the dispersible type of pine wood nematode. However, there are relatively few reports on the research and application of indoor culture of the third-instar larvae of the dispersible type of pine wood nematode. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a simple and quick method for cultivating large quantities and stable third-instar larvae of the pine wood nematode.

[0006] Solution for solving the problem

[0007] In a first aspect, the present invention provides a method for culturing third-instar larvae of the pine wood nematode, the method comprising the following steps:

[0008] (1) Obtain reproductive second-instar larvae of pine wood nematode;

[0009] (2) Inoculate the reproductive second-instar larvae of pine wood nematode into the culture medium and culture them;

[0010] (3) Obtain the third instar larvae of the pine wood nematode.

[0011] Preferably, the cultivation in step (2) includes a first cultivation stage and a second cultivation stage, wherein the temperature of the second cultivation stage is controlled to be lower than the temperature of the first cultivation stage.

[0012] Preferably, the temperature of the first culture stage is controlled at 20-30°C, more preferably 22-28°C, and even more preferably 22-26°C.

[0013] Preferably, the temperature of the second culture stage is controlled at 10-20°C, more preferably 12-18°C, and even more preferably 12-16°C.

[0014] Preferably, the duration of the first culture stage is controlled to be 10 to 20 days, more preferably 10 to 18 days, even more preferably 12 to 16 days, and most preferably 12 to 15 days.

[0015] Preferably, the duration of the second culture stage is controlled to be 40-60 days, more preferably 40-55 days, even more preferably 45-55 days, and most preferably 45-50 days.

[0016] Preferably, the cultivation in step (2) is carried out in a dark environment.

[0017] Preferably, in step (2), the concentration of the reproductive second-instar larvae of pine wood nematode is 80-120 larvae / μL, more preferably 85-110 larvae / μL, more preferably 90-110 larvae / μL, and most preferably 95-105 larvae / μL.

[0018] Preferably, the specific steps for obtaining the reproductive second-instar larvae of pine wood nematode in step (1) are as follows: after inoculating and culturing Botrytis cinerea in PDA culture medium, pine wood nematodes are inoculated into PDA culture medium and cultured, pine wood nematode eggs are harvested and the eggs are hatched to obtain the reproductive second-instar larvae of pine wood nematode.

[0019] More preferably, the culture temperature of the *Botrytis cinerea* is controlled at 22–26°C.

[0020] More preferably, the culture time of the *Botrytis cinerea* is controlled to be 2 to 8 days.

[0021] More preferably, the temperature for culturing the pine wood nematode is controlled at 22–26°C.

[0022] More preferably, the culture time of the pine wood nematode is controlled to be 5 to 7 days.

[0023] More preferably, the incubation temperature is controlled at 22–26°C.

[0024] More preferably, the incubation period is controlled to be 20 to 30 hours.

[0025] More preferably, the cultivation of Botrytis cinerea, the cultivation of pine wood nematodes, and the hatching of the nematode eggs are controlled separately and independently in a dark environment.

[0026] Preferably, the culture method further includes preparing the culture medium, the preparation comprising the following steps:

[0027] S1. Mix corn kernels with water and boil;

[0028] S2. The boiled corn kernels obtained in the dispensing step S1 are autoclaved to obtain corn culture medium.

[0029] S3. Inoculate the corn culture medium with Botrytis cinerea and culture it until the corn culture medium is covered with white mycelium, thus obtaining the culture medium.

[0030] More preferably, in step S1, the mass-to-volume ratio of the corn kernels to the water is 0.8–1.5 g: 1 mL.

[0031] More preferably, the boiling time is controlled to be 5 to 15 minutes.

[0032] More preferably, in step S3, the culture temperature is controlled to be 22-26°C.

[0033] More preferably, in step S3, the culture time is controlled to be 12 to 16 days.

[0034] Secondly, the present invention provides an application of the culture method described in the first aspect in the indoor culture of dispersible third-instar larvae of pine wood nematode.

[0035] The effects of the invention

[0036] The method for culturing dispersive third-instar larvae of pine wilt disease provided by this invention is simple, fast, and can obtain a large number of dispersive third-instar larvae of pine wilt disease. It greatly solves the technical problems in the existing technology, such as the limitation of field collection of dispersive third-instar larvae of pine wilt disease in the low-temperature season, diseased host pine trees, identification and collection of pine wilt nematodes in the host pine trees, and whether the pine wilt nematodes have formed dispersive third-instar larvae. It provides technical support for in-depth research on pine wilt disease. Attached Figure Description

[0037] Figure 1 A schematic diagram showing white mycelium of Botrytis cinerea covering the entire plate;

[0038] Figure 2A schematic diagram showing the mycelium of Botrytis cinerea on a plate after pine wood nematodes have finished feeding on it;

[0039] Figure 3 A schematic diagram showing a corn culture medium covered with white mycelium;

[0040] Figure 4 A schematic diagram showing the disappearance of Botrytis cinerea hyphae 14 days after inoculation with pine wood nematode inoculation medium;

[0041] Figure 5 Microscopic image of pine wood nematodes washed off after being cultured in a medium for 49 days;

[0042] Figure 6 Microscopic image of third-instar larvae of pine wood nematode after being heated and killed in the inoculation medium. Detailed Implementation

[0043] In response to the problem that existing technologies rely on the natural environment to obtain the third instar larvae of pine wilt disease, the inventors, through extensive and in-depth research, discovered that by first propagating and culturing the reproductive second instar larvae of pine wilt disease in a corn culture medium, and then continuously culturing the culture medium under low temperature conditions, a large number and stable third instar larvae of pine wilt disease can be obtained indoors, providing technical support for in-depth research on pine wilt disease.

[0044] In a first aspect, the present invention provides a method for culturing third-instar larvae of the pine wood nematode, the method comprising the following steps:

[0045] (1) Obtain reproductive second-instar larvae of pine wood nematode;

[0046] (2) Inoculate the reproductive second-instar larvae of pine wood nematode into the culture medium and culture them;

[0047] (3) Obtain the third instar larvae of the pine wood nematode.

[0048] In some implementations, the cultivation in step (2) includes a first cultivation stage and a second cultivation stage, wherein the temperature of the second cultivation stage is controlled to be lower than the temperature of the first cultivation stage.

[0049] In some embodiments, the temperature of the first culture stage is controlled to be 20-30°C, such as 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc.

[0050] In some embodiments, the temperature of the first culture stage is controlled at 22–28°C.

[0051] In some embodiments, the temperature of the first culture stage is controlled at 22–26°C.

[0052] In some embodiments, the temperature of the second culture stage is controlled to be 10-20°C, for example, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, etc.

[0053] In some embodiments, the temperature of the second culture stage is controlled at 12–18°C.

[0054] In some embodiments, the temperature of the second culture stage is controlled at 12–16°C.

[0055] In some implementations, the duration of the first culture stage is controlled to be 10 to 20 days, such as 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, etc.

[0056] In some implementations, the duration of the first culture phase is controlled to be 10 to 18 days.

[0057] In some implementations, the duration of the first culture phase is controlled to be 12 to 16 days.

[0058] In some implementations, the duration of the first culture phase is controlled to be 12 to 15 days.

[0059] In some implementations, the duration of the second culture phase is controlled to be 40 to 60 days, such as 42 days, 44 days, 46 days, 48 ​​days, 50 days, 52 days, 54 days, 56 days, 58 days, 60 days, etc.

[0060] In some implementations, the second culture phase is controlled to last for 40 to 55 days.

[0061] In some implementations, the second culture phase is controlled for 45 to 55 days.

[0062] In some implementations, the second culture phase is controlled to last for 45 to 50 days.

[0063] In some implementations, the cultivation described in step (2) is carried out in a dark environment.

[0064] In some embodiments, in step (2), the concentration of the reproductive second-instar larvae of pine wood nematode is 80 to 120 larvae / μL, for example 82 larvae / μL, 84 larvae / μL, 86 larvae / μL, 88 larvae / μL, 90 larvae / μL, 92 larvae / μL, 94 larvae / μL, 96 larvae / μL, 98 larvae / μL, 100 larvae / μL, 102 larvae / μL, 104 larvae / μL, 106 larvae / μL, 108 larvae / μL, 110 larvae / μL, 112 larvae / μL, 114 larvae / μL, 116 larvae / μL, 118 larvae / μL, 120 larvae / μL, etc.

[0065] In some embodiments, in step (2), the concentration of the reproductive second-instar larvae of the pine wood nematode is 85-110 larvae / μL.

[0066] In some embodiments, in step (2), the concentration of the reproductive second-instar larvae of the pine wood nematode is 90-110 larvae / μL.

[0067] In some embodiments, in step (2), the concentration of the reproductive second-instar larvae of the pine wood nematode is 95-105 larvae / μL.

[0068] In some embodiments, in step (2), the concentration of the reproductive second-instar larvae of the pine wood nematode is 95-105 larvae / μL, and the inoculation amount is 80-120μL.

[0069] In some embodiments, the specific steps for obtaining the reproductive second-instar larvae of pine wood nematode in step (1) are as follows: after inoculating and culturing Botrytis cinerea in PDA culture medium, pine wood nematodes are inoculated into PDA culture medium and cultured, pine wood nematode eggs are harvested and the eggs are hatched to obtain the reproductive second-instar larvae of pine wood nematode.

[0070] In some embodiments, the temperature for culturing the *Botrytis cinerea* is controlled at 22–26°C, for example, 22°C, 23°C, 24°C, 25°C, 26°C, etc.

[0071] In some embodiments, the temperature for culturing the *Botrytis cinerea* is controlled at 24–26°C.

[0072] In some embodiments, the culture time of the *Botrytis cinerea* is controlled to be 2 to 8 days, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, etc.

[0073] In some embodiments, the culture time of the *Botrytis cinerea* is controlled to be 2 to 7 days.

[0074] In some embodiments, the culture time of the *Botrytis cinerea* is controlled to be 3 to 6 days.

[0075] In some embodiments, the temperature for culturing the pine wood nematodes is controlled to be 22–26°C, for example, 22°C, 23°C, 24°C, 25°C, 26°C, etc.

[0076] In some embodiments, the temperature for culturing the pine wood nematodes is controlled at 24–26°C.

[0077] In some embodiments, the culture time of the pine wood nematode is controlled to be 5 to 7 days until a large number of eggs are observed under a microscope.

[0078] In some embodiments, the concentration of the pine wood nematode is 8 to 12 nematodes / μL, for example 9 nematodes / μL, 10 nematodes / μL, 11 nematodes / μL, 12 nematodes / μL, etc.

[0079] In some embodiments, the incubation temperature is controlled to be 22-26°C, for example, 22°C, 23°C, 24°C, 25°C, 26°C, etc.

[0080] In some embodiments, the incubation temperature is controlled to be 24–26°C.

[0081] In some implementations, the incubation period is controlled to be 20 to 30 hours, such as 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, etc.

[0082] In some implementations, the incubation period is controlled to be 20 to 26 hours.

[0083] In some embodiments, the culture of the *Botrytis cinerea* is controlled to be carried out in a dark environment.

[0084] In some implementations, the culture of the pine wood nematodes is controlled to be carried out in a dark environment.

[0085] In some implementations, the hatching of the insect eggs is controlled to take place in a dark environment.

[0086] In some embodiments, the culture method further includes preparing the culture medium, the preparation comprising the following steps:

[0087] S1. Mix corn kernels with water and boil;

[0088] S2. The boiled corn kernels obtained in the dispensing step S1 are autoclaved to obtain corn culture medium.

[0089] S3. Inoculate the corn culture medium with Botrytis cinerea and culture it until the corn culture medium is covered with white mycelium, thus obtaining the culture medium.

[0090] In some embodiments, in step S1, the mass-to-volume ratio of the corn kernels to the water is 0.8 to 1.5 g: 1 mL, for example, 0.9 g: 1 mL, 1.0 g: 1 mL, 1.1 g: 1 mL, 1.2 g: 1 mL, etc.

[0091] In some embodiments, the corn kernels are dried corn kernels.

[0092] In some embodiments, the boiled corn kernels obtained in step S1 are dispensed into Erlenmeyer flasks.

[0093] In some embodiments, the boiling time is controlled to be 5 to 15 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, etc.

[0094] In some embodiments, the boiling time is controlled to be 8 to 12 minutes.

[0095] In some embodiments, in step S3, the culture temperature is controlled to be 22-26°C, for example, 22°C, 23°C, 24°C, 25°C, 26°C, etc.

[0096] In some embodiments, in step S3, the temperature of the culture is controlled to be 24–26°C.

[0097] In some implementations, in step S3, the culture time is controlled to be 12 to 16 days, such as 12 days, 13 days, 14 days, 15 days, 16 days, etc.

[0098] The prepared pine wood nematode inoculation medium can provide food for the mass reproduction of pine wood nematodes, and at the same time provide a suitable space for the formation of dispersive third-instar larvae. After 14 days of culture, dispersive third-instar larvae begin to form, and the number reaches its peak at 49 days.

[0099] The present invention provides a method for indoor culture of dispersible third-instar larvae of pine wood nematode. This method is simple, fast, and can obtain a large number of dispersible third-instar larvae of pine wood nematode. It greatly solves the technical problems in the existing technology, such as the limitation of field collection of dispersible third-instar larvae of pine wood nematode in the low-temperature season, diseased host pine trees, identification and collection of pine wood nematodes from host pine trees, and whether pine wood nematodes have formed dispersible third-instar larvae. This provides technical support for in-depth research on pine wood nematode disease.

[0100] Secondly, the present invention provides an application of the culture method described in the first aspect in the indoor culture of dispersible third-instar larvae of pine wood nematode.

[0101] Thirdly, the present invention also provides a method for morphological identification of third-instar larvae of the pine wood nematode.

[0102] In some embodiments, the average body length of the third instar larvae of the pine wood nematode is about 640 μm. The top of the head has obvious constriction, the stylet is slender, there is no obvious reproductive organ structure, the tail is blunt and rounded and "finger" shaped, without a tail tip, the worm body has a thick cuticle, and except for the head, the body is filled with a large number of fat body particles. After being killed by heating, the morphology is "(".

[0103] the term

[0104] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0105] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0106] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0107] To enable those skilled in the art to clearly understand the above-described implementation details and operations of the present invention, and to demonstrate the significant advancements in the embodiments and applications of the present invention, the following examples illustrate the above technical solutions.

[0108] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0109] Unless otherwise specified, the materials and reagents used in the following examples are commercially available or synthesized using known methods.

[0110] PDA medium was purchased from BD (Becton, Dickinson and Company) in the United States.

[0111] The *Botrytis cinerea* strain and *Pinus wiltii* strain used in this application are both deposited in the Pine Wilt Research Laboratory of the Institute of Forest Ecology, Environment and Nature Conservation, Chinese Academy of Forestry.

[0112] Example 1

[0113] This embodiment provides a method for the cultivation and collection of reproductive second-instar larvae of pine wood nematode.

[0114] Activated *Botrytis cinerea* was inoculated into the center of several 9cm diameter solid PDA culture plates and incubated in the dark at 25°C for 5 days. 100μL of pine wood nematode suspension (10 nematodes / μL) was inoculated onto plates contaminated with *Botrytis cinerea* and incubated in the dark at 25°C for 5-7 days. When a portion of the culture medium was cut off, rinsed with sterile water, and a large number of nematode eggs were observed under a microscope, the pine wood nematodes were eluted from the culture medium with sterile water and placed in a clean glass petri dish. The dish was then incubated in the dark at 25°C for 1 hour. After standing, the nematode eggs will stick to the glass culture dish. Gently remove the nematode suspension and wash with sterile water 1-2 times. Then add an appropriate amount of sterile water to the culture dish and place the obtained pine wood nematode eggs in a 25℃ constant temperature incubator for 24 hours in the dark. After the eggs hatch into reproductive second-instar larvae of pine wood nematode, transfer the nematode suspension to a 15mL centrifuge tube and centrifuge at 5000rpm for 2 minutes at room temperature. Then remove the supernatant to obtain a large number of synchronously cultured reproductive second-instar larvae of pine wood nematode. Figure 1 A schematic diagram showing the white mycelium of Staphylococcus aureus covering the entire plate is shown; Figure 2 A schematic diagram is shown showing the pine wood nematode feeding on Staphylococcus aureus hyphae on a plate.

[0115] All inoculations with Botrytis cinerea must be performed under aseptic conditions, specifically under aseptic conditions next to an alcohol lamp in a laminar flow hood that has been sterilized by ultraviolet light for half an hour.

[0116] Example 2

[0117] This embodiment provides a method for preparing a culture medium for inoculating pine wood nematodes.

[0118] Weigh 200g of dried corn kernels, wash and dry them, then add 200mL of purified water and boil for 10 minutes. After the corn kernels have completely absorbed the water, cool appropriately and take about 40g of the boiled corn kernels, dispensing them into 100mL Erlenmeyer flasks to prepare corn culture medium. Cover the corn culture medium with breathable sealing film, autoclave at 121℃ for 30 minutes, and let it cool before use.

[0119] Take a PDA solid plate covered with Botrytis cinerea, punch holes in the plate to obtain Botrytis cinerea mycelial blocks, pick up 3-4 mycelial blocks with an inoculation needle and inoculate them into corn culture medium, place them in a constant temperature incubator at 25℃ and incubate in the dark for 14 days to obtain corn culture medium covered with white mycelium, which is the pine wood nematode inoculation medium. Figure 3 A schematic diagram is shown of a corn culture medium covered with white mycelium.

[0120] Example 3

[0121] This embodiment provides a method for culturing third-instar larvae of the pine wood nematode.

[0122] Collected second-instar reproductive nematode larvae were mixed with an appropriate amount of sterile water to prepare a suspension containing approximately 100 larvae / μL. 100 μL of this larval suspension was then inoculated into the pine nematode inoculation medium and cultured in the dark at 25℃ for 14 days. After feeding on the mycelium, the pine nematodes established a high-density and highly active population, and the white mycelium in the inoculation medium disappeared. The inoculation medium was then transferred to incubators at 25℃, 20℃, and 15℃ in the dark. Every 7 days, three corn kernels (0.6g±0.1g) were picked from the medium to wash away the nematodes, which were then observed and counted under a microscope. The results showed that the highest proportions of dispersive third-instar larvae formed in the pine nematode inoculation medium at 25℃ and 20℃ were 8% and 15%, respectively. When the pine wood nematode inoculation medium was cultured at 15℃ for 14 days, the formation of dispersive third-instar larvae began. After 14 days, the number continued to increase. By day 49, the dispersive third-instar larvae accounted for 51% of the total number of nematodes in the medium, reaching a peak of about 5100 larvae / g. Figure 4 A schematic diagram shows the disappearance of Botrytis cinerea hyphae 14 days after inoculation with pine wood nematode inoculation medium. Figure 5 The image shows a microscopic examination of pine wood nematodes washed off after 49 days of culture in an inoculated medium.

[0123] Example 4

[0124] This embodiment provides a method for identifying the third instar larvae of the spreading pine wood nematode.

[0125] The pine wood nematode used in this invention is a pine wood nematode that has been preserved after molecular biological identification. Therefore, this embodiment mainly describes the morphological identification of the third instar larvae of the spreading type of pine wood nematode.

[0126] The identification criteria for the third instar larvae of the spreading pine wood nematode under a microscope are as follows: the average body length is approximately 640 μm, the head tip shows obvious contraction, the stylet is slender, there are no obvious reproductive organs, the tail is blunt and rounded and "finger-shaped", and there is no tail tip. The larvae have a thick cuticle, and except for the head, the body is filled with a large number of fat body granules. After being killed by heating, the larvae are shaped like "(".

[0127] Microscopic examination revealed that the dispersive third-instar larvae in the pine wood nematode inoculation culture medium of this application conformed to the morphological identification description. This demonstrates that it is feasible to use the method of this invention to culture dispersive third-instar larvae of pine wood nematodes indoors, and that these larvae can be cultured indoors and obtained in large quantities in the laboratory. Figure 6 The image shows a microscopic examination of third-instar larvae of the pine wood nematode inoculation medium after heat killing.

[0128] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for culturing third-instar larvae of the pine wood nematode, characterized in that, The cultivation method includes the following steps: (1) Obtain reproductive second-instar larvae of pine wood nematode; (2) Inoculate the reproductive second-instar larvae of pine wood nematode into the culture medium and culture them; (3) Obtain the third instar larvae of the pine wood nematode; The cultivation in step (2) includes a first cultivation stage and a second cultivation stage, wherein the temperature of the second cultivation stage is controlled to be lower than the temperature of the first cultivation stage; The temperature of the first culture stage is controlled at 20~30℃, and the time of the first culture stage is 10~20 days; The temperature of the second culture stage is controlled at 10~20℃, and the culture time of the second culture stage is 40~60 days.

2. The cultivation method according to claim 1, characterized in that, The temperature of the first culture stage is controlled at 22~28℃.

3. The cultivation method according to claim 2, characterized in that, The temperature of the first culture stage is controlled at 22~26℃.

4. The cultivation method according to claim 1, characterized in that, The temperature of the second culture stage is controlled at 12~18℃.

5. The cultivation method according to claim 4, characterized in that, The temperature of the second culture stage is controlled at 12~16℃.

6. The cultivation method according to claim 1, characterized in that, The duration of the first culture stage is controlled to be 10-18 days.

7. The cultivation method according to claim 6, characterized in that, The duration of the first culture stage is controlled to be 12-16 days.

8. The cultivation method according to claim 7, characterized in that, The duration of the first culture stage is controlled to be 12-15 days.

9. The cultivation method according to claim 1, characterized in that, The second culture stage is controlled to last for 40 to 55 days.

10. The cultivation method according to claim 9, characterized in that, The second culture stage is controlled to last 45-55 days.

11. The cultivation method according to claim 10, characterized in that, The second culture stage is controlled to last 45-50 days.

12. The cultivation method according to any one of claims 1 to 11, characterized in that, The cultivation described in step (2) is carried out in a dark environment.

13. The cultivation method according to claim 1, characterized in that, In step (2), the concentration of the reproductive second-instar larvae of the pine wood nematode is 80-120 larvae / μL.

14. The cultivation method according to claim 13, characterized in that, The concentration of the reproductive second-instar larvae of the pine wood nematode was 85-110 individuals / μL.

15. The cultivation method according to claim 14, characterized in that, The concentration of the reproductive second-instar larvae of the pine wood nematode was 90-110 individuals / μL.

16. The cultivation method according to claim 15, characterized in that, The concentration of the reproductive second-instar larvae of the pine wood nematode was 95-105 individuals / μL.

17. The cultivation method according to claim 1, characterized in that, The specific steps for obtaining the reproductive second-instar larvae of pine wood nematode in step (1) are as follows: after inoculating and culturing Botrytis cinerea in PDA culture medium, pine wood nematodes are inoculated into PDA culture medium and cultured, pine wood nematode eggs are harvested and hatched to obtain the reproductive second-instar larvae of pine wood nematode.

18. The cultivation method according to claim 17, characterized in that, The culture temperature of the *Botrytis cinerea* was controlled at 22-26°C.

19. The cultivation method according to claim 17, characterized in that, The culture time of the *Botrytis cinerea* is controlled to be 2-8 days.

20. The cultivation method according to claim 17, characterized in that, The culture temperature of the pine wood nematode was controlled at 22~26℃.

21. The cultivation method according to claim 17, characterized in that, The culture time for the pine wood nematode is controlled to be 5-7 days.

22. The cultivation method according to claim 17, characterized in that, The incubation temperature is controlled at 22~26℃.

23. The cultivation method according to claim 17, characterized in that, The incubation period is controlled to be 20-30 hours.

24. The cultivation method according to claim 17, characterized in that, The cultivation of *Botrytis cinerea*, the cultivation of *Pinus fibrosum*, and the hatching of the eggs are controlled separately and independently in a dark environment.

25. The cultivation method according to claim 1, characterized in that, The cultivation method further includes preparing the culture medium, the preparation of which includes the following steps: S1. Mix corn kernels with water and boil; S2. The boiled corn kernels obtained in the dispensing step S1 are autoclaved to obtain corn culture medium. S3. Inoculate the corn culture medium with Botrytis cinerea and culture it until the corn culture medium is covered with white mycelium, thus obtaining the culture medium.

26. The cultivation method according to claim 25, characterized in that, In step S1, the mass-to-volume ratio of the corn kernels to the water is 0.8~1.5g:1mL.

27. The cultivation method according to claim 25, characterized in that, In step S1, the boiling time is controlled to be 5-15 minutes.

28. The cultivation method according to claim 25, characterized in that, In step S3, the culture temperature is controlled to be 22~26℃.

29. The cultivation method according to claim 25, characterized in that, In step S3, the culture time is controlled to be 12-16 days.

30. The application of the culture method according to any one of claims 1 to 29 in the indoor culture of dispersible third-instar larvae of pine wood nematode.

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