Method for researching resistance mechanism of larch to pine wood nematodes based on Chinese pine control

Abstract

The invention discloses a method for researching the resistance mechanism of larch to pine wood nematodes based on Chinese pine control, and relates to the technical field of biological control. The method for researching the resistance mechanism of larch to pine wood nematodes based on pinus tabulaeformis contrast comprises the seven steps that the larch and the pinus tabulaeformis are inoculated with the pine wood nematodes and are manually pruned, so that healing parts are formed at the pruned parts of the larch and the pinus tabulaeformis; according to the method, larch and Chinese pine healing positions are classified, the bursaphelenchus xylophilus proliferation quantity is counted, in the aspect of physiological indexes, after larch is infected with bursaphelenchus xylophilus, the internal physiological metabolism process of larch can be well maintained balanced, the change of each physiological index is relatively small, and the analysis of flavone content and polyphenol content shows that the larch and Chinese pine healing positions can be found in the aspect of the bursaphelenchus xylophilus proliferation quantity. The larch has an inhibiting effect on feeding and growth of pine wood nematodes, and the characteristic provides important theoretical basis and practical basis for development of pine wood nematode prevention and control technologies and cultivation of insect-resistant tree species.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to a method for studying the resistance mechanism of larch to pine wood nematodes based on Chinese pine as a control. Background Art

[0002] Pine wood nematode (Bursaphelenchus xylophilus) is currently the number one pest that harms my country's forestry production and forest ecological environment. The disease caused by pine wood nematode is called pine wood nematode disease or pine wilt disease because it spreads rapidly, has strong reproductive capacity, and kills trees quickly. Disease-resistant breeding is one of the important ways to prevent and control diseases. For pine wood nematode disease, disease-resistant gene banks and seed gardens have been established. However, the production cycle of disease-resistant seeds is long and the yield is limited. Therefore, how to use plant resistance to prevent and control pine wood nematodes is of great significance.

[0003] Among them, the "Preparation method of the preparation of antibacterial active ingredients of carbonaceous bacteria for preventing and controlling pine dieback disease" disclosed in the application number "CN101897345B" has solved the various disadvantages of using chemical control, which has brought high benefits to forestry production but easily caused environmental pollution and destruction of the natural balance of the ecosystem.

[0004] Although it is now clear that some pine wood has a certain resistance to pine wood nematodes, the frequent transportation of seedlings has caused localized pine seed control. How to identify and find a pine wood that is resistant to pine wood nematodes has become a key issue to be solved. Drugs and manual control measures for pine wood nematodes are difficult, labor-intensive, inefficient and costly, and difficult to implement, and basically remain at the theoretical level. Therefore, it is necessary to use a method to study the resistance mechanism of larch to pine wood nematodes based on Pinus tabulaeformis as a control. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for studying the resistance mechanism of larch to pine wood nematodes based on Pinus tabulaeformis as a control, thereby solving the existing problems.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for studying the resistance mechanism of larch to pine wood nematodes based on Pinus tabulaeformis as a control is used, comprising the following steps:

[0007] Step 1: Extraction of pine wood nematodes as raw materials for subsequent experiments;

[0008] Step 2: co-culturing pine wood nematodes and Botrytis cinerea to serve as experimental raw materials;

[0009] Step 3, inoculating larch and Chinese pine with pine wood nematodes;

[0010] Step 4, extracting the flavonoids content of larch;

[0011] Step 5, extracting the polyphenol content of Pinus tabulaeformis;

[0012] Step 6: Manually prune the inoculated larch and Chinese pine, so that healing areas are formed at the pruning sites of the larch and Chinese pine;

[0013] Step 7: Classify the healing sites of larch and Chinese pine, separate the nematodes using Baermann funnel, and count the number of pine wood nematodes proliferating.

[0014] Preferably, the step 1 includes the following steps:

[0015] S1. Sample collection

[0016] Select trees with typical symptoms of pine wilt disease from naturally infected larch forests, collect branch and trunk samples of diseased trees using saws or scissors, and select tissues close to the lesions. Cut the collected larch samples into small sections of 1-2 cm;

[0017] S1.2, Baermann funnel separation,

[0018] Place the larch sample in a Baermann funnel, add a certain amount of water to the top of the funnel, immerse the sample in water, and control the ambient temperature of the Baermann funnel to be 25°C. Leave it for 24-48 hours to allow the nematodes to escape from the sample tissue and enter the collection tube through the filter paper at the bottom of the funnel, thereby obtaining a nematode suspension.

[0019] S1.3, Nematode purification,

[0020] Transfer the nematode suspension collected by the Baermann funnel to a centrifuge tube and centrifuge at 3000 rpm for 5-10 minutes to allow the nematodes to settle to the bottom of the tube. Discard the supernatant, add an appropriate amount of sterile water to resuspend the nematodes, and repeat the centrifugation 2-3 times to remove impurities.

[0021] Preferably, the step 2 includes the following steps:

[0022] S2. Cultivate PDA.

[0023] Prepare a potato dextrose agar (PDA) medium, inoculate Botrytis cinerea onto the medium under sterile conditions, and place the culture dish in a constant temperature incubator at 25°C for 3-5 days until the Botrytis cinerea grows all over the culture dish;

[0024] S2.1. Co-culture

[0025] The purified pine wood nematodes were inoculated into a culture dish full of Botrytis cinerea, and different inoculation density gradients were set, with 50, 100, and 150 nematodes per square centimeter;

[0026] S2.2, Experimental data recording,

[0027] The number and vitality of nematodes were recorded. At different time points after co-culture (1d, 3d, 5d, 7d, 9d, 14d, and 21d), multiple fields of view were randomly selected from each culture dish, the number of pine wood nematodes was counted under a microscope, and the vitality, motility, and morphological integrity of the nematodes were observed.

[0028] Preferably, in the step three, 1-year-old twigs, large branches and trunks are selected from healthy Chinese pine and larch respectively to ensure that the samples are fresh and free from diseases and insect pests. The larch is inoculated, and samples are taken at designated time points of 1d, 3d, 5d, 7d, 9d, 14d and 21d. At the same time, uninoculated Chinese pine at the same time point is taken as a control, and the prepared pine wood nematode suspension is inoculated into different parts of the tree including but not limited to twigs, large branches and trunks using a sterile syringe.

[0029] Preferably, in step 4, the sample is mixed with ethanol in a certain proportion, loaded into an ultrasonic extractor, and the ultrasonic wave is turned on to utilize the mechanical vibration and cavitation effect generated by the ultrasonic wave to destroy the cell wall and promote the release of flavonoid compounds. After the extraction is completed, the mixed solution is allowed to stand to allow the solid particles to precipitate or the solid and liquid are separated by filtration.

[0030] Preferably, in step five, the polyphenol content of Pinus tabulaeformis is extracted by selecting large branches from Pinus tabulaeformis, removing dead leaves and impurities, and then cutting the sample into small pieces to increase the surface area, and then drying to remove moisture, and selecting ethanol as the extraction solvent, the ratio of sample weight 1g / mL: solvent volume 10g / mL, after the sample powder and the solvent are evenly mixed, they are placed in a container suitable for ultrasonic extraction for solid-liquid separation.

[0031] Preferably, in step six, the scissors and pruning shears are soaked in a disinfectant 0.1% potassium permanganate solution for 15-20 minutes, and then rinsed with sterile water to prevent the introduction of pathogens during the pruning process and affect the formation of the healing site. For larch and Chinese pine, representative parts are selected on the tender branches, large branches and trunks for pruning. When pruning, ensure that the incision is smooth and minimize damage to surrounding tissues. The incision area should not exceed 1 / 3 of the cross-sectional area of ​​the branch or trunk. After pruning, the incision is immediately smeared with a disinfectant for disinfection to prevent infection by pathogens. A mixed solution of indoleacetic acid and cytokinin is then used to smear the wound, and then the incision is wrapped to maintain the humidity of the incision.

[0032] Preferably, in step 7, the healing sites of the Chinese pine and larch are observed and recorded, wherein the recovery levels of the healing sites of the Chinese pine and larch are classified into the following three categories:

[0033] Level 1: severe water staining and complete healing;

[0034] Level 2: Severe water staining, slight collapse at the healing site, and obvious browning;

[0035] Level 3: Severe water stains, obvious browning at the healing site, and slight collapse at the healing site.

[0036] Preferably, in the step seven, the healing part of the 1-day Chinese pine twig is white after TTC staining, has lost vitality and has no growth characteristics, and the number of pine wood nematodes separated and counted by Baermann funnel is ≥12,000, which has a high sensitivity to pine wood nematodes and does not have good resistance. The healing part of the 1-day larch twig is light red after TTC staining, has obvious growth characteristics, and the number of pine wood nematodes separated and counted by Baermann funnel is ≤6,500, which has a high resistance to pine wood nematodes.

[0037] Beneficial Effects

[0038] The present invention provides a method for studying the resistance mechanism of larch to pine wood nematodes using a Chinese pine control. Compared with the prior art, it has the following beneficial effects:

[0039] 1. The present invention provides a method for studying the resistance mechanism of larch to pine wood nematodes based on Pinus tabulaeformis as a control. The larch and Pinus tabulaeformis are inoculated with pine wood nematodes and manually pruned to form healing sites at the pruned sites of the larch and Pinus tabulaeformis. The healing sites of the larch and Pinus tabulaeformis are classified and the nematodes are separated by a Baermann funnel. The number of proliferation of pine wood nematodes is counted. It can be seen that the healing sites of the larch after inoculation with pine wood nematodes are light red after TTC staining, have obvious growth characteristics, and the number of proliferation of pine wood nematodes is ≤6500. The healing sites of the pine branches after inoculation with pine wood nematodes are white after TTC staining, lose vitality and have no growth characteristics, and the number of proliferation of wood nematodes is ≥12000. Through a series of studies and experimental data analysis It can be seen that larch shows significant resistance characteristics when facing the invasion of pine wood nematodes, while Chinese pine shows high sensitivity to the action of pine wood nematodes. In terms of physiological indicators, after being infected with pine wood nematodes, the internal physiological metabolic process of larch can maintain a good balance, and the changes in various physiological indicators are relatively small. In contrast, the physiological indicators of Chinese pine showed more obvious fluctuations. The analysis of flavonoid content and polyphenol content showed that larch has an inhibitory effect on the feeding and growth of pine wood nematodes, while the chemical components with resistance effects in Chinese pine are relatively few or low in content. This characteristic provides an important theoretical basis and practical basis for the development of pine wood nematode control technology and the cultivation of insect-resistant tree species.

[0040] 2. The present invention provides a method for studying the mechanism of resistance of larch to pine wood nematodes using a control of Chinese pine. In the experiment, after the larch and Chinese pine were inoculated with pine wood nematodes, the changes in their polyphenol content and flavonoid content were detected respectively. The results showed that after the larch was inoculated with pine wood nematodes, the polyphenol content and flavonoid content first decreased, then returned to normal and increased; while after the Chinese pine was inoculated with pine wood nematodes, the phenol content and flavonoid content continued to decrease. Based on the changes in the above chemical components, it can be concluded that larch has resistance to pine wood nematodes during its growth, while Chinese pine lacks such resistance. This difference provides a key theoretical basis and practical direction for the control of pine wood nematodes and the cultivation of insect-resistant tree species. By further studying the chemical components related to resistance in larch and their mechanisms of action, it is expected to develop more effective pine wood nematode control technology and provide scientific guidance for the selection of insect-resistant tree species. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the flavonoid content of larch of the present invention;

[0042] Figure 2 This is a schematic diagram of the flavonoid content of Pinus tabulaeformis of the present invention;

[0043] Figure 3 This is a schematic diagram of the polyphenol content of larch of the present invention;

[0044] Figure 4 This is a schematic diagram of the polyphenol content of Pinus tabulaeformis of the present invention;

[0045] Figure 5 It is a schematic diagram of the experimental process of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1

[0048] See also Figure 1-Figure 5 The present invention provides two technical solutions:

[0049] The first implementation method: a method for studying the resistance mechanism of larch to pine wood nematodes based on Pinus tabulaeformis as a control, comprising the following steps:

[0050] Step 1: Extraction of pine wood nematodes as raw materials for subsequent experiments;

[0051] Step 2: co-culturing pine wood nematodes and Botrytis cinerea to serve as experimental raw materials;

[0052] Step 3, inoculating larch and Chinese pine with pine wood nematodes;

[0053] Step 4, extracting the flavonoids content of larch;

[0054] Step 5, extracting the polyphenol content of Pinus tabulaeformis;

[0055] Step 6: Manually prune the inoculated larch and Chinese pine, so that healing areas are formed at the pruning sites of the larch and Chinese pine;

[0056] Step 7: Classify the healing sites of larch and Chinese pine, separate the nematodes using Baermann funnel, and count the number of pine wood nematodes proliferating.

[0057] The extraction of pine wood nematodes includes the following steps:

[0058] S1. Sample collection

[0059] Select trees with typical symptoms of pine wilt disease from naturally infected larch forests. Typical symptoms of pine wilt disease include but are not limited to needle discoloration and wilting. Use saws or scissors to collect branch and trunk samples of diseased trees, and select tissues close to the lesions. Cut the collected larch samples into small sections of 1-2 cm.

[0060] S1.2, Baermann funnel separation,

[0061] Place the larch sample in a Baermann funnel and add a certain amount of water to the top of the funnel to immerse the sample in water. Control the ambient temperature of the Baermann funnel to 25°C and leave it for 24-48 hours to allow the nematodes to escape from the sample tissue and enter the collection tube through the filter paper at the bottom of the funnel, thereby obtaining a nematode suspension. Observe the nematode suspension at the bottom of the collection funnel under a microscope and identify it based on its morphological characteristics: body length and stylet length, to ensure that the nematodes collected are pine wood nematodes.

[0062] S1.3, Nematode purification,

[0063] Transfer the nematode suspension collected by the Baermann funnel to a centrifuge tube, use a centrifuge and control the speed to 3000 rpm for 5-10 minutes to allow the nematodes to settle to the bottom of the tube, discard the supernatant, add an appropriate amount of sterile water to resuspend the nematodes, repeat the centrifugation 2-3 times to remove impurities, and observe the purified nematodes under a microscope to ensure the purity and activity of the nematodes;

[0064] The co-cultivation of pine wood nematode and Botrytis cinerea includes the following steps:

[0065] S2. Cultivate PDA.

[0066] Prepare a potato dextrose agar (PDA) medium, inoculate Botrytis cinerea onto the medium under sterile conditions, and place the culture dish in a constant temperature incubator at 25°C for 3-5 days until the Botrytis cinerea grows all over the culture dish;

[0067] S2.1. Co-culture

[0068] The purified pine wood nematodes were inoculated into a culture dish full of Botrytis cinerea, and different inoculation density gradients were set, with 50, 100, and 150 nematodes per square centimeter. Multiple replicates were set for each gradient, and the inoculated culture dish was returned to a constant temperature incubator and cultured at 25°C;

[0069] S2.2, Experimental data recording,

[0070] Record the number and vitality of nematodes, randomly select multiple fields of view from each culture dish at different time points (1d, 3d, 5d, 7d, 9d, 14d, and 21d) after co-culture, count the number of pine wood nematodes under a microscope, and observe the vitality, movement ability, and morphological integrity of the nematodes, record the changes in the number of pine wood nematodes at different inoculation densities and different culture times, draw a curve of the change in the number of nematodes over time, and the reproduction status, including but not limited to observing the reproduction status of pine wood nematodes during co-culture, recording the number of eggs produced, and the number of larvae hatched;

[0071] Inoculate pine wood nematodes. Select 1-year-old, 2-year-old, and 3-year-old twigs, large branches, and trunks from healthy Chinese pine and larch, respectively, to ensure that the samples are fresh and free of pests and diseases. Inoculate larch, and take samples at designated time points of 1d, 3d, 5d, 7d, 9d, 14d, and 21d. At the same time, take uninoculated Chinese pine at the same time point as a control. Use a sterile syringe to inoculate the prepared pine wood nematode suspension into different parts of the tree, including but not limited to twigs, large branches, and trunks.

[0072] Extract the flavonoids content of larch, ensure that the sample is fresh, clean it to remove surface impurities, cut the sample into small pieces or powder according to research needs to increase the surface area and improve the extraction efficiency, and choose ethanol as the extractant, which can effectively dissolve flavonoids and is relatively mild and friendly to heat-sensitive flavonoids. Extract the flavonoids content of larch, mix the sample with ethanol in a certain proportion, put it into an ultrasonic extractor, turn on the ultrasonic wave, use the mechanical vibration and cavitation effect it produces to destroy the cell wall and promote the release of flavonoids. The temperature is controlled between 40-50℃ to maintain the extraction efficiency while protecting the flavonoids from being destroyed by high temperature. The time is controlled between 30 and 60 minutes to ensure full extraction while avoiding component degradation caused by over-extraction. After the extraction is completed, let the mixed solution stand to allow solid particles to precipitate or separate solids and liquids by filtration, and then concentrate the extract. Use a rotary evaporator to reduce the volume of the solvent to obtain a concentrated flavonoid extract.

[0073] To extract the polyphenol content of Pinus tabulaeformis, select large branches from Pinus tabulaeformis, remove dead leaves and impurities, and then cut the samples into small pieces to increase the surface area. Then dry them to remove moisture, and choose ethanol as the extraction solvent. The ratio is 1g / mL sample weight: 10g / mL solvent volume. After the sample powder and the solvent are mixed evenly, they are placed in a container suitable for ultrasonic extraction for solid-liquid separation, and filter paper or filter extraction liquid is used to remove residual solid matter;

[0074] Select healthy larch and Chinese pine plants that have been inoculated with pine wood nematodes, place the experimental plants in a sterile environment to adapt them for a period of time, and ensure that the plants are in a relatively stable physiological state after being inoculated with pine wood nematodes;

[0075] Soak scissors and pruning shears in a disinfectant 0.1% potassium permanganate solution for 15-20 minutes, then rinse with sterile water to prevent the introduction of pathogens during pruning and affect the formation of healing sites. For larch and Chinese pine, select representative sites on tender branches, large branches and trunks for pruning. When pruning, ensure that the incision is smooth and minimize damage to surrounding tissues. The incision area should not exceed 1 / 3 of the cross-sectional area of ​​the branch or trunk. After pruning, immediately apply disinfectant to the incision to prevent pathogen infection, then use a mixed solution of indoleacetic acid and cytokinin to apply to the wound, and then wrap the incision to keep it moist.

[0076] The treated larch and Chinese pine plants were placed in an incubator set at 25 ± 2°C, 70%-80% humidity and 12-16 hours of light per day, and the formation of healing sites at the incisions was regularly observed at different times after inoculation, such as 1d, 3d, 5d, 7d, 9d and 14d;

[0077] The healing part of the 1-day-old Chinese pine twigs was white after TTC staining, and had lost vitality and no growth characteristics. The number of pine wood nematodes separated and counted by Baermann funnel was ≥12,000, indicating that it was highly sensitive to pine wood nematodes and did not have good resistance. The healing part of the 1-day-old larch twigs was light red after TTC staining, and had obvious growth characteristics. The number of pine wood nematodes separated and counted by Baermann funnel was ≤6,500, indicating that it had high resistance to pine wood nematodes.

[0078] The second implementation method is mainly different from the first implementation method in that the healing sites of Pinus tabulaeformis and Larch are observed and recorded, wherein the recovery levels of the healing sites of Pinus tabulaeformis and Larch are classified into the following three categories:

[0079] Level 1: severe water staining and complete healing;

[0080] Level 2: Severe water staining, slight collapse at the healing site, and obvious browning;

[0081] Grade 3: Severe waterlogging, obvious browning at the healing site, and slight collapse at the healing site; samples were taken from the healing sites of Pinus tabulaeformis and Larch, and the healing sites of Pinus tabulaeformis and Larch were observed and analyzed.

[0082] Class, using 14d, 1d of Chinese pine and larch healing place ;

[0083] The healing part of the 1d pine twig is seriously water-soaked, with obvious browning and slight collapse at the healing part, which is the third level.

[0084] 1d The healing part of larch twigs was seriously waterlogged, with slight collapse and obvious browning at the healing part, which was rated as Level 2.

[0085] On the 14th day, the healing part of the large branch of Pinus tabulaeformis was seriously water-soaked, with slight collapse and obvious browning, which was rated as Level 2.

[0086] 14d The healing part of the larch branch was seriously waterlogged, and the healing part was intact and rated as level one.

[0087] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0088] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0089] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for studying the resistance mechanism of larch to pine wood nematodes based on Pinus tabulaeformis, characterized in that: The following steps are involved: Step 1: Extraction of pine wood nematodes as raw materials for subsequent experiments; Step 2: co-culturing pine wood nematodes and Botrytis cinerea to serve as experimental raw materials; Step 3, inoculating larch and Chinese pine with pine wood nematodes; Step 4, extracting the flavonoids content of larch; Step 5, extracting the polyphenol content of Pinus tabulaeformis; Step 6: Manually prune the inoculated larch and Chinese pine, so that healing areas are formed at the pruning sites of the larch and Chinese pine; Step 7: Classify the healing sites of larch and Chinese pine, separate the nematodes using Baermann funnel, and count the number of pine wood nematodes proliferating.

2. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Chinese pine according to claim 1, characterized in that: The step one includes the following steps: S1. Sample collection: Select trees with typical symptoms of pine wilt disease from naturally infected larch forests, collect branch and trunk samples of diseased trees using saws or scissors, and select tissues close to the lesions. Cut the collected larch samples into small sections of 1-2 cm; S1.2, Baerman funnel separation: Place the larch sample in a Baermann funnel, add a certain amount of water to the top of the funnel, immerse the sample in water, and control the ambient temperature of the Baermann funnel to be 25°C. Leave it for 24-48 hours to allow the nematodes to escape from the sample tissue and enter the collection tube through the filter paper at the bottom of the funnel, thereby obtaining a nematode suspension. S1.

3. Nematode purification: Transfer the nematode suspension collected by the Baermann funnel to a centrifuge tube and centrifuge at 3000 rpm for 5-10 minutes to allow the nematodes to settle to the bottom of the tube. Discard the supernatant, add an appropriate amount of sterile water to resuspend the nematodes, and repeat the centrifugation 2-3 times to remove impurities.

3. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Chinese pine according to claim 1, characterized in that: The step 2 includes the following steps: S2. Cultivate PDA: Prepare a potato dextrose agar (PDA) medium, inoculate Botrytis cinerea onto the medium under sterile conditions, and place the culture dish in a constant temperature incubator at 25°C for 3-5 days until the Botrytis cinerea grows all over the culture dish; S2.

1. Co-culture: The purified pine wood nematodes were inoculated into a culture dish full of Botrytis cinerea, and different inoculation density gradients were set, with 50, 100, and 150 nematodes per square centimeter; S2.

2. Experimental data recording: The number and vitality of nematodes were recorded. At different time points after co-culture (1d, 3d, 5d, 7d, 9d, 14d, and 21d), multiple fields of view were randomly selected from each culture dish, the number of pine wood nematodes was counted under a microscope, and the vitality, motility, and morphological integrity of the nematodes were observed.

4. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Chinese pine according to claim 1, characterized in that: In the step three, 1-year-old, 2-year-old and 3-year-old twigs, large branches and trunks are selected from healthy Chinese pine and larch, respectively, to ensure that the samples are fresh and free of pests and diseases. The larch is inoculated, and samples are taken at designated time points of 1d, 3d, 5d, 7d, 9d, 14d and 21d. At the same time, uninoculated Chinese pine at the same time point is taken as a control, and the prepared pine wood nematode suspension is inoculated into different parts of the tree, including twigs, large branches and trunks, using a sterile syringe.

5. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Chinese pine according to claim 1, characterized in that: In step 4, the sample is mixed with ethanol in a certain proportion, loaded into an ultrasonic extractor, and ultrasound is turned on to utilize the mechanical vibration and cavitation effect generated by the ultrasound to destroy the cell wall and promote the release of flavonoid compounds. After the extraction is completed, the mixed solution is allowed to stand to allow solid particles to precipitate or the solid and liquid are separated by filtration.

6. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Chinese pine according to claim 1, characterized in that: In the step 5, the polyphenol content of Pinus tabulaeformis is extracted, large branches are selected from Pinus tabulaeformis, dead leaves and impurities are removed, and the sample is cut into small pieces to increase the surface area, and then dried to remove moisture, and ethanol is selected as the extraction solvent, and the ratio is 1g / mL sample weight: 10g / mL solvent volume. After the sample powder and the solvent are evenly mixed, they are placed in a container suitable for ultrasonic extraction for solid-liquid separation.

7. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Pinus tabulaeformis according to claim 1, characterized in that: In the step six, the scissors and pruning shears are soaked in a disinfectant 0.1% potassium permanganate solution for 15-20 minutes, and then rinsed with sterile water to prevent the introduction of pathogens during the pruning process and affect the formation of the healing site. For larch and Chinese pine, representative parts are selected on the tender branches, large branches and trunks for pruning. When pruning, ensure that the incision is smooth and minimize damage to surrounding tissues. The incision area should not exceed 1 / 3 of the cross-sectional area of ​​the branch or trunk. After pruning, the incision is immediately smeared with a disinfectant to disinfect it to prevent infection by pathogens. A mixed solution of indoleacetic acid and cytokinin is then used to smear the wound, and then the incision is wrapped to maintain the humidity of the incision.

8. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Chinese pine according to claim 1, characterized in that: In the step 7, the healing sites of the Chinese pine and larch are observed and recorded, wherein the recovery levels of the healing sites of the Chinese pine and larch are classified into the following three categories: Level 1: severe water staining and complete healing; Level 2: Severe water staining, slight collapse at the healing site, and obvious browning; Level 3: Severe water stains, obvious browning at the healing site, and slight collapse at the healing site.

9. The method for studying the resistance mechanism of larch to pine wood nematodes using a control of Chinese pine according to claim 1, characterized in that: In the step seven, the healing part of the 1-day Chinese pine twig was stained white by TTC, lost vitality and had no growth characteristics, and the number of pine wood nematodes separated and counted by Baermann funnel was ≥12,000, which had high sensitivity to pine wood nematodes and did not have good resistance. The healing part of the 1-day larch twig was stained light red by TTC, had obvious growth characteristics, and the number of pine wood nematodes separated and counted by Baermann funnel was ≤6,500, which had high resistance to pine wood nematodes.

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