Research method for resistance mechanism of larch to pine wood nematodes based on Korean pine contrast

Abstract

The invention relates to the technical field of biology, in particular to a Korean pine control-based method for researching the resistance mechanism of larch to pine wood nematodes, which comprises the following steps: S1, extracting pine wood nematodes: separating to-be-tested nematodes from naturally infected larch by using a funnel; s2, pine wood nematode culture: utilizing botrytis cinerea to co-culture pine wood nematode as a test raw material; s3, selecting tree species: selecting twigs, boughs and trunks of healthy larch with tree ages of 1 year, 2 years and 3 years respectively, and selecting Korean pine with the same requirements as a contrast; s4, sampling tree species: sampling larch and Korean pine inoculated for 1 day, 3 days, 5 days, 7 days, 9 days, 14 days and 21 days; by analyzing the chemical components of the Korean pine and the larch in different stages before, during and after the bursaphelenchus xylophilus infection, the purpose of researching the pathogenesis of the Korean pine and the larch by local pine nematodes in Liaoning is achieved, so that the purpose of effectively preventing the bursaphelenchus xylophilus is achieved.

Description

Technical Field

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

[0002] Pine wood nematodes are currently the number one pest that harms my country's forestry production and forest ecological environment. The disease caused by pine wood nematodes is called pine wood nematode disease or pine wilt disease. Due to its rapid spread, strong reproductive capacity, and rapid tree death, pine trees can die as soon as 40 days after infection, and pine forests can be destroyed 3-5 years after infection. The economic losses caused to my country exceed 10 billion yuan each year;

[0003] According to the public number: CN118489482A, a high-efficiency breeding method for a new germplasm of larch with high resistance to pine caterpillars is disclosed. This technology discloses "a high-efficiency breeding method for a new germplasm of larch with high resistance to pine caterpillars, including the steps of collecting larch high-resistant scion, selecting and processing rootstocks, grafting breeding and post-grafting management. In the process of breeding high-resistant scion seedlings, scientific seed winter storage, sowing seedlings and seedling management are used to provide guarantees for obtaining a large number of strong high-resistant scion shoots. The scion uses young branches grown on seedlings for 10-15 days, and the rootstock is selected from 10-15-day-old seedlings with straight and thick stems and full terminal buds. , with highly adaptable larch seedlings with well-developed root systems and no diseases, pests or damage as rootstocks, so that the grafted joints can heal quickly and shorten the breeding cycle. After grafting, they are first grown in a very stable plant growth room for about 2 months to wait for the scion to survive. During the transition period, they are transferred to a greenhouse for 1 year to become strong seedlings. Finally, they are further hardened in outdoor open-air test sites to make the seedlings fully lignified, thereby obtaining a large number of highly resistant larch seedlings for artificial afforestation. The technical solutions have the technical effects of "reducing the probability of pest occurrence from the source, improving the insect resistance of larch itself, and improving forest quality, growth rate and wood utilization efficiency";

[0004] By investigating the resistance of Korean pine, Chinese pine and larch to pine wood nematodes in the forest areas of Liaodong region, artificial injection of pine wood nematodes was carried out on Korean pine, Chinese pine and larch of different ages, and the resistance of the three tree species to pine wood nematodes was studied. It was found that both Korean pine and Chinese pine could be infected and die, while larch showed a certain resistance to pine wood nematode disease. The infection degree of small trees was light, and large trees were not infected. This shows that the study of its insect resistance mechanism is of great significance for the prevention and control of pine wood nematodes. In view of this situation, there is an urgent need to establish a method system specifically for the study of the mechanism of larch resistance to pine wood nematodes, aiming to effectively prevent and control the occurrence of pine wood nematode diseases. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for studying the resistance mechanism of larch to pine wood nematodes based on red pine as a control, which has the characteristics of studying the pathogenic mechanism of local pine nematodes in Liaoning to red pine and larch by analyzing the chemical components of red pine and larch at different stages before, during and after infection with pine wood nematode disease, thereby achieving the purpose of effectively preventing pine wood nematode disease.

[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 Korean pine as a control, comprising the following steps:

[0007] S1, pine wood nematode extraction: the tested nematodes were isolated from naturally infected larch using a funnel;

[0008] S2, pine wood nematode culture: pine wood nematodes co-cultured with Botrytis cinerea were used as experimental raw materials;

[0009] S3, tree species selection: select healthy larch branches, large branches and trunks with the ages of 1, 2 and 3 years, and select Korean pine with the same requirements as the control;

[0010] S4, tree species sampling: larch and Korean pine were sampled 1, 3, 5, 7, 9, 14 and 21 days after inoculation;

[0011] S5, sampling and analysis: using liquid chromatography, mass spectrometry and high performance liquid chromatography to analyze its components and find the unique chemical components of larch;

[0012] S6, analysis of the growth and development of pine wood nematodes: observe the growth and development of pine wood nematodes in larch and evaluate the colonization ability of nematodes in larch;

[0013] S7, transcriptome sequencing: The pine wood nematodes in larch were recovered for transcriptome sequencing, and the pine wood nematodes in the fungus-feeding stage were used as a control for analysis.

[0014] Preferably, in S1, a glass funnel is selected and a latex tube is connected to the bottom, and a water-stop clamp for controlling the outflow of liquid is installed on the latex tube; a sample containing nematodes is collected from naturally diseased larch, wrapped with gauze and placed in a glass funnel, and water is added to the wrapped sample in the funnel until the sample is moistened; due to the hydrotaxis of the pine wood nematodes and their own weight, the pine wood nematodes leave the sample and swim in the water until they settle in the latex tube at the bottom of the funnel; a collecting vessel is placed under the latex tube and the water-stop clamp is opened to extract and collect the pine wood nematodes in the water.

[0015] Preferably, in S2, the Botrytis cinerea hyphae block is inoculated onto a sterilized PDA culture medium plate, the culture dish is turned over so that the bottom of the dish faces upward, and cultured in a 25°C constant temperature box for 5 to 7 days until the hyphae are fully grown; then the pine wood nematodes isolated from naturally infected larch are washed out with sterile water and inoculated onto a culture medium containing Botrytis cinerea, and the pine wood nematodes grow and reproduce using Botrytis cinerea as food to simulate the growth environment under natural conditions.

[0016] Preferably, in S3, by comparing larch and Korean pine samples of different ages, the differences between the two tree species in growth, development and adaptability are understood; and by comparative analysis of young branches, large branches and trunks, the changing patterns and physiological characteristics of different parts of the trees during their growth process are understood.

[0017] Preferably, in S4, the growth of larch and Korean pine after different inoculation times is monitored by regular sampling, and the effect of inoculation on the growth and development of larch and Korean pine, as well as the duration of the inoculation effect, is evaluated by comparing samples after different inoculation times.

[0018] Preferably, in S5, the complex components in larch can be effectively separated by liquid chromatography and high performance liquid chromatography, and mass spectrometry can provide the molecular weight and structural characteristics of the components, providing key information for the identification of the components.

[0019] Preferably, in S6, healthy larch and Korean pine are selected as experimental samples, and the samples are pre-treated by pruning branches and removing pests and diseases; pine wood nematodes are then inoculated using a standardized method, and the inoculation amount is ensured to be consistent; the number, morphology and activity of pine wood nematodes in larch and Korean pine are regularly observed, and the growth curves and key indicators of the reproduction rate of the nematodes in the two types of pine trees are recorded.

[0020] Preferably, in S7, RNA is extracted from the recovered pine wood nematodes and a sequencing library is constructed, and the library is sequenced using a high-throughput sequencing platform to obtain transcriptome data, thereby clarifying the molecular mechanism by which larch affects the growth and fat accumulation of pine wood nematodes; based on the transcriptome sequencing results and in combination with existing research, irritating chemicals that cause pine wood nematodes to be unable to feed normally are screened, and the effects of these chemicals on the feeding behavior of pine wood nematodes are verified through experiments.

[0021] The invention provides a method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control.

[0022] Compared with the prior art, it has the following beneficial effects:

[0023] 1. By taking samples regularly, the growth of larch and Korean pine after different inoculation times is monitored. By comparing samples after different inoculation times, the effects of inoculation on the growth and development of larch and Korean pine, as well as the duration of the inoculation effect, are evaluated. Liquid chromatography and high-performance liquid chromatography can then be used to effectively separate the complex components in larch. Mass spectrometry analysis can provide the molecular weight and structural characteristics of the components, providing key information for the identification of the components.

[0024] 2. By comparing samples of larch and Korean pine of different ages, we can understand the differences in growth, development and adaptability between the two tree species; and by comparing and analyzing young branches, large branches and trunks, we can understand the changing patterns and physiological characteristics of different parts of trees during their growth process, and by analyzing the chemical composition of healthy and diseased larch and Korean pine of different ages, we can find out the pathogenic mechanism of larch in Liaodong region against pine wood nematodes.

[0025] 3. Through RNA extraction and sequencing library construction of recovered pine wood nematodes, the library was sequenced using a high-throughput sequencing platform to obtain transcriptome data, and the molecular mechanism by which larch affects the growth and fat accumulation of pine wood nematodes was clarified; based on the transcriptome sequencing results and combined with existing research, the irritating chemicals that prevent pine wood nematodes from feeding normally were screened, and the effects of these chemicals on the feeding behavior of pine wood nematodes were verified through experiments, and the pine wood nematodes in the fungus-feeding stage were used as a control for analysis to find the irritating chemicals that prevent pine wood nematodes from feeding normally. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flowchart of the research steps of the pine wood nematode resistance mechanism of the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] See also Figure 1 The present invention provides a technical solution for a method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control: a method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control, comprising the following steps:

[0029] S1, pine wood nematode extraction: the tested nematodes were isolated from naturally infected larch using a funnel;

[0030] S2, pine wood nematode culture: pine wood nematodes co-cultured with Botrytis cinerea were used as experimental raw materials;

[0031] S3, tree species selection: select healthy larch branches, large branches and trunks with the ages of 1, 2 and 3 years, and select Korean pine with the same requirements as the control;

[0032] S4, tree species sampling: larch and Korean pine were sampled 1, 3, 5, 7, 9, 14 and 21 days after inoculation;

[0033] S5, sampling and analysis: using liquid chromatography, mass spectrometry and high performance liquid chromatography to analyze its components and find the unique chemical components of larch;

[0034] S6, analysis of the growth and development of pine wood nematodes: observe the growth and development of pine wood nematodes in larch and evaluate the colonization ability of nematodes in larch;

[0035] S7, transcriptome sequencing: The pine wood nematodes in larch were recovered for transcriptome sequencing, and the pine wood nematodes in the fungus-feeding stage were used as a control for analysis.

[0036] In this implementation plan, by analyzing the chemical composition of red pine and larch at different stages before, during and after infection with pine wilt disease, the purpose of studying the pathogenicity mechanism of local pine nematodes in Liaoning to red pine and larch is achieved, thereby achieving the purpose of effectively preventing pine wilt disease.

[0037] Specifically, in S1, a glass funnel is selected and a latex tube is connected to the bottom, and a water-stop clamp for controlling the outflow of liquid is installed on the latex tube; a sample containing nematodes is collected from naturally diseased larch, wrapped with gauze and placed in a glass funnel, and water is added to the wrapped sample in the funnel until the sample is moistened; the pine wood nematodes leave the sample and swim in the water due to the hydrotaxis and their own weight until they settle in the latex tube at the bottom of the funnel; a collecting vessel is placed under the latex tube and the water-stop clamp is opened to extract and collect the pine wood nematodes in the water.

[0038] In this embodiment, the funnel separation method utilizes the hydrophilicity of nematodes to achieve rapid separation of nematodes from plant tissues; moreover, the nematodes are less damaged during the operation, which is beneficial to maintaining the activity of the nematodes.

[0039] Specifically, in S2, the mycelium block of Botrytis cinerea is inoculated onto a sterilized PDA culture medium plate, the culture dish is turned over so that the bottom of the dish faces upward, and it is placed in a 25°C constant temperature box for culture for 5 to 7 days until the mycelium is fully grown; then the pine wood nematodes isolated from naturally infected larch are washed out with sterile water and inoculated onto a culture medium containing Botrytis cinerea. The pine wood nematodes grow and reproduce using Botrytis cinerea as food to simulate the growth environment under natural conditions.

[0040] In this embodiment, using Botrytis cinerea to co-cultivate pine wood nematodes can quickly obtain a large number of nematode samples to meet experimental needs; and because Botrytis cinerea is a natural food source for pine wood nematodes, using it for co-cultivation can ensure the accuracy and reliability of the experimental results.

[0041] Specifically, S3 compares larch and Korean pine samples of different ages to understand the differences in growth, development and adaptability between the two tree species; and through comparative analysis of young branches, large branches and trunks, understands the changing patterns and physiological characteristics of different parts of trees during growth.

[0042] In this embodiment, by comparing larch and Korean pine samples of different ages, we can have a deeper understanding of the physiological changes and growth mechanisms of trees at different growth stages, including nutrient absorption, water transport, photosynthesis, etc.

[0043] Specifically, in S4, the growth of larch and Korean pine after different inoculation times was monitored by regular sampling, and the effect of inoculation on the growth and development of larch and Korean pine, as well as the duration of the inoculation effect, was evaluated by comparing samples after different inoculation times.

[0044] In this embodiment, early sampling 1 to 3 days after inoculation can evaluate the plant's immediate response to pine wood nematodes, including the activation of defense mechanisms and initial resistance manifestations; mid-term sampling 7 to 14 days after inoculation can be used to observe whether the resistance of larch and Korean pine gradually increases, remains stable, or declines after inoculation with pine wood nematodes, which helps to understand the dynamic process of plant-nematode interaction; late sampling 14 to 21 days after inoculation can evaluate the plant's sustained resistance to pine wood nematodes, understand whether the resistance mechanism is stable and effective, and whether there is a phenomenon of resistance decline.

[0045] Specifically, in S5, liquid chromatography and high-performance liquid chromatography can effectively separate the complex components in larch, and mass spectrometry analysis can provide the molecular weight and structural characteristics of the components, providing key information for the identification of the components.

[0046] In this embodiment, through component analysis, the content and proportion of various chemical components in larch can be understood. Liquid chromatography and high-performance liquid chromatography can effectively separate the complex components in larch through different chromatographic columns and mobile phase combinations. This efficient separation capability enables researchers to clearly observe the position of each component in the chromatogram, providing a basis for subsequent component identification; mass spectrometry can provide the molecular weight and structural characteristics of each component in larch, which is key information for component identification. Through the mass spectrum, researchers can infer the chemical structure of the components and further understand their possible functions and mechanisms of action; through liquid chromatography, high-performance liquid chromatography and mass spectrometry, the relationship between each component and resistance in larch can be deeply understood. For example, certain specific components may have the effect of inhibiting the growth or reproduction of pine wood nematodes, thus becoming a key factor in larch resistance.

[0047] Specifically, S6 selected healthy larch and Korean pine as experimental samples, and pre-treated the samples by pruning branches and removing pests and diseases. Then, a standardized method was used to inoculate pine wood nematodes, and the inoculation amount was ensured to be consistent. The number, morphology and activity of pine wood nematodes in larch and Korean pine were regularly observed, and the growth curves and key indicators of the reproduction rate of the nematodes in the two pine trees were recorded.

[0048] In this embodiment, by observing the growth and development of pine wood nematodes in larch, the resistance mechanism of larch to pine wood nematodes can be revealed, providing a theoretical basis for disease-resistant breeding; and by evaluating the colonization ability of nematodes in larch, the resistance level of larch to pine wood nematodes can be quantified, providing a basis for formulating prevention and control measures.

[0049] Specifically, in S7, RNA was extracted and sequencing libraries were constructed for the recovered pine wood nematodes, and the libraries were sequenced using a high-throughput sequencing platform to obtain transcriptome data, thereby clarifying the molecular mechanism by which larch affects the growth and fat accumulation of pine wood nematodes. Based on the transcriptome sequencing results and in combination with existing research, irritating chemicals that prevent pine wood nematodes from feeding normally were screened, and the effects of these chemicals on the feeding behavior of pine wood nematodes were verified through experiments.

[0050] In this embodiment, firstly, the transcriptome data of pine wood nematodes and fungus-feeding stage pine wood nematodes in larch are obtained, and through comparative analysis, differentially expressed genes related to larch resistance can be found; secondly, differentially expressed genes related to pine wood nematode growth, fat accumulation and feeding behavior are found, and these genes may be involved in regulating the adaptive changes of pine wood nematodes in larch; then, irritating chemicals that may cause pine wood nematodes to be unable to feed normally are screened out, and through experimental verification, the specific effects of these chemicals on the feeding behavior of pine wood nematodes can be confirmed.

[0051] 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", "comprises" or any other variations 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.

[0052] 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 Korean pine, characterized in that: The following steps are involved: S1, pine wood nematode extraction: the tested nematodes were isolated from naturally infected larch using a funnel; S2, pine wood nematode culture: pine wood nematodes co-cultured with Botrytis cinerea were used as experimental raw materials; S3, tree species selection: select healthy larch branches, large branches and trunks with the ages of 1, 2 and 3 years, and select Korean pine with the same requirements as the control; S4, tree species sampling: larch and Korean pine were sampled 1, 3, 5, 7, 9, 14 and 21 days after inoculation; S5, sampling and analysis: using liquid chromatography, mass spectrometry and high performance liquid chromatography to analyze its components and find the unique chemical components of larch; S6, pine wood nematode growth and development analysis: observe the growth and development of pine wood nematodes in larch and evaluate the colonization ability of nematodes in larch; S7, transcriptome sequencing: The pine wood nematodes in larch were recovered for transcriptome sequencing, and the pine wood nematodes in the fungus-feeding stage were used as a control for analysis.

2. The method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control according to claim 1, characterized in that: In S1, a glass funnel is selected and a latex tube is connected to the bottom, and a water-stop clamp for controlling the outflow of liquid is installed on the latex tube; a sample containing nematodes is collected from naturally diseased larch, wrapped with gauze and placed in a glass funnel, and water is added to the wrapped sample in the funnel until the sample is moistened; the pine wood nematodes leave the sample and swim in the water due to their hydrotaxis and their own weight until they settle in the latex tube at the bottom of the funnel; a collecting vessel is placed under the latex tube and the water-stop clamp is opened to extract and collect the pine wood nematodes in the water.

3. The method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control according to claim 1, characterized in that: In S2, the mycelium block of Botrytis cinerea is inoculated onto a sterilized PDA culture medium plate, the culture dish is turned over so that the bottom of the dish faces upward, and it is placed in a 25°C constant temperature box for culture for 5 to 7 days until the mycelium is fully grown; then the pine wood nematodes isolated from naturally infected larch are washed out with sterile water and inoculated onto a culture medium containing Botrytis cinerea. The pine wood nematodes grow and reproduce using Botrytis cinerea as food to simulate the growth environment under natural conditions.

4. The method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control according to claim 1, characterized in that: In S3, by comparing larch and Korean pine samples of different ages, we can understand the differences in growth, development and adaptability between the two tree species; and by comparing and analyzing young branches, large branches and trunks, we can understand the changing patterns and physiological characteristics of different parts of trees during their growth process.

5. The method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control according to claim 1, characterized in that: In S4, the growth of larch and Korean pine after different inoculation times was monitored by regular sampling, and the impact of inoculation on the growth and development of larch and Korean pine, as well as the duration of the inoculation effect, was evaluated by comparing samples after different inoculation times.

6. The method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control according to claim 1, characterized in that: In S5, liquid chromatography and high-performance liquid chromatography can effectively separate the complex components in larch, and mass spectrometry analysis can provide the molecular weight and structural characteristics of the components, providing key information for the identification of the components.

7. The method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control according to claim 1, characterized in that: In S6, healthy larch and Korean pine were selected as experimental samples, and the samples were pre-treated by pruning branches and removing pests and diseases. Then, pine wood nematodes were inoculated using a standardized method, and the inoculation amount was ensured to be consistent. The number, morphology and activity of pine wood nematodes in larch and Korean pine were regularly observed, and the growth curves and key indicators of the reproduction rate of the nematodes in the two pine trees were recorded.

8. The method for studying the resistance mechanism of larch to pine wood nematodes based on Korean pine as a control according to claim 1, characterized in that: In S7, RNA was extracted from the recovered pine wood nematodes and a sequencing library was constructed. The library was sequenced using a high-throughput sequencing platform to obtain transcriptome data and clarify the molecular mechanism by which larch affects the growth and fat accumulation of pine wood nematodes. Based on the transcriptome sequencing results and in combination with existing research, irritating chemicals that prevent pine wood nematodes from feeding normally were screened, and the effects of these chemicals on the feeding behavior of pine wood nematodes were verified through experiments.

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