A method for precisely obtaining pine wood nematodes from inoculated pine trees

Abstract

This invention discloses a method for accurately obtaining pine wood nematodes from inoculated pine trees, relating to the field of pest control technology. The method includes the following steps: (1) collecting pine wood nematodes and preparing a nematode suspension; (2) making a 45° oblique cut on the pine bark at the inoculation point using a sterilized scalpel blade, with the wound reaching the phloem; (3) placing defatted cotton into the wound and sealing the bottom of the wound with Parafilm sealing film wrapped around the pine trunk; (4) dripping the nematode suspension onto the defatted cotton using a pipette; (5) at a specific time after inoculation, cutting the pine branches and twigs below the inoculation point to separate and collect the pine wood nematodes, and performing data statistics and analysis. This invention confirms that a method can accurately obtain the vast majority of pine wood nematodes in inoculated pine trees by collecting only branches and twigs within 20 cm below the inoculation point.

Description

Technical Field

[0001] This invention relates to the field of pest control technology, and more specifically to a method for accurately obtaining pine wilt nematodes from inoculated pine trees. Background Technology

[0002] Pine wilt nematode is a globally quarantined forest pest that causes pine wilt disease after infecting parasitic pine trees, causing devastating damage to the pine industry worldwide. In the early stages, pine needles wilt and turn yellow; as the disease progresses, the needles turn reddish-brown, eventually leading to the death of the entire pine tree. The pathogenesis of pine wilt disease is complex, involving the pine nematode, the host pine tree, microorganisms, climate, and human factors, and currently, there are no scientifically effective control methods. In the laboratory, artificial inoculation is a common experimental method for studying the pathogenesis and control techniques of pine wilt nematode. However, the migration direction and activity characteristics of pine wilt nematodes within pine trees after inoculation are unclear. Therefore, accurately locating the migration direction and aggregation behavior of pine wilt nematodes in pine trees will provide methodological support for conducting indoor inoculation experiments.

[0003] Research on the damage mechanism of pine wilt nematode and the control technology of pine wilt disease requires inoculating pine wilt nematodes into pine trees, isolating the nematodes from the pine trees within a specific time after inoculation, and statistically analyzing the number of pine wilt nematodes in the pine trees to compare the reproductive and parasitic abilities of pine wilt nematodes in different pine trees, thereby inferring the resistance of different pine trees to pine wilt nematode disease.

[0004] Therefore, providing an experimental method for accurately obtaining pine wood nematodes from inoculated pine trees and accurately analyzing inoculation data is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for accurately obtaining pine wood nematodes from inoculated pine trees.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for accurately obtaining pine wood nematodes from inoculated pine trees includes the following steps:

[0008] (1) Collect pine wood nematodes and prepare a nematode suspension;

[0009] (2) Use a sterilized scalpel blade to make a 45° oblique cut on the pine bark at the inoculation point, with the wound reaching the phloem;

[0010] (3) Place the degreased cotton into the wound, and wrap the sealing film around the pine trunk to seal the bottom of the wound;

[0011] (4) Use a pipette to drop the nematode suspension onto a piece of defatted cotton;

[0012] (5) At a specific time after inoculation, cut the pine branches and twigs within 20 cm below the inoculation point, separate and collect the pine wood nematodes, and conduct data statistics and analysis.

[0013] Furthermore, the pine wood nematodes mentioned in step (1) are artificially cultured in the laboratory, and the collection method is to separate and collect them using the Behman funnel method.

[0014] Furthermore, the concentration of the nematode suspension in step (1) is 1000 nematodes / mL.

[0015] Furthermore, in step (2), the pine tree is planted in a net house, and the inoculation point is at least 20 centimeters away from the first branch of the pine tree.

[0016] Further, in step (4), each pine tree is inoculated with 1,000 pine wood nematodes.

[0017] Furthermore, the specific time after inoculation in step (5) is 28 days, and the collection method is to separate and collect the samples using the Behmann funnel method.

[0018] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The traditional method involves cutting all branches and trunks of pine trees inoculated with pine wood nematodes into pieces and then separating and collecting the nematodes through a Behman funnel. This invention clarifies the behavioral characteristics of pine wood nematodes after inoculation, showing that they mainly migrate downwards and aggregate within the pine tree, centered on the inoculation point. It also confirms a method that can accurately obtain the vast majority of pine wood nematodes in inoculated pine trees by collecting only the trunk below the inoculation point.

[0020] This invention determines that when conducting experiments on pine trees inoculated with pine wood nematodes indoors, only pine branches within 20 cm below the inoculation point need to be collected. After separation using the Behman funnel method, more than 95% of the pine wood nematodes in the pine tree can be obtained. Compared with conventional methods of isolating the entire pine tree, this method significantly reduces workload, saves working time, and shortens the experimental process. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The attached figure shows a comparison of the differences in the number of pine wood nematodes in different parts of the pine tree after inoculation. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] (1) Artificially cultured pine wood nematodes were separated and collected by the Behman funnel method and prepared into a nematode suspension with a concentration of 1000 nematodes / mL.

[0026] (2) A total of 45 two-year-old pine seedlings in the net house were divided into 3 groups: the group above the inoculation point, the group below the inoculation point, and the whole plant survey group, with 15 plants in each group.

[0027] The area within 20 cm above the inoculation point is the "above inoculation point" group, and the area within 20 cm below the inoculation point is the "below inoculation point" group. All branches and trunks of the entire plant are considered the whole plant.

[0028] The inoculation point should be at least 20 centimeters away from the first branch. Use a sterilized scalpel blade to make a 45° oblique cut on the pine bark, with a wound depth of about 1 centimeter.

[0029] (3) Place sterile degreased cotton in the wound and wrap it around the pine trunk with Parafilm to seal the bottom of the wound.

[0030] (4) Using the same inoculation method, 1,000 pine wood nematodes were inoculated into each pine tree. 1 mL of pine wood nematode suspension was taken with a pipette and slowly dripped onto defatted cotton.

[0031] (5) 28 days after inoculation, pine branches were collected from each group according to the experimental group. The pine branches or twigs were cut into 1 cm long sections. After being cut into pieces, the pine wood nematodes were separated and collected using the Behman funnel method. Data statistics and analysis were carried out to compare the number of pine wood nematodes in different parts.

[0032] The Behman funnel method for isolating pine wood nematodes: Place a funnel with a diameter of 10-15 cm on a stand, attach a 10 cm section of rubber tubing to the bottom, and connect a 1.5 ml centrifuge tube to the rubber tubing. Fill the funnel with water and squeeze the rubber tubing to expel the air. Cut pine branches into small pieces, wrap them in kitchen paper towels, and place them in the funnel filled with water. Let them stand for 24 hours, then remove the centrifuge tube and count the nematodes under a microscope.

[0033] V. The experimental results are as follows:

[0034] Table 1: Comparison of pine wilt nematode numbers in different parts of pine trees after inoculation

[0035]

[0036] Table 1 and Figure 1 The results showed that in the pine wilt nematode inoculation experiment, 28 days after inoculation, the number of nematodes isolated from the main trunk within 20 cm above the inoculation point was extremely low, showing a significant difference compared to the group isolated from ...

[0037] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for accurately obtaining pine wood nematodes from inoculated pine trees, characterized in that, Includes the following steps: (1) Collect pine wood nematodes and prepare a nematode suspension; (2) Use a sterilized scalpel blade to make a 45° oblique cut on the pine bark at the inoculation point, with the wound reaching the phloem; (3) Place the degreased cotton into the wound, and wrap the sealing film around the pine trunk to seal the bottom of the wound; (4) Use a pipette to drop the nematode suspension onto a piece of defatted cotton; (5) At a specific time after inoculation, cut the pine branches and twigs within 20 cm below the inoculation point, separate and collect the pine wood nematodes, and conduct data statistics and analysis.

2. The method according to claim 1, characterized in that, The pine wood nematodes mentioned in step (1) are artificially cultured in the laboratory, and the collection method is to separate and collect them using the Behman funnel method.

3. The method according to claim 1, characterized in that, The concentration of the nematode suspension in step (1) is 1000 nematodes / mL.

4. The method according to claim 1, characterized in that, In step (2), the pine trees are planted in a net house, and the inoculation point is at least 20 centimeters away from the first branch of the pine tree.

5. The method according to claim 1, characterized in that, Step (4) Inoculate each pine tree with 1000 pine wood nematodes.

6. The method according to claim 1, characterized in that, The specific time after inoculation in step (5) is 28 days, and the collection method is to separate and collect the samples using the Behmann funnel method.

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