Method for accurately obtaining pine wood nematodes from inoculated pine trees

Abstract

The invention discloses a method for accurately obtaining pine wood nematodes from inoculated pine trees, and relates to the technical field of pest control. Comprising the following steps: (1) collecting pine wood nematodes, and preparing a nematode suspension; (2) obliquely cutting the skin of the pine tree at 45 degrees by using a sterilized surgical blade at an inoculation point, wherein the wound depth reaches the phloem; (3) putting degreased cotton into the wound, and winding the wound along the trunk of the pine tree by using a Parafilm sealing film to tightly seal the bottom of the wound; (4) dripping the nematode suspension onto the degreased cotton by using a pipettor; and (5) in specific time after inoculation, cutting pine tree branches and branches below an inoculation point into pieces, separating and collecting pine wood nematodes, and performing data statistics and analysis. According to the method, the vast majority of pine wood nematodes in the inoculated pine tree can be accurately obtained only by collecting branches and trunks within 20 cm below the inoculation point.

Description

Technical Field

[0001] The present invention relates to the technical field of pest control, and more particularly to a method for accurately obtaining pine wood nematodes from inoculated pine trees. Background Art

[0002] The pine wilt nematode is a globally quarantined forest pest. It infects parasitic pine trees and causes pine wilt disease, which has a devastating impact on the pine industry worldwide. In the early stages of the disease, pine needles wilt and turn yellow. As the disease progresses, the needles turn reddish-brown, and eventually the entire pine tree dies. The pathogenic mechanism of pine wilt disease is complex, involving pine wilt nematodes, host pine trees, microorganisms, climate, and human factors. To date, there are still no scientifically effective prevention and control methods. In the laboratory, artificial inoculation is a common experimental method to study the pathogenic mechanism and prevention and control techniques of pine wilt nematodes. However, the migration direction and activity characteristics of pine wilt nematodes in 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 the implementation of indoor pine wilt inoculation experiments.

[0003] The study of the damage mechanism of pine wood nematodes and the prevention and control technology of pine wood nematode disease requires inoculating pine wood nematodes into pine trees and separating them from the pine trees within a specific time after inoculation. By statistically analyzing the number of pine wood nematodes in pine trees, the reproduction and parasitic abilities of pine wood nematodes in different pine trees can be compared, thereby inferring the resistance of different pine trees to pine wood nematodes.

[0004] Therefore, providing an experimental method for accurately obtaining pine wood nematodes from inoculated pine trees and accurately analyzing the inoculation data are issues that need to be urgently addressed 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] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0008] (1) Collecting pine wood nematodes and preparing a nematode suspension;

[0009] (2) Use a sterilized surgical blade to cut the pine bark at a 45° angle at the inoculation site, with the wound reaching the phloem;

[0010] (3) Place absorbent cotton into the wound and wrap it around the pine tree trunk with sealing film to seal the bottom of the wound tightly;

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

[0012] (5) At a specific time after inoculation, pine trunks and branches within 20 cm below the inoculation point were cut into pieces, and pine wood nematodes were separated and collected for data statistics and analysis.

[0013] Furthermore, the pine wood nematode in step (1) is artificially cultured in a laboratory, and the collection method is separation and collection using the Behmann funnel method.

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

[0015] Furthermore, the pine trees in step (2) are planted in a mesh house, and the inoculation point is at least 20 cm away from the first fork of the pine trees.

[0016] Furthermore, 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 separation and collection using the Behmann funnel method.

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

[0019] The traditional method is to cut all the branches and trunks of the pine tree inoculated with pine wood nematodes into pieces and then separate and collect the pine wood nematodes through a Behmann funnel. The present invention clarifies the behavioral characteristics of pine wood nematodes after inoculation, that is, they migrate and gather mainly downward with the inoculation point as the center within the pine tree body, and confirms a method that only collects the main trunk below the inoculation point to accurately obtain most of the pine wood nematodes in the inoculated pine tree.

[0020] This study demonstrates that when conducting indoor experiments on pine trees inoculated with pine wood nematodes, more than 95% of the pine wood nematodes can be recovered by simply collecting pine branches within 20 centimeters below the inoculation point and separating them using the Behmann funnel method. Compared to conventional methods for whole-tree separation, this method significantly reduces workload, saves time, and shortens the experimental process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0022] Figure 1 The attached figure shows the comparison of the number of pine wood nematodes in different parts of the pine trees after inoculation. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0024] Example 1

[0025] (1) The cultured pine wood nematodes were separated and collected using the Behmann funnel method, and a nematode suspension with a concentration of 1000 nematodes / mL was prepared;

[0026] (2) There were 45 two-year-old elliottii seedlings in the net house, which were divided into three groups: the group above the inoculation point, the group below the inoculation point, and the whole-plant investigation group, with 15 plants in each group.

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

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

[0029] (3) Place sterilized absorbent cotton in the wound and wrap Parafilm around the pine tree trunk to seal the bottom of the wound.

[0030] (4) Using the same inoculation method, inoculate 1000 pine wood nematodes into each pine tree. Use a pipette to draw 1 mL of pine wood nematode suspension and slowly drip it onto the absorbent cotton.

[0031] (5) 28 days after inoculation, pine branches were collected from each group according to the experimental groups. The pine branches or twigs were cut into small pieces of 1 cm in length. After cutting, the pine wood nematodes were separated and collected using the Behmann funnel method. The data were statistically analyzed and the number of pine wood nematodes in different parts was compared.

[0032] To isolate pine wood nematodes, use the Behmann funnel method: Place a 10-15 cm diameter funnel on a stand. Attach a 10 cm piece of rubber tubing to the bottom, then connect a 1.5 ml centrifuge tube to the tubing. Fill the funnel with water and squeeze the tubing to expel any air. Chop pine branches, wrap them in paper towels, and place them in the water-filled funnel. Let them sit for 24 hours, then remove the tube and count the nematodes under a microscope.

[0033] 5. The experimental results are as follows:

[0034] Table 1: Comparison of the number of pine wood nematodes in different parts of pine trees after inoculation

[0035]

[0036] Table 1 and Figure 1 The results showed that in the pine wood nematode inoculation experiment, 28 days after inoculation, the number of nematodes isolated from the trunk within 20 cm above the inoculation point was extremely small, showing a significant difference from the number of nematodes isolated from the trunk within 20 cm below the inoculation point and the whole-plant survey (P < 0.05). However, the number of nematodes isolated from the trunk within 20 cm below the inoculation point was not significantly different from that isolated from the whole-plant survey. This shows that in laboratory pine wood nematode inoculation experiments, the vast majority (95%) of pine wood nematodes in inoculated pine trees can be recovered by collecting the trunk within 20 cm below the inoculation point or the entire trunk below the inoculation point and performing Behmann funnel separation.

[0037] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to 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: The following steps are involved: (1) Collecting pine wood nematodes and preparing a nematode suspension; (2) Use a sterilized surgical blade to cut the pine bark at a 45° angle at the inoculation site, with the wound reaching the phloem; (3) Place absorbent cotton into the wound and wrap it around the pine tree trunk with sealing film to seal the bottom of the wound tightly; (4) Use a pipette to drop the nematode suspension onto absorbent cotton; (5) At a specific time after inoculation, pine trunks and branches within 20 cm below the inoculation point were cut into pieces, and pine wood nematodes were separated and collected for data statistics and analysis.

2. The method according to claim 1, characterized in that The pine wood nematodes in step (1) are artificially cultured in a laboratory, and the collection method is separation and collection using the Behmann 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, wherein The pine trees in step (2) are planted in a net house, and the inoculation point is at least 20 cm away from the first fork of the pine trees.

5. The method according to claim 1, wherein Step (4) inoculate 1000 pine wood nematodes per pine tree.

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 separation and collection using the Behmann funnel method.

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