JP3963461B2 - Nematode segregation method - Google Patents

Description

  The present invention relates to a method for separately collecting nematodes according to food habits, sizes, and chemotaxis. This method can be used to identify or control plant parasitic nematodes that damage crops because it can isolate only certain nematodes from the various nematodes that inhabit the soil and have mobility. It is.

  As a general method of collecting miscellaneous nematodes that inhabit the soil and have the ability to move, the Bellman method (Non-patent Document 1) and the two-layer centrifugal suspension method (Non-Patent Document 2) can be mentioned. In the Bellman method, the collected soil is filled with gravity water and capillary water to negate the effect of surface tension, which plays an important role in the movement of nematodes, and the movement of nematodes occurs only in the gravitational action and is submerged in the water. It uses the sinking. In the two-layer centrifuge method, the nematode having a lighter specific gravity than the soil is collected on the sucrose surface by centrifuging the soil in a centrifuge tube containing sucrose. As a method for separating and collecting specific types of nematodes from the group of miscellaneous nematodes collected by these methods, specialists observe the morphological characteristics of nematodes and visually observe the target nematodes under a microscope. A method is known in which a single object is adsorbed and taken out at the tip of a fine object. In addition, with regard to collection for the purpose of preventing damage to plant parasitic nematodes whose host is agricultural products, a method of attracting and collecting the target nematodes by planting captured crops in the field is known.

As a method for controlling plant parasitic nematodes, methods using soil fumigants, steam disinfection, hot water soil irrigation, irrigation, solar heat disinfection, nematode parasitic bacteria infection, and the like have been developed. However, soil health (plant production capacity) is maintained by the balance of soil biota, and the presence of soil organisms is indispensable. However, in order to control disease nematodes, soil organisms can be protected with hot water and fumigants. Because it kills and destroys its balance, there is a high possibility of damaging soil health. Therefore, it is considered desirable to control only certain plant parasitic nematodes, and methods for controlling specific nematodes have been developed, such as field control methods with absolute parasitic bacteria and soil methionine treatment methods. .
Edited by Shoji Fukaya, Shojiro Ishii, Teruo Yamazaki: Insect experiment method, published by Japan Plant Protection Association, p494 (1959). Kazuo Takagi: Separation of soil nematodes by the two-layer centrifuge method, Journal of Japanese Society of Applied Entomology, pp108-110 (1970).

  The nematode collection method by the Bellman method and the two-layer centrifugation method only collects miscellaneous nematodes that inhabit the soil and have the ability to move, and collects specific nematodes separately. I can't. In order to extract specific nematodes from nematodes collected by these methods, specialized techniques and knowledge are required, and thus there is a problem that efficient separate collection is difficult.

  On the other hand, in agriculture, it is indispensable to confirm the presence of nematodes parasitic on crops on a per-field basis from the viewpoint of minimizing the damage caused by parasitism. There is a problem that it is difficult to identify the collected nematodes unless they are experts.

  In order to solve the problems of the technology as described above, the present invention makes use of the fact that the ability and mobility of nematodes are affected by the fine structure and soil water movement, The present invention provides a method for separating a specific type of nematode from a group of insects, or a method for collecting and removing a specific type of nematode without destroying the balance of soil biota.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor performed a characteristic behavior according to the type of liquid flow existing in the fine pores of the medium in which the nematode is present. I found it through experiments. In other words, the life of nematodes having different sizes and migration abilities depending on the food habits and growth stages depends on the fine structure such as the pore network of the habitat from the point of food search, and at the same time, Inferring that it is also affected by liquid movement, we conducted a verification experiment. In the verification process, it was discovered that the response of nematodes to liquid movement in the microstructure depends on the size, mobility, and chemotaxis of nematodes. By taking into account that the moving ability and size are different from those of other nematodes with food habits, we found that the above characteristics of nematodes are effective as a means to roughly classify nematodes, and this book Completed the invention.

That is, the present invention includes the following (1) to (7).
(1) It is characterized by filling the pores of a porous medium containing nematodes with a liquid, causing the liquid to flow in a certain direction, and collecting nematodes separately using the response behavior of the nematodes to the flow of the liquid as an index. How to collect nematodes to separate.
(2) The nematode according to (1), wherein the response action of the nematode is an action of moving along the flow of the liquid or an action of resisting the flow of the liquid and trying to stay on the spot. Separation collection method.
(3) The nematode according to (1) or (2), wherein the porous medium is any one of soil, sand, particulate matter accumulation, fiber porous, or fine perforations or uneven processed products. Separate collection method.
(4) The means for causing the liquid to flow in a certain direction is a means for locally evaporating the liquid component in the porous medium, a means for moving the liquid component in the porous medium due to a difference in water level, and a minute flow rate to the porous medium Means for connecting a control pump and supplying or discharging liquid, means for bringing a water-absorbing polymer, adsorbent paper, semipermeable membrane, fiber porous, or plant root into contact with the porous medium and sucking the liquid component of the porous medium The method for separating and collecting nematodes according to any one of (1) to (3), wherein:
(5) A method for removing a specific nematode characterized by contacting a water-absorbing material with soil and causing water in the soil to flow in the direction of the water-absorbing material, and then removing only the water-absorbing material or the water-absorbing material and surrounding soil. .
(6) The method for removing a specific nematode according to (5), wherein the specific nematode is a plant parasitic nematode.
(7) The method for removing specific nematodes according to (5) or (6), wherein the water-absorbing material is a water-absorbing polymer, adsorbent paper, semipermeable membrane, fiber porous, or plant root.

  Hereinafter, the present invention will be described in detail.

  The method for separating and collecting nematodes according to the present invention fills the pores of a porous medium containing nematodes with a liquid, causes the liquid to flow in a certain direction, and uses nematode response behavior to the liquid flow as an indicator. It is characterized by collecting separately.

  The porous medium may be anything as long as it has pores with dimensions that allow the nematode to move, but considering the distribution of the body width of general nematodes, the pore cross-sectional diameter is 0.02 mm. It is preferably composed of fine pores of about 0.2 mm, and the pore gaps are connected in a network. Examples of such a medium include soil, sand, particulate matter accumulation, fiber porous, fine perforation or uneven processed material (preferably manufactured by a technique capable of mechanical processing with an accuracy of 0.1 mm or less). it can.

  The liquid that fills the pores may be any type as long as it does not prevent nematode survival, but is preferably an aqueous solution. Examples of such liquids include distilled water, tap water, aqueous sucrose solution, and physiological saline.

  The type of nematode to be separated and collected may be any type as long as it has the ability to move inside the porous medium at any point in its life cycle. Further, the nematode may be naturally grown inside the medium or artificially introduced from the outside into the medium, but is preferably a plant parasitic nematode. Examples of plant parasitic nematodes include root-knot nematodes, cyst nematodes, and nematode nematodes.

  The means for causing the liquid to flow in a certain direction is not particularly limited, but means for locally evaporating the liquid component in the porous medium, means for moving the liquid component in the porous medium due to the difference in water level, Connect the flow control pump to bring the liquid supply or removal means, water-absorbing polymer, adsorbent paper, semipermeable membrane, fiber porous, or plant root into contact with the porous medium and suck the liquid component of the porous medium It is preferable to use means or the like. Examples of the micro flow rate control pump include a micro syringe pump and a diaphragm pump. Examples of water-absorbing polymers include starch-polyacrylonitrile hydrolyzate, poly (sodium acrylate) cross-linked product, cellulose cross-linked product, filter paper as an example of adsorbent paper, copper ferrocyanide precipitation film, cellophane film, polyamide Polysulfone hollow fiber membrane, cellulose hollow fiber membrane, polyurethane hollow fiber membrane as examples of membrane, cellulose acetate membrane, fiber porous, sweet potato, carrot, radish, burdock, eggplant, tomato, marigold, buckwheat etc. as examples of plant root It can be illustrated.

  The nematode response behavior to the liquid flow includes, for example, behavior that moves along the flow of the liquid, behavior that resists the flow of the liquid and tries to stay there.

  The above-described method for separating and collecting nematodes can be applied to a method for removing specific nematodes. That is, a method for removing a specific nematode characterized by contacting a water-absorbing material with soil and allowing water in the soil to flow in the direction of the water-absorbing material, and then removing only the water-absorbing material or the water-absorbing material and surrounding soil. Provided.

  Examples of the water-absorbing material used here include the above-described water-absorbing polymer, adsorbent paper, semipermeable membrane, fiber porous, and plant root.

  This method removes nematodes that move along the flow of water. As shown in the examples described later, Meloidogyne incognita, which is one of plant parasitic nematodes, moves along the flow of water, and such properties are presumed to be commonly observed in plant parasitic nematodes. Therefore, this method is considered to be effective as a method for removing plant parasitic nematodes.

  Hereinafter, one embodiment of the method for removing the specific nematode of the present invention will be described.

  The soil in which nematodes are present is made to contain water sufficiently by irrigation or flooding. Next, the water-absorbing polymer molded into a cylindrical shape is embedded in the soil. At this time, since the water in the pores of the soil moves in the direction of the water-absorbing polymer, nematodes such as root-knot nematodes, which tend to move in synchronization with the direction of water movement, are present in the soil. When doing so, the nematode moves in the direction in which the water-absorbing polymer exists. By leaving it for a sufficiently long time, for example, 24 hours, root-knot nematodes existing around the water-absorbing polymer are collected in the vicinity of the water-absorbing polymer due to the slow movement of water caused by the water-absorbing polymer. The root-knot nematode can be removed from the soil by taking out the soil in the vicinity of the water-absorbing polymer together with the water-absorbing polymer.

  Further, by enclosing the extracted water-absorbing polymer and soil in a filter and immersing them in a container filled with water, only root-knot nematodes can be extracted at the bottom of the container by the self-weight sedimentation action of nematodes.

  Since the method for separating and collecting nematodes of the present invention can separate only specific nematodes from the various nematodes that inhabit the soil, it is possible to identify and control plant parasitic nematodes that damage crops. Is available.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1]
Using the apparatus shown in FIG. 1, the response behavior of the infested larvae of the insect parasitic nematode Steinernema carpocapsae and the infected larvae of the plant parasitic nematode Meloidogyne incognita to the water flow was examined.

  The apparatus used for the experiment is composed of a lid 1, a flat plate 2, and a container 3. In the central portion of the upper surface of the flat plate 2, there is a region where a plurality of fine concave structures are formed, and the periphery is uniformly concave. (Figure 2). The concave structure is a recess having a uniform depth as shown in FIG. 3 (depth: 0.05 mm), and the recesses are arranged vertically and horizontally in the region. When the flat plate 2 is inserted and the lid 1 is brought into close contact with the container 3, the upper part of the concave structure is closed by the lid 1, and a maze-like structure is formed. The lid 1 has three through holes, and these holes are connected to the upper surface of the flat plate 2.

  Approximately 30 nematodes were sucked together with 5 μL of distilled water with a micropipette, and the nematodes were introduced from the injection hole 5 into the region where the concave structure was formed. After introduction of the nematode, a micro syringe pump was connected to the injection hole 5 and distilled water was injected at a flow rate of 5 μL / min, while distilled water was discharged from the discharge holes 4 and 6 on the left and right of the injection hole 5. In this state, when the distilled water is continuously injected for 10 minutes, the movement of the nematode is stored in the computer as a moving image, and then only the moving nematode image is extracted and superimposed from the image by performing image processing. A nematode moving pattern image was obtained. The movement pattern image of Steinernema carpocapsae is shown in FIG. 4, and the movement pattern of Meloidogyne incognita is shown in FIG. In addition, the circular figure seen in the center of the figure is the injection hole 5.

  As shown in FIG. 4, since the movement pattern of Steinernema carpocapsae is localized around the injection hole 5, the nematode strongly resists the flow and continues to remain in the region where the concave structure is formed. Indicates that there are many. On the other hand, as shown in FIG. 5, in the movement pattern of Meloidogyne incognita, many trajectories of individuals moving from the periphery of the injection hole 5 to the outside of the region where the concave structure is formed were observed. This shows that there are few individuals against the flow and many individuals move along the direction of the flow.

  From the above, by controlling the water flow in the area where the concave structure is formed, the difference in the behavior of the nematode with respect to the liquid flow is utilized, and the nematode and the flow remaining in the area where the concave structure is formed are used. It was shown that it is possible to separate into nematodes that move along.

[Example 2]
Using a glass bead packed column as a typical soil, the plant parasitic nematode Meloidogyne incognita and the insect parasitic nematode Steinernema carpocapsae were introduced at the same time, and an experiment was conducted to separate both nematode species using water movement caused by water absorption by filter paper. went.

  In the experiment, ten glass bead-packed columns in which 2 g of glass beads (average diameter of about 0.4 mm) were packed in a Pasteur pipette (total length: 14 mm, maximum inner diameter: 5 mm, minimum inner diameter: 1 mm) were used. Distilled water suspension of Meloidogyne incognita (MI) infectious larvae (average concentration: 0.8 head / μL) 50 μL, and Steinernema carpocapsae (SC) infectious larvae distilled water suspension (average concentration: 0.6 head / μL) Inject 50 μL into the rear end of the column (pipette) at the same time, and immerse it in a resin centrifuge tube containing 1.5 mL of distilled water with the end of the column (pipette) standing upright to create an environment with a temperature of 25 ° C and a humidity of 60%. Left for 24 hours. After leaving, the resin centrifuge tube was removed, 20 μL of distilled water was collected from the narrowed bottom of the centrifuge tube with a micropipette, and the number of MI and SC contained in the collected distilled water was counted using a stereomicroscope. This was repeated twice, and the total for each nematode species was taken to obtain the number of heads for each nematode species that passed through each column.

  Usually, in the above state, due to the spontaneous movement of the introduced nematodes and the influence of gravity, most nematodes pass through the column downward and are discharged from the tip after 24 hours. Further, since the nematode species have almost the same size, there is no difference in the ability to pass through the gap between the glass beads. In this experiment, five columns were left open as a control group (no suction), and the remaining five columns were wrapped in a cylindrical filter paper (diameter 3 mm, length 50 mm) from the rear end of the column to the tip of the filter paper. It inserted so that it might contact glass beads, and capillary water contained in beads was aspirated and evaporated (treatment group: with suction). FIG. 6 shows the average number of passing heads and standard error of both nematode species in the treatment group and the control group.

  As shown in Fig. 6, in the case of no aspiration, the average number of passing heads (approximately 16 and 24 respectively) for both SC and MI was about half of the average number of heads introduced (30 and 40 respectively from the average concentration). there were. On the other hand, in the case of with suction, the average number of passing heads for SC was almost half (about 14 heads), which was not different from the case without suction, whereas the average number of passing heads for MI was about 3 and 10 minutes. It was 1. This result shows that the number of MI passing heads was greatly reduced by capillary water suction with filter paper. SC was passed through the column without being affected by suction, whereas MI was strongly affected by suction. Tend to stay inside the column. In addition, since the water absorption speed of the filter paper in this experimental environment is 1.54 μL / min in actual measurement, the flow velocity at the column tip (inner diameter 1 mm) is estimated to be 0.49 mm / min. On the other hand, when there is no obstacle, the speed at which the nematode settles in water due to its own weight is 5 mm / min or more, which is much larger than the flow rate generated by water absorption of the filter paper.

  From the above findings, it was shown that insect parasitic nematodes and plant parasitic nematodes mixed in a porous medium (in this case, glass bead packed column) can be separated using capillary water absorption. At the same time, Example 1 shows that MI and SC are approximately the same size, but SC has a greater motor capacity than MI, so it is much more capable of resisting water flow than MI. Therefore, it is considered that the separation mechanism is not simply due to the flow resistance of water, but due to the MI taking movement behavior according to the flow of water.

The figure which represented typically the apparatus used in Example 1. FIG. The dimension drawing of the flat plate in which the concave structure was formed. The dimension drawing of the concave structure currently formed on the flat plate. The figure which shows the movement pattern image of Steinernema carpocapsae. The figure which shows the movement pattern image of Meloidogyne incognita. The figure which shows the relationship between a capillary water suction process and the number of passing nematodes.

Explanation of symbols

1 Lid 2 Flat plate 3 Container 4 Discharge hole 5 Injection hole 6 Discharge hole

Claims (6)

The pore part of the porous medium containing the nematode is filled with the liquid, the liquid flows in a certain direction, moves along the flow of the nematode liquid with respect to the flow of the liquid, or resists the flow of the liquid, A method for separating and collecting nematodes, characterized by separating and collecting nematodes using as an index the behavior of staying in the field . The method for separating and collecting nematodes according to claim 1 , wherein the porous medium is any one of soil, sand, particulate matter accumulation, fiber porous, or fine perforations or uneven processed products. The means for causing the liquid to flow in a certain direction is a means for locally evaporating the liquid component in the porous medium, a means for moving the liquid component in the porous medium due to the difference in water level, and a micro flow rate control pump for the porous medium. Means for connecting, supplying or discharging liquid, water absorbing polymer, adsorbent paper, semipermeable membrane, fiber porous, or plant root contacting the porous medium and sucking the liquid component of the porous medium The method for separating and collecting nematodes according to claim 1 or 2 .   A method for removing specific nematodes, comprising bringing a water-absorbing material into contact with soil and allowing water in the soil to flow in the direction of the water-absorbing material, and then removing only the water-absorbing material or the water-absorbing material and surrounding soil. The method for removing specific nematodes according to claim 4 , wherein the specific nematodes are plant parasitic nematodes. The method for removing specific nematodes according to claim 5 , wherein the water-absorbing material is a water-absorbing polymer, adsorbent paper, semipermeable membrane, fiber porous, or plant root.