JP5831731B2 - Method for separating soil nematodes - Google Patents

Description

  The present invention relates to the analysis of soil nematodes in agriculture and horticulture.

Analysis of soil microorganisms including nematodes is an important issue in crop cultivation. In general, it is very difficult to control soil diseases compared to the above-ground diseases, and the current situation where large amounts of fumigant disinfectants and other powerful pesticides are administered to the soil is a concern for safety to the human body and the environment. Yes. Since the analysis of soil pathogens is not performed sufficiently, the current situation is that the countermeasures must be based on simplified methods such as sterilization and preventive pesticide treatment of the whole soil. One of the reasons is that it is difficult for farmers to diagnose soil diseases themselves, and they have to rely on specialists.
On the other hand, it is considered to reduce the usage of pesticides through more detailed analysis such as density measurement of soil nematodes and parasitism of natural enemy microorganisms and to confirm the control effect of organic materials and natural enemy microorganisms. Furthermore, the microbial flora in the soil is enriched to prevent the occupation of specific pathogenic microorganisms, and the cultivation of crops that do not rely on chemical pesticides has been carried out. Today, the efficiency of soil microbial analysis methods, including nematodes, has been implemented. And refinement are increasingly important issues.

The analysis method of soil nematodes is generally a method of separating nematodes from soil and analyzing them, but recently, a method has been proposed for detection and quantification by compacting soil data and extracting nucleic acids (patents) Reference 1).
JP 2009-195147 A

  As a method for separating the nematode from the soil, a Bellman method that uses the activity of the nematode itself and a two-layer centrifugal suspension method that physically separates using a difference in specific gravity are generally performed.

  The Bellman method (FIG. 1) is simple and most widely used. However, the separation rate is low compared with the centrifugal separation method, and the fluctuation of the number of separated insects is slightly large (Non-patent Document 1).

The nematodes that move in the soil soaked in water fall down step by step with their own weight, and as time passes, they pass through the Japanese paper and tissue laid under the soil and emerge in the water. This method does not collect nematodes with low motility, but has the advantage that there is little contamination with soil particles that obstruct observation.
1. 20 g of soil is placed in a mesh dish with tissue paper and placed on a bellman funnel filled with water.
2. After 72 hours, collect the tube with the nematode.

  Double-layer centrifugal suspension method. Centrifugation methods that have been studied overseas are methods in which light organic substances and the like are removed by a first centrifugation with water, and nematodes are separated by a second centrifugation with a specific gravity solution (non-patented). Reference 2). On the other hand, the two-layer centrifugal suspension method is very efficient because nematodes can be separated by one centrifugation (Non-patent Document 3). The separation rate is about 60% for root-knot nematode second stage larvae, and the fluctuation of the number of separated insects is relatively small at about 10% for CV. Another advantage of this method is that it can be used to separate inactive nematodes and developmental stages (Non-Patent Document 4). However, nematode eggs and egg sac are not separable like active nematodes because they tend to settle due to adhesion of soil particles. The amount of soil that can be tested is small. Separation rate tends to decrease in soils rich in organic matter and clay. The separation rate of large nematodes is low. In sandy soil, nematodes are crushed during the soil agitation process and the separation rate tends to decrease (Non-Patent Document 5, (FIG. 2): Non-Patent Document 1).

Since nematodes are heavier than water (specific gravity is about 1.05), using sugar water with a high specific gravity instead of water can lift only the nematodes themselves. This method has the advantage that the recovery rate is high and can be done in a short time, but nematodes may be weakened by the influence of sugar water.
1. Put 10g soil and 20ml water in a 50ml centrifuge tube and stir well.
2. Insert the pipette to the bottom of the centrifuge tube and gently add 10 ml of 40% sugar water.
3. Centrifuge for 1 minute at 1,500 rpm. Mud settles to the bottom and nematodes gather at the border between water and sugar water.
4). Scrape the supernatant with a 20-40 μm sieve and collect the nematodes remaining on the sieve. Or a nematode is sucked up with a pipette from the boundary line of water and sugar water (nonpatent literature 1).
“FY2009 Vegetable Textbook for Intractable Pest Control (B)”, Hokkaido Agricultural Research Center Jenkins, W.M. R. (1964) Pl. Dis. Reptr. 48,372-381 Kazuo Takagi (1970) Response Kun 14,108-110 Minagawa, N .; (1979) Appl. Entmol. Zool. 14, 469-477, Sano Yoshikazu (1975) Journal of Nematode Research Institute 5, 41-47. “Nematode Experimental Method” published by the Japanese Nematode Society, March 2004

  None of the separation methods can separate all nematodes in the soil, and only a certain part is always extracted, but it is operated by understanding the characteristics of each and using the method suitable for the purpose. Is the reality. The Bellman method is simple but takes 2-3 days to separate. Nematode eggs, egg folds and inactive nematodes are not isolated and may underestimate the number and type of nematodes.

  The double-layer centrifugal suspension method is an excellent method that can separate the nematode phase of the soil because it can be separated in several tens of minutes, can also separate dead nematodes and egg sac, and has a high separation rate. However, the frequency of use is still low among specialists because it requires a centrifuge and further technical skill.

  Conventional methods cannot easily and quickly separate many types of nematodes from soil, and it is difficult for those who do not have specialized skills and knowledge to separate nematodes.

  A specific gravity liquid and water or a flocculant solution are formed in a soft tube to form an interface, and the soil suspension is dropped from above, so that soil particles settle to the bottom of the specific gravity liquid, and soil microorganisms including nematodes Collect at the interface of water or flocculant solution and specific gravity. Next, the tube was left to stand, and the tube was wound up or pulled with an applicator to collect water or the flocculant solution and the specific gravity solution, whereby microorganisms containing most nematodes in the soil could be separated. Soil residues remain in the tube. The aqueous solution containing microorganisms was easily recovered by nylon mesh. All operations were completed in about 10 minutes, and the separation rate of nematodes was equivalent to that of the double-layer centrifugal suspension method, and that the separation rate of large nematodes was higher than that of the Bellman method and the double-layer centrifugal suspension method, the present invention was completed. did.

  Using simple tools, soil microorganisms can be quickly separated in about 10 minutes, and no mechanical device or power source is required. This is a technique that enables even a person who does not have specialized techniques and knowledge about nematodes to easily and quickly isolate nematodes and allows the farmers themselves to diagnose harmful nematodes. It is also useful for the isolation and diagnosis of Xiphinema spp., A harmful nematode that is important for quarantine for plant and bonsai export industries.

It is the figure which showed the outline | summary of the tube method. It is the figure which showed the outline | summary of the tube method. It is the figure which showed the outline | summary of the Bellman method. It is the figure which showed the outline | summary of the two-layer centrifugal suspension method.

  Use a transparent or translucent soft tube. The material of the tube is, for example, a plastic or a polymer such as polyethylene, polypropylene, vinyl chloride, or Teflon, but any material can be used as long as it can withstand the operation of putting water into the tube and pressing it to push out the water.

  The size of the tube can be properly used according to the amount of soil to be tested. When the crop parasitic nematodes are separated and their types and numbers are measured and used for soil diagnosis, 20 to 100 g of soil is often used, in which case the diameter is 25 mm to 35 mm and the length is 700 mm to 1200 mm. A size of 30 μm or more can be suitably used. You can also change the ratio of diameter to length. The thickness can be changed as long as the material can withstand the pressure of water.

  Pinch cock 1 is attached by injecting a specific gravity solution into a polytube with the bottom sealed. A sucrose aqueous solution is usually used to adjust the specific gravity solution, but sodium chloride, magnesium sulfate, zinc sulfate, calcium chloride and the like can also be used. The specific gravity of the nematode is about 1.05, and the specific gravity of the specific gravity liquid can be 1.05 or more to about 1.3. More preferably, a 20% to 40% sucrose aqueous solution can be used. Next, an aqueous solution of water or soil flocculant is injected and the pinch cock 2 is attached. When using a flocculant, it can be used as long as soil particles can be settled regardless of organic or inorganic type. The amount of the specific gravity liquid and water or the flocculant liquid is preferably about 100 ml to 300 ml.

  A solution obtained by suspending 20 to 100 g of test soil in about 100 to 400 ml of water is injected. When the pinch cock is removed in the order of 1, 2, soil particles settle from the soil suspension, pass through water or a flocculant solution, transfer to sucrose solution, and settle to the bottom. The specific gravity and water or diluted flocculant solution form an interface. A nematode solution is obtained by allowing the tube to stand sideways to settle the soil, attaching a thin tube to the outlet and winding the tube from the end or pulling it with an applicator to drain the flocculant solution and the specific gravity solution. Leave the soil in the tube. The amount of the separated soil is about 1.5 times that of the test air-dried soil, and the acquired nematode solution is the total remaining amount. The nematode solution is passed through a nylon mesh of about 10 μm to 200 μm, and the nematode is collected and washed with water. A small amount of water was taken in a sample dish and counted with a biological microscope. By changing the mesh opening, nematodes can be collected separately for different sizes.

  Embodiments of the present invention will be described based on examples. The present invention is not limited to this form.

  Two types of nematode-contaminated soil were prepared and analyzed and compared in four iterations each using the method of the present invention (tube method) and the double-layer centrifugal suspension method (Table 1).

Tube Method A pinch cock 1 is prepared by injecting 200 ml of a 25% aqueous sucrose solution into a polyethylene tube (diameter 30 mm, length 1000 mm, thickness 50 μm) whose bottom is sealed. Next, 200 ml of water is poured into the pinch cock 2. Suspend 50 g of the test soil in 200 ml of water and inject the solution. (FIG. 1). When the pinch cock is removed in the order of 1, 2, soil particles settle from the soil suspension, pass through the aqueous layer, transfer to the sucrose solution, and settle to the bottom. The sucrose solution and the aqueous layer form an interface. When the tube is left to stand to settle the soil, a thin tube is attached to the outlet and the tube is wound up from the end, the aqueous layer and sucrose solution flow out, and the soil remains in the wound tube. The obtained nematode solution was 570 ml, and the residual soil was 78 g including a polyethylene tube. The nematode solution is passed through a 50 μm nylon mesh to collect nematodes and washed with water. All operations were completed in about 10 minutes. A small amount of water was taken in a sample dish and counted with a biological microscope.

(Comparative example 1) Two-layer centrifugation method 25 g of test soil was dispersed in 100 ml of 2% sodium hexametaphosphate solution. The dispersion was sonicated for 5 minutes, then added to 85 ml of 50% sucrose solution in a 250 ml centrifuge tube, and subjected to two-layer centrifugation at 500 rpm for 1 minute using a centrifuge. Nematodes were collected from the clean water using a 20 micron nylon mesh. The collected nematodes were counted with a biological microscope and converted per 50 g of soil.

  The results are shown in Table 1. The root-knot nematode is a typical nematode that harms crops, and the giant nematode and the nematode are important nematodes for export quarantine of plants and bonsai. The tube method used a simpler instrument than the double-layer centrifuge suspension method, and was able to perform nematode separation equivalent to or better than the double-layer centrifuge method quickly while being a simpler operation.

Claims (4)

A specific gravity liquid and water or a soil flocculant solution are added to the tube to form an interface, and by dropping the soil suspension from the top, the soil particles settle to the bottom of the specific gravity liquid, and nematodes are water or soil flocculant. and wherein the gathering at the interface of the solution and the specific gravity liquid, a method of separating the soil or al nematodes. The method of claim 1 tube Ru is clear der soft in claim 1. The method according to claim 1, wherein the specific gravity liquid is a sucrose liquid . The method according to claim 1, wherein the soil flocculant solution is a magnesium chloride solution .

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