JPH08173189A - Method for simply identifying nematode - Google Patents

Abstract

PURPOSE: To enable to simply and rapidly perform the identification of the kind of the nematode and the density of the nematode for each its kind in soil even in the level of an actual place by dyeing a natural enemy bacterium against the nematode and subsequently allowing the dyed bacterium to live in the nematode. CONSTITUTION: The method for simply identifying the nematode comprises dyeing a natural enemy bacterium (e.g. Pasteuria penetrans against Meloidogyne) against the nematode, allowing the dyed bacterium to live in the nematode, and subsequently observing the pigments of spores and their number. The natural enemy bacterium is preferably dyed with first green FCF, acridine orange, methyl violet, brilliant blue G, aniline blue, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention identifies the type of target nematodes in controlling biologically and cultivated plant parasitic nematodes that cause great damage to agricultural crops, and It relates to a method for counting density.

[0002]

2. Description of the Related Art Soil nematodes, especially root-knot nematodes, have become a serious inhibitory factor in the production of field crops such as tobacco. In Japan, the damage caused by three species of sweet potato nematode, nematode nematode, and nematode nematode is the largest, and not only these nematode nematodes harm the crops alone.
It is a big problem because it causes soil infection and complex infection.

In order to control these diseases efficiently, it is necessary to accurately grasp the species and density of root-knot nematodes in soil. This is because the root range and ecological characteristics of root-knot nematodes differ depending on the species, and therefore the crop rotation system, natural enemy use, and cultivated control methods differ. The following methods have been conventionally used to identify the root-knot nematode species.

The first method is morphological observation with a scanning electron microscope or an optical microscope. This method is to discriminate species by observing the pattern (perineal pattern) around the perineum of adult females and each part of the 2nd stage larvae and adult males. This method requires time and labor for sample preparation. However, there is a problem in that the final determination of the species requires skill.

As the second method, there is a discriminant host method. In this method, six types of cotton, tobacco, pepper, watermelon, peanut, and tomato are used for species identification of root-knot nematodes. This method does not require any special technique, but has a drawback that it requires a temperature-controllable greenhouse and it takes about 2 months to make a judgment. The third method is an isozyme method. This method is a method in which a protein extract of an adult female is electrophoresed, various isozyme stains are performed, and a band specific to the species of root-knot nematode is detected, and in particular, 2 of esterase and malate dehydrogenase are detected. Types are used. However, a large amount of protein extract can be collected from female adult 1
It is suitable for judging species from the head and above, but not suitable for judging species in the 2nd stage larva.

The fourth method is a PCR (polymerase chain reaction) method. This method specifically amplifies part of the root-knot nematode DNA and determines the species based on the difference in the electrophoretic pattern, and has been reported in many foreign literatures. However, this technique requires special equipment and expensive reagents, and is not practical at the local level.

By the way, as a natural enemy bacterium of root-knot nematodes, Pasteuria penetran (Pasteuria penetran)
s) is known. This Pasteuria penetrans has a very high host specificity, and a large number of second-stage larvae of root-knot nematodes of the same species attach to each other via this bacterium to form an aggregate. There is a method of easily identifying a species by combining a strain of Pasteuria penetrance and a root-knot nematode to be identified and by which combination the aggregated mass was formed. However, this requires a large number of second-stage larvae of the same root-knot nematode,
Identification is difficult when the number of root-knot nematodes separated by the Berman method is small. In addition, this technique cannot measure the density of each root-knot nematode.

[0008]

The object of the present invention is to solve the drawbacks of the conventional methods for identifying nematodes and to identify the species of nematodes in soil easily and quickly and at the local level. It is to provide a simple identification method capable of measuring the density of each insect type.

[0009]

Means for Solving the Problems As a result of earnest research in view of the above-mentioned problems, the present inventors have made the stained nematode natural enemy bacteria parasitize the nematode, and determine the pigment and the number of spores attached to the nematode. By observing, it was found that the type of nematode and the density of each type can be easily measured, and the present invention was completed.

That is, the present invention is characterized by staining nematode natural enemy bacteria that parasitize nematodes specifically, and identifying the type of nematode using the stained nematode natural enemy bacteria. Is a simple identification method. Hereinafter, the present invention will be described in detail.
The nematode natural enemy bacteria used in the present invention may be any bacteria as long as they specifically parasitize nematodes.
An example of such a bacterium is a bacterium belonging to the genus Pasteuria, among which the root-knot nematode (Meloidogyne) is mentioned.
Genus) for Pasteuria
penetrans) can be preferably used. In addition, in addition, Pasteuria thoynei (Pasteuria thoynei) for Negusaaresenchu (Pratylenchus genus), Pasteuria nishizawae (Pasteuria nishizawae) for cyst nematode (Heterodera genus, Globodera genus)
Etc. can be used.

[0012] Pasteuria penetrans has a very high host specificity and does not parasitize organisms other than nematodes, and the host nematodes are almost limited to the nematodes of the species or their genus. . In addition, Pasteuria penetrans is extremely resistant to drought, high temperature, and drugs, and has advantages not found in other natural enemies. In the present invention, any kind of nematode can be identified as long as the nematode natural enemy bacteria can specifically parasitize it. Examples of such nematodes include root-knot nematodes, and among them, sweet potato root-knot nematodes, Javane root-knot nematodes, and fox-knot nematodes can be preferably identified.

[0012] Specific combinations of these nematodes and the nematode natural enemy bacteria include combinations of sweet potato mung bean nematode and pastureia penetrans derived from sweet potato mung bean nematode, combination of Javanese mown beetle nematode and pasteuria penetrans derived from Javane catb nematode, Also, a combination of P. pertussis and Pasteuria penetrance derived from P. pertussis is known.

In the present invention, nematode natural enemy bacteria are stained to identify nematodes.
When a penetrans is used, the Pasteuria penetrans may be subjected to ultrasonic treatment before dyeing. By performing this ultrasonic treatment, the number of Pasteuria penetrans attached to the root-knot nematode increases, which facilitates identification. Moreover, even if there are root-knot nematodes belonging to the same species from different collection sources, they can be identified without losing the host specificity. This ultrasonic treatment may be carried out by a conventional method on a suspension of Pasteuria penetrance in ice.

Further, the number of Pasteuria penetrans attached to root-knot nematodes can be increased by subjecting Pasteuria penetrans to enzymatic treatment with protease, and this enzyme treatment is combined with the above ultrasonic treatment. May be. The dyeing may be carried out by a conventional method, but preferably at 20 to 40 ° C. for 16 to 24 hours. As the dye used in this dyeing, fast green FCF, acridine orange, methyl violet, brilliant blue G, aniline blue, or a mixture thereof is preferable. These dyes can stain nematode natural enemy bacteria well, are difficult to be decolored even by washing, and have little adverse effect on bacteria.

The amount of the dye used is not particularly limited as long as it can stain nematode natural enemy bacteria sufficiently, but it is preferably used in a saturated amount in consideration of staining efficiency. As a solvent used for dyeing, an aqueous ethanol solution having a concentration of 10 to 30% by weight,
10-500 mM phosphate buffer containing 10-30 wt% ethanol, 0.5-1 containing 10-30 wt% ethanol.
0% by weight ammonium oxalate aqueous solution, 10-500 mM citrate buffer containing 10-30% by weight ethanol, 10-
10-500 mM Tris-HCl containing 30% by weight ethanol
Buffer or 10 to 10 containing phenol at a concentration of 1 to 5% by weight
A 30% by weight acetic acid solution is preferred. By using these solvents, even when minute nematode natural enemy bacteria such as Pasteuria penetrance are stained, it is possible to stain the individual bacteria vividly.

Particularly preferred solvents in combination with Fast Green FCF are 10 to 30% by weight aqueous ethanol solution, 0.5 to 1.0% by weight aqueous ammonium oxalate solution containing 10 to 30% by weight ethanol, and 10 to 30%. Mention may be made of 10-500 mM citrate buffer containing ethanol in a concentration by weight%. Preferred solvents in combination with Acridine Orange include 10-500 mM phosphate buffer, 10-30% ethanol containing 10-30% w / w concentration.
0.5-1.0 wt% ammonium oxalate aqueous solution containing 10 wt% ethanol, 10-500 mM citrate buffer containing 10-30 wt% ethanol, and 10-500 containing 10-30 wt% ethanol. mM Tris-HCl buffer, and particularly preferred solvents include 10 to 500 mM phosphate buffer containing 10 to 30% by weight of ethanol and 0.5 to 1.0% by weight containing 10 to 30% by weight of ethanol. Examples include an ammonium oxalate aqueous solution and a 10 to 500 mM Tris-HCl buffer solution containing 10 to 30% by weight of ethanol.

As a preferred solvent in combination with methyl violet, an aqueous ethanol solution having a concentration of 10 to 30% by weight, and an ethanol solution having a concentration of 10 to 30% by weight are used.
Examples include 0.5 mM to 1.0% by weight ammonium oxalate aqueous solution containing 0 mM phosphate buffer and 10% to 30% by weight ethanol, and particularly preferable solvent is 10 to 500% containing 10% to 30% by weight ethanol. mM phosphate buffer, and 10
An aqueous solution of 0.5 to 1.0 wt% ammonium oxalate containing ethanol at a concentration of -30 wt% is included.

A particularly preferred solvent in combination with Brilliant Blue G is an aqueous ethanol solution having a concentration of 10 to 30% by weight. Preferred solvents in combination with aniline blue include 10-30% by weight acetic acid solution containing 1-5% by weight phenol. When staining is completed, washing is usually performed. This is the container used for dyeing (eg Eppendorf tube)
This is because if the pigment remains inside, it affects nematode natural enemy bacteria that have been stained with another staining solution, and the original color cannot be clearly observed in subsequent microscopic observation. Washing may be performed by a conventional method, for example, centrifugal washing or the like. In the case of centrifugal washing, it is preferable to carry out 3 to 5 times, and it is preferable to use distilled water as the washing liquid.

In the present invention, nematodes are identified using the nematode natural enemy bacteria stained as described above. In addition, in order to use the stained nematode natural enemy bacteria at the local level, pH 4.0-pH
A general stabilizer such as a citrate phosphate buffer solution of about 8.0 may be added. In addition, it is preferable to add a surfactant such as Triton X100 to a final concentration of about 0.01 to 0.001% to allow the nematodes floating on the surface of the solution to sink into the solution.

The nematodes can be separated from the soil by a conventional method, for example, the Balemann method. Once the nematode suspension is obtained, it is mixed with the stained nematode natural enemy bacteria. At this time, a buffer such as MCILVAINE buffer (citrate phosphate buffer), potassium phosphate buffer, etc.
It is preferable to maintain the pH at 4.0 to 8.0 when the ultrasonic treatment is not performed in the above case by adding the composition in a weight percentage, and it is preferable to maintain the pH at 6.0 to 8.0 when the ultrasonic treatment is performed. .

Once mixed, preferably 4-24 at 20-30 ° C.
Let stand for about an hour. Then, the type of nematode can be easily identified by determining the color of the nematode natural enemy bacteria attached to the nematode with a microscope of about 100 to 400 times. At this time, the density of each type of nematodes can be easily known by measuring the number of nematodes to which the nematode natural enemy bacteria are attached.

[0022]

【Example】

(Example 1) (1) Collection and culture of Pasteuria penetrance A. Pasteuria penetrance derived from the sweetpotato nematode, which is specific to the sweetpotato nematode, was collected on March 6, 1991, in a tobacco continuous cropping field in Chosei Village, Chiba Prefecture. Tobacco seedlings were planted in this soil and managed in a greenhouse for 2 months or more. Adult females infected with Pasteuria penetrans parasitic on tobacco roots were extracted and ground to obtain spores. The second-stage larvae of a population grown from a single egg sac of the sweet potato root-knot nematode that lived in this soil were mixed with a spore suspension, and spores were attached to the body surface of the second-stage larvae, which were then applied to tobacco seedlings. I inoculated. After controlling in a greenhouse for 2 months or longer, adult females infected with Pasteuria penetrance parasitic on tobacco roots were isolated and ground to obtain spores. Subculture was performed by this repetition. The mature spores thus obtained were stored at 5 ° C. and tested.

[0023] The obtained sweet potato Nematoda, Michu
P. penetrans derived from ncognita (hereinafter MIPP (Choseimura)
Say. ) Was rejected to the Institute of Biotechnology, Institute of Biotechnology, because it cannot be artificially cultured in vitro. B. Pasteurian penetrance derived from Javanese root nematode which is specific to Javanese root nematode On September 2, 1992, the soil of the tobacco continuous cultivation field in Sakuto-cho, Okayama prefecture was collected. Tobacco seedlings were planted in this soil and managed in a greenhouse for 2 months or longer. Adult females infected with Pasteuria penetrance parasitic on tobacco roots were extracted and ground to obtain spores. The second-stage larvae of a population grown from one egg stalk of the Japanese root-knot nematode that lived in this soil was mixed with the spore suspension, and spores were attached to the body surface of the second-stage larvae, which was then applied to tobacco seedlings. I inoculated. The plant was kept in a greenhouse for 2 months or longer, and a female adult infected with Pasteuria penetrans parasitic on the tobacco root was extracted and ground to obtain spores. Subculture was performed by this repetition. The mature spores thus obtained were stored at 5 ° C. and tested.

Obtained Javanese rootworm M.javan
The ica-derived P. penetrans (hereinafter referred to as MJPP (Okayama 2)) was rejected by the Institute of Biotechnology, Institute of Industrial Science, since it could not be artificially cultured in vitro. C. Pesturia penetrance derived from Pseudomonas aeruginosa, which is specific to Pseudomonas aeruginosa, on September 28, 1991, the soil of a tobacco continuous cultivation field in Showa-cho, Akita Prefecture was collected. Tobacco seedlings were planted in this soil and managed in a greenhouse for 2 months or more. Adult females infected with Pasteuria penetrans parasitic on tobacco roots were extracted and ground to obtain spores. The second-stage larvae of a population grown from a single egg squirrel of the foxglove, which lived in this soil, was mixed with the spore suspension,
Spores were attached to the surface of the larvae of the second stage and inoculated into tobacco seedlings. After controlling in a greenhouse for 2 months or longer, adult females infected with Pasteuria penetrance parasitic on tobacco roots were isolated and ground to obtain spores. Subculture was performed by this repetition. The mature spores thus obtained are stored at 5 ° C,
I tried it.

The obtained P. penetrans derived from M. hapla (hereinafter referred to as MHPP (Showa Town))
Was unable to be artificially cultured in vitro, so it was rejected by the Institute of Biotechnology, Institute of Industrial Science and Technology. (2) Staining and washing of Pasteuria penetrance MIPP (Choseimura), MJPP (Okayama 2) and MHPP
So that you can see the difference between each spore in (Showa Town),
They were dyed with different dyes. The dye and solvent of the dyeing solution used and the color of the dyeing solution are shown below.

MIPP: Brilliant Blue G (manufactured by Wako Pure Chemical Industries, Ltd.), 20% by weight saturated solution of ethanol, blue MJPP: Acridine Orange (manufactured by Wako Pure Chemical Industries, Ltd.),
Tris-hydrochloric acid buffer solution (pH 9.0) with 20 wt% ethanol content, 50 mM, saturated concentration solution, yellow MHPP: methyl violet (manufactured by Wako Pure Chemical Industries, Ltd.)
, A diluted solution of a saturated solution of 0.8% ammonium oxalate solution containing 20% by weight ethanol in 1/4, and for the purple staining operation, 10 μl of Pasteuria penetrans and 40 μl of each staining solution in a 0.5 ml Eppendorf tube. , Was mixed well, and allowed to stand at 25 ° C. 24 hours later, sterile water
Washing was performed by adding 450 μl and centrifuging at 15,000 rpm for 30 minutes three times. The number of spores was counted with a hemocytometer, and the concentration was adjusted with sterile water before the test. (3) Identification of root-knot nematodes Root-knot nematodes to be identified include sweet-potato root-knot nematodes from Chosei-mura, Chiba Prefecture (hereinafter referred to as sweet-potato root-knot nematodes (Chosei-mura)), and Japanese root-knot nematodes from Sakuto-cho, Okayama Prefecture (hereinafter referred to as Javane-kob nematode (Okayama 2)). 2) larvae of Kitanekobushi nematode (hereinafter referred to as Kitanekobushi nematode (Showamachi)) derived from Showa-cho, Akita Prefecture, which hatched at 25 ° C. within 3 days.

96-well microwell plate (Nunk)
50 ml each of MIPP (Choseimura) stained with Brilliant Blue G, MJPP stained with acridine orange (Okayama 2) and MHPP stained with methyl violet (Showa-cho), and the MCILVAINE buffer of pH 6.0 ( 50 μl of citric acid / phosphate buffer solution was mixed to make 200 μl per well. The final concentration of each Pasteuria penetrance was 5 × 10 ⁴ cells / ml.

Add 2% of 0.1% Triton X100 to each well
Then, 5 sweet potato root-knot nematodes (Choseimura), 5 Japanese root-knot nematodes (Okayama 2) and 5 Kitane-kob nematodes (Showa-machi) were inoculated by needles one by one. Then, leave the mixture at 25 ° C for 24 hours, transfer the mixture to a slide glass 24 hours later, put a cover glass on it, and cover all inoculated root-knot nematodes with an optical microscope 100-40.
Each animal was observed at 0-fold and the attached spores were counted by color.
Table 1 shows the results obtained by averaging the number of spores attached per head by color.

[0029]

[Table 1]

As is clear from Table 1, each pasteuria
Penetrans has a high host specificity even when stained, so by observing the color attached to root-knot nematodes,
The type of nematode can be easily identified. (Example 2) MIPP (Choseimura), MJPP (Okayama 2)
And each spore of MHPP (Showamachi) were subjected to ultrasonic treatment. Specifically, a suspension of each spore (concentration: 2 × 10 ⁵
(/ Piece / ml) under ice cooling for 5, 10, 20 and 30 minutes, ultrasonic destruction device (TA-5287 type, horn: 7φ, stainless steel, resonance frequency: 19.5 kHz, horn tip maximum amplitude: 30 μ)
Ultrasonic treatment was performed using m pp, manufactured by Mitamura Giken Kogyo Co., Ltd.

After that, 100 μl of Pasteuria Penetrans
And 50 μl of pH 6.0 MCILVAINE buffer (citrate / phosphate buffer) or sterile water, and 50 μl of sterile water containing 50 to 100 second-stage larvae of root-knot nematodes that have hatched within 3 days at 25 ° C.
And were mixed to make 200 μl per well. The final concentration of each Pasteuria penetrance was 1 × 10 ⁵ cells / ml. In addition, when spores were stained and when they were not stained,
Since there was no significant difference in the number of attached spores to nematodes, spores were not stained for the test.

The combination of nematode and pasturea penetrance is sweet potato moth nematode (Choseimura)
And MIPP (Choseimura), Javanebet nematode (Okayama 2) and MJPP (Okayama 2), Kitanekob nematode (Showa-machi) and MHPP (Showa-machi). Then, let stand at 25 ℃, after 4 hours transfer this mixture to a slide glass,
Twenty randomly selected root-knot nematodes were covered with a cover glass and observed one by one with an optical microscope of 100 to 400 times to count the number of attached spores. The experiment was repeated 3 times. Also, the same experiment was performed when the ultrasonic treatment was not performed. The results are shown in Table 2 as an index with the average number of adhesions not treated with ultrasonic waves as 1.00.

[0033]

[Table 2]

As is clear from Table 2, ultrasonic treatment increased the number of Pasteuria penetrans attached to root-knot nematodes. (Example 3) MIPP (Choseimura), MJPP (Okayama 2)
And each spore of MHPP (Showamachi) were subjected to ultrasonic treatment for 30 minutes in the same manner as in Example 2. After that, each spore was stained in the same manner as in Example 1, and each of the sweet potato mung bean nematode: Javane mung beetle nematode: Kitanebek nematode per well was 1 head: 1 head: 1 head, 5 heads:
Each of the root-knot nematodes was inoculated with a needle so that the number of the animals was 5: 5 and 10: 10: 10 (pasturia penetrans final concentration: 4 × 10 ⁴ cells / ml).

In the same manner as in Example 1, the number of spores attached to each root-knot nematode was counted by color, and the color of the most attached spores per head was determined to identify nematodes. The results are shown in Table 3.

[0036]

[Table 3]

As is clear from Table 3, each stained spore adhered host-specifically to various inoculated root-knot nematodes.
In addition, the number of inoculated sweet potato Monk root nematodes: Javane root gall nematode: Kitanekob nematode: 1 head
The number of root-knot nematodes could be accurately identified from the adherent-stained spores even if the number of heads was as small as 10 and as many as 10:10:10.

(Example 4) MIPP (Choseimura), MJP
The spores of P (Okayama 2) and MHPP (Showamachi) were subjected to ultrasonic treatment for 30 minutes in the same manner as in Example 2.
Then, in the same manner as in Example 1, 50 μl each of MIPP (Shoseimura) stained with Brilliant Blue G, MJPP (Okayama 2) stained with acridine orange, and MHPP (Showa-cho) stained with methyl violet, pH 8. 50 μl of MCILVAINE buffer solution (citrate / phosphate buffer solution) of 0 was mixed in a 96-well microwell plate (Nunk) to give 200 μl per well. The final concentration of each Pasteuria penetrans was 1 × 10 ⁵ cells / ml.

Add 2% of 0.1% Triton X100 to each well
l, followed by inoculation with 10 needles of Sweet potato Nematode (Chouseimura), 10 of Japanese Nekobushi (Okayama 2) and 10 of Kitanekobu nematode (Showa-machi), each with a needle. Then, let stand at 25 ℃, after 4 hours and 12 hours, transfer the mixture to a slide glass, cover it with a cover glass, observe all inoculated root-knot nematodes with an optical microscope 100 to 400 times for each animal, and attach it. The spores were counted by color. The experiment was repeated 3 times. The nematodes were identified by determining the color of the most attached spores attached per head. The results are shown in Table 4.

[0040]

[Table 4]

As is clear from Table 4, the number of root-knot nematodes adhered to the root-knot nematodes was large even after 4 hours, and it was possible to identify the kind of root-knot nematodes from each stained spore. Further, after 12 hours, the number of adhered substances increased, so that the identification became easier and more reliable. (Embodiment 5) 1 of each of the Pasteuria penetrans which was not ultrasonicated and the Pasteuria penetrans which was ultrasonicated for 30 minutes under ice cooling in the same manner as in Embodiment 2.
00 μl and 50 μl of MCILVAINE buffer (citrate / phosphate buffer) or sterile water of pH 4.0, pH 6.0 and pH 8.0, 25
50 stage 2 larvae of root-knot nematodes within 3 days after hatching at ℃
Mix with 100 μl of sterile water (50 μl) and add 20 per well.
The volume was 0 μl. The final concentration of each Pasteuria penetrance was 1 × 10 ⁵ cells / ml. There was no significant difference in the number of spores attached to nematodes between the case where the spores were stained and the case where the spores were not stained. Therefore, the test was performed without staining the spores.

The combination of nematode and pasturea penetrance is sweet potato caterpillar nematode (Choseimura)
And MIPP (Choseimura), Javanebet nematode (Okayama 2) and MJPP (Okayama 2), Kitanekob nematode (Showa-machi) and MHPP (Showa-machi). Then, let stand at 25 ℃, after 4 hours transfer this mixture to a slide glass,
Twenty randomly selected root-knot nematodes were covered with a cover glass and observed one by one with an optical microscope of 100 to 400 times to count the number of attached spores. The experiment was repeated 3 times. The results are shown in Table 5.

[0043]

[Table 5]

As is clear from Table 5, the optimum pH of the adhesion activity was different between the case where the ultrasonic treatment was carried out and the case where the ultrasonic treatment was not carried out. (Example 6) 100 μl of Pasteuria penetrans which was sonicated for 30 minutes under ice cooling in the same manner as in Example 2 so that the final concentrations were 2 ^-2 , 2 ^-3 , 2 ^-4 and 2 ^-5. Adjusted pH
8.0M CILVAINE buffer (citrate / phosphate buffer) 50μ
l was further mixed with 50 μl of sterilized water containing 50 to 100 second-stage larvae of root-knot nematodes that had hatched at 25 ° C. within 3 days to make 200 μl per well. The final concentration of each Pasteuria penetrance was 1 × 10 ⁵ cells / ml. There was no significant difference in the number of spores attached to nematodes between the case of staining spores and the case of non-staining, so that the test was performed without staining spores.

The combination of nematode and pasteuria penetrance is sweet potato caterpillar nematode (Choseimura)
And MIPP (Choseimura), Javanekobsenshu (Okayama 2) and MJPP (Okayama 2), Kitanekobsenchu (Showa-machi) and MHPP (Showa-machi). Then, let stand at 25 ℃, after 4 hours transfer this mixture to a slide glass,
Twenty randomly selected root-knot nematodes were covered with a cover glass and observed individually with a light microscope of 100 to 400 times to count the number of attached spores. The experiment was repeated 3 times. The results are shown in Table 6.

[0046]

[Table 6]

As is clear from Table 6, the buffer solution was ^{added in the} range of 2 ^-2 .
Even when diluted to 2 ^-5 and used, the number of adhered substances was not significantly affected. (Example 7) 100 μl of Pasteuria penetrans which was sonicated for 30 minutes under ice cooling in the same manner as in Example 2 and pH 8.0
50 μl of MCILVAINE buffer (citrate / phosphate buffer)
Was mixed with 50 μl of sterilized water containing 50 to 100 second-stage larvae of root-knot nematodes hatched within 3 days at 25 ° C. to make 200 μl per well. The final concentration of each Pasteuria penetrance was 1 × 10 ⁵ cells / ml. There was no significant difference in the number of spores attached to nematodes between the case where the spores were stained and the case where the spores were not stained. Therefore, the test was performed without staining the spores.

[0048] The combination of nematode and pasturea penetrance is sweet potato moth nematode (Choseimura)
And MIPP (Choseimura), Javanekobsenshu (Okayama 2) and MJPP (Okayama 2), Kitanekobsenchu (Showa-machi) and MHPP (Showa-machi). After that, 15, 20,
After standing still at 25 and 30 ℃, 4 hours later, this mixture was transferred to a slide glass, covered with a cover glass, and 20 randomly selected root-knot nematodes were observed with an optical microscope 100 to 400 times for each head. The number of attached spores was counted. Experiment 3
Repeated times. The results are shown in Table 7.

[0049]

[Table 7]

As is clear from Table 7, the rate of attachment to root-knot nematodes was highest when it was allowed to stand at 25 to 30 ° C. (Example 8) 100 μl of Pasteuria penetrans dyed in the same manner as in Example 1 and 50 μl of MCILVAINE buffer solution (citric acid / phosphate buffer solution) having a pH of 8.0 (however, MIPP
For Nagasei Village, pH 7.0 was used. ) And 3 at 25 ℃
50 μl of sterilized water containing 50 to 100 second stage larvae of each population of root-knot nematodes that had hatched within a day was mixed to make 200 μl per well. The final concentration of Pasteuria penetrans tested was 1 × 10 ⁵ cells / ml.

MIP as a pasture pene transformer
P (Choseimura), MJPP (Okayama 2) and MHPP (Showacho) were used without ultrasonic treatment and with ultrasonic treatment for 30 minutes under ice cooling as in Example 2. did. The populations of root-knot nematodes include sweet potato root-knot nematodes (Chosei-mura), sweet-potato root-knot nematodes (hereinafter referred to as sweet-potato root-knot nematodes (Miura-mura)) derived from Miura-mura, Ibaraki prefecture, Javanese root-knot nematode (Okayama 2), and deer-crown rootworm from Dejima-mura, Ibaraki prefecture. (Hereafter, it will be referred to as Javanese rootworm (Mito 5).)
, Kitanekob nematode (Showa-machi) and Kitanekob nematode (hereinafter referred to as Kitanekob nematode (Hachinohe 6)) derived from Kanagi-cho, Aomori prefecture were used.

After that, the mixture was allowed to stand at 25 ° C., and 4 hours later, this mixed solution was transferred to a slide glass, covered with a cover glass,
Twenty randomly selected root-knot nematodes were observed with a light microscope of 100 to 400 times for each head, and the number of attached spores was counted. The experiment was repeated 3 times. Table 8 shows the results.

[0053]

[Table 8]

As is clear from Table 8, attachment of spores of Pasteuria penetrans to the same species as the collection source of Pasteuria penetrans was observed, and almost no attachment of spores to different species of roots nematodes was observed. . In addition, spores can be sufficiently attached to root-knot nematodes without ultrasonic treatment, but the number of attachment of Pasteuria penetrans to root-knot nematodes is increased by sonication, and host specificity is lost. There wasn't. Therefore, ultrasonic treatment is effective in the present invention.

Example 9 Soil collected from various places (Table 9)
20 g, the nematodes were separated by the Baleman method at 25 ° C. for 1 day. Brilliant Blue G was prepared in the same manner as in Example 1 with respect to the Pasteuria penetrans that was sonicated for 30 minutes under ice cooling in the same manner as in Example 2.
50 μl each of MIPP (Choseimura), MJPP stained with acridine orange (Okayama 2) and MHPP stained with methyl violet (Showamachi), and a 50 μl suspension containing all nematodes separated by the Balemann method. 15 μl of MCILVAINE buffer (citrate / phosphate buffer) having a pH of 8.0 was mixed to make 215 μl per well of a 96-well microwell plate (Nunk). The final concentration of each Pasteuria penetrance was 1 × 10 ⁵ cells / ml.

Further, 2 μl of 0.1% Triton X100 was added and the mixture was allowed to stand at 25 ° C. After 12 hours, the mixed solution was transferred to a slide glass, covered with a cover glass, and all the inoculated root-knot nematodes were observed with an optical microscope at a magnification of 100 to 400 for each animal, and the adhered spores were counted by color. The nematodes were identified by determining the color of the most attached spores attached per head. The results are shown in Table 9.

[0057]

[Table 9]

As is clear from Table 9, even if the target root-knot nematode was the root-knot nematode separated from the actual soil by the Balemann method, it could be identified. In addition, it was possible to identify the type of each one, even when only one was isolated or when the types of root-knot nematodes were mixed. Furthermore, the attachment of dyed spores to the self-active nematodes and Negaresus nematodes, which were separated at the same time as the root-knot nematodes, was hardly observed, and they specifically attached to the root-knot nematodes.

[0059]

According to the present invention, it is possible to easily and quickly identify the species of nematodes in soil and measure the density of each species, even at the local level.

Claims (7)

[Claims] 1. A simple method for identifying a nematode, which comprises staining nematode natural enemy bacteria that parasitize nematodes specifically, and identifying the type of nematode using the stained nematode natural enemy bacteria. . 2. The species of the nematode is identified by observing the color of the nematode natural enemy bacterium adhered to the nematode by making the stained nematode natural enemy bacterium parasitic on the nematode. A simple method for identifying a nematode according to claim 1. 3. The simple identification method of a nematode according to claim 1, wherein the nematode natural enemy bacterium is a bacterium belonging to the genus Pasteuria. 4. The simple method for identifying a nematode according to claim 1, wherein the nematode is a root-knot nematode. 5. The simple method for identifying a nematode according to claim 4, wherein the species of root-knot nematode is identified by using Pasteuria penetrance. 6. The nematode natural enemy bacteria are fast green FCF, acridine orange, methyl violet,
The simple identification method for a nematode according to claim 1, which is stained with at least one dye selected from Brilliant Blue G and aniline blue. 7. The simple method for identifying a nematode according to claim 1, wherein the nematode natural enemy bacteria before staining are subjected to ultrasonic treatment.