JPH06165670A - Method for multiplying natural enemy microorganism of nematode - Google Patents

Abstract

PURPOSE:To provide the improved medium used for the multiplication of microorganisms and suitable for artificially culturing the natural enemy microorganisms of nematodes by adding the extract of the nematodes. CONSTITUTION:The extract of a nematode and/or nutritive ingredients contained in the extract of the nematode or chemical ingredients are added to a medium for the multiplication of the microorganisms to produce the medium suitable for the multiplication of the enemy microorganisms of the nematode.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the production of natural enemy microorganisms of nematodes, which are useful as a means for controlling plant parasitic nematodes which damage crops.

[0002]

2. Description of the Related Art Conventionally, chemical control agents such as fumigants have been used to control nematodes living in soil. However, due to concern about environmental pollution, there is a demand for a method for controlling nematode damage by using natural enemies of nematodes instead of chemical agents.

[0003] Among the natural enemy microorganisms of nematodes, the bacteria of the genus Pasteria, which is classified as an absolute parasite, proliferates using only the host nematode as a host, and therefore can be put to practical use as a suitable microorganism that does not disturb the biological environment of the soil. Is desired.

As a method for multiplying the present microorganism, a method of inoculating a soil of a plant with a nematode in which the microorganism is parasitized and recovering the microorganism propagated at the root of the plant is used. However, the growth efficiency of microorganisms by this method is not sufficient for agricultural applications.

For this reason, attempts have been made in the past to directly cultivate this microorganism by means of microbial culture, but it has not been successful to date.

[0006] Williams et al. Investigated the culture of this microorganism using an artificial medium and reported that the artificial culture was not successful (Journal of Applied Bacteriology (19
89) 67, 145-156).

Bishop et al. Reported that the growth of this microorganism was observed in a medium containing a disrupted liquid of a specific microorganism (Thermoactinomyces vulgaris) (Bioc
ontrol Science & Technology (1991) 1.101-104) reported that lysis of this microorganism occurred after growth and no spores were obtained.

[0008]

DISCLOSURE OF THE INVENTION The present invention is to provide a medium suitable for artificially culturing a Pasteria bacterium, which is regarded as an absolute parasite because it is difficult to culture.

[0009]

Means for Solving the Problems The present inventor has diligently studied the composition of a medium used for artificial culture of a Pasteurian bacterium, and as a result, by adding a nematode extract to a culture medium of a microorganism, The present invention was completed by the discovery that they grow well and form a large number of spores.

The present invention is a method for growing a nematode natural enemy microorganism using a medium for artificially culturing Pastera bacteria, which is obtained by adding (A) nematode extract to (B) microorganism culture medium. Is.

The nematode extract used in the present invention can be prepared from autoactive nematodes, phagocytic nematodes and plant parasitic nematodes. For example, extracts of nematodes such as natural hosts such as meloidine, heterodera, and platinencus, and nematodes such as tervatrix and senoradestis, which are easy to culture artificially, are used.

In the present invention, nematode extract is added as a practical method for supplying nutrients lacking in a general medium. Therefore, carbohydrates, proteins, glycoproteins, amino acids and fatty acids, glycerides contained in nematode extracts,
Similar effects can be obtained by adding chemical components such as glycolipids and steroids.

The culture medium of the microorganism used in the present invention is
It can be adjusted with reference to the known medium composition. For example, an artificial culture medium suitable for culturing mycoplasma, spiroplasma, etc. is used.

An artificial culture medium is prepared by mixing these medium components and adjusting the amount of nutrients such as sugar and the osmotic pressure of the medium. A suitable osmotic pressure is the same condition as a natural parasitic environment.

The medium of the present invention thus obtained is
It contains nutrients necessary for the growth of natural enemy microorganisms existing in the body of nematodes, and is suitable as a medium for artificial culture of natural enemy microorganisms.

[0016]

EXAMPLES The present invention will now be described in more detail with reference to examples.

Preparation of Nematode Crush Solution As a nematode crush solution, a Cenora detis elegans crush solution was prepared by the following method.

Soybean peptone (Difco) 30 g, yeast extract (Difco) 30 g, glucose (Wako Pure Chemical Industries)
10 g, MES (Wako Pure Chemical Industries, Ltd.) 4.3 g, distilled water 950
After dissolving in ml and adjusting the pH to 5.2 with acetic acid, autoclave sterilization was performed. 1.0 g of pig-derived hemoglobin (Tokyo Kasei) was dissolved in 20 ml of distilled water, sterilized by filtration, and added to the medium after autoclaving to prepare a medium for nematode culture.

80,000 nematodes were added to this medium at 20 ° C.
And aired for 16 days. Nematodes that had grown to 20 million were collected by sinking by centrifugation at 3500 rpm, and suspended in distilled water to wash nematodes. Centrifugation again gave 13 g of fresh nematodes.

The nematodes were freeze-thawed, 10 ml of distilled water was added, and the cells were crushed for 1 minute with a cell disrupter (Branson, Sonifier). The disrupted liquid was further homogenized with a homogenizer (Inai Seieido Co., Ltd.) at 1500 rpm for 5 minutes. The homogenized solution was centrifuged at 16000 rpm for 20 minutes to remove the precipitate, and sterilized by filtration to prepare 16.8 ml of nematode disrupted solution.

Preparation of Artificial Culture Medium As a basal medium, SP-4 medium for spiroplasma culture was used as an American Type Culture Collection Medium Handbook (ATCC Medium handbook, pp49,
1st Ed., 1984). SP-4 medium is PP
LO broth (Difco) 3.5 g, tryptone (Difco) 10.0 g, peptone (Difco) 5.3
g, glucose (Wako Pure Chemical Industries, Ltd.) 5.0 g, distilled water 615
After dissolving in ml and adjusting the pH to 6.2, autoclave sterilization was performed, and then CMRL1066 medium (Gibco) 50 ml, yeast extract (Gibco) 35 ml,
It was prepared by adding 100 ml of yeast rate (Difco), 170 ml of fetal bovine serum (Sigma), and 20 ml of phenol red (Sigma).

75 ml of SP-4 medium and 25 m of nematode crushed liquid
1 was mixed, pH was adjusted to 6.2 with caustic soda, and sterile filtration was performed to prepare an artificial culture medium.

Example 1 An artificial culture medium (500 μl) was placed in an incubator, and Pasteria penetrance in the growth phase obtained from 67 female adults of meloidogyne incognita was added, and the mixture was heated to 28 ° C. in a carbon dioxide incubator. I kept it. After 8 days from the start of the culture, the Pasteria penetrans was collected using a centrifuge and observed for an increase in the growth phase.

Table 1 shows the observed numbers of each phase of Pasteria penetrans at the start of culture and after 8 days.

Table 1 Number of nascent morphology of Pasteria bacteria per adult female Adult Early growth Late leaf Four leaf stage Culture start 3400 0 08 days after 14500 3700 4800

Example 2 800 μl of the same artificial culture medium as in Example 1 was placed in an incubator, and pastoria penetrans in the growth phase obtained from 70 female adults of meloidogyne incognita was added,
The temperature was kept at 28 ° C in a carbon dioxide gas incubator. After 10 days from the start of the culture, the bacteria of the genus Pasteria were collected using a centrifuge and observed for sporulation. Table 2 shows the observed numbers of each phase of Pasteria penetrans at the start of culture and after 10 days in this experiment.

Table 2 Number of nascent morphology of Pasteria bacteria per female adult Growth period Early late Late four leaves Bilobular stage Sporulation stage Start of culture 2610 1460 740 160 50 50 After 10 days 1480 200 100 30 4490

Comparative Example 1 The same experiment as in Example 1 was conducted using an artificial culture medium prepared without adding the nematode mixed liquid. Table 3 shows the observed numbers of each phase of Pasteria penetrans at the start of culture and after 7 days.

Table 3 Number of nascent morphology of Pasteria bacteria per adult female: early growth phase late foliage four leaf phase start of culture 4640 0 14 7 days after 3220 14 0

[0030]

EFFECTS OF THE INVENTION The artificial culture medium used in the present invention, which is characterized by adding a nematode extract, is a natural enemy of nematodes by imparting nutrient components which are lacking in the conventional medium for culturing microorganisms. It is suitable as a medium for the growth of microorganisms, particularly Pasteria spp.

Claims (3)

[Claims] 1. A method for proliferating a natural enemy microorganism of a nematode using an artificial culture medium, which comprises adding (A) a nematode extract to (B) a medium for growing a microorganism. 2. The method for growing a natural enemy microorganism of a nematode according to claim 1, wherein the natural enemy microorganism of the nematode is a bacterium of the genus Pastoria. 3. The method for growing a nematode natural enemy microorganism according to claim 1, wherein nutrients and / or chemical components contained in the nematode extract are used as an alternative to (A).