JP3509056B2 - Microbial material that suppresses the growth of harmful soil nematodes and its production method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a soil improving material containing a novel microorganism having an anti-nematode activity against harmful soil nematodes as an active ingredient and a method for producing the same.

[0002]

2. Description of the Related Art Most of harmful soil nematodes parasitize most crops, and the damage in continuous cultivation and facility cultivation is serious, and poses a serious threat to stable production of agricultural crops. In Japan, it is said that about 20 billion yen is consumed annually by soil fumigants in order to avoid these damages. However, many problems have so far been reported regarding the use of these nematicides, such as environmental damage, ecological damage, and safety to humans. Against this background, consumers are rapidly increasing their interest in food safety and environmental protection, and development of new crop cultivation technology and pest control technology that do not rely on pesticides is desired.

In order to meet this demand, biological control of harmful nematodes using natural enemy microorganisms such as nematode-trapping bacteria and nematoparasitic bacteria has been actively attempted. However, since these microorganisms are absolute parasites with high host-specificity, it is difficult to mass-produce them by artificial culture, and there is a problem that the effect cannot be expected unless the strains of bacteria are properly used according to the type of nematode. confronting.

[0004]

DISCLOSURE OF THE INVENTION The inventors of the present invention searched for a microorganism which has an antagonistic property against harmful soil nematodes, is easy to handle such as culturing, and is highly safe. It is provided as a soil improving material which can be stabilized or proliferated in soil by being fixed in the soil.

[0005]

In order to achieve the above object, the present inventors have prepared in soil microorganisms a bacterium that produces a substance that suppresses the motor activity of the second-stage larvae of the sweet potato nematode nematode, a kind of harmful soil nematode. 5 strains belonging to the genus Streptomyces sp. Having the deposit numbers shown in Table 1 below, identification numbers NA-494, NA-3
It succeeded in separating 69, NA-359, NA-303, and NA-150. Furthermore, by fixing these strains to an appropriate carrier and applying them as a soil improving material, we succeeded in reducing the damage of Sweet potato Nematode.

[0006]

[Table 1]

The culture properties of each strain of the present invention are shown in Table 2 below.
6 is as follows.

[0008]

[Table 2]

[0009]

[Table 3]

[0010]

[Table 4]

[0011]

[Table 5]

[0012]

[Table 6]

The physiological and biochemical properties of each strain are shown in the table below.
7 is as follows.

[0014]

[Table 7]

[0015]

EXAMPLES Example 1 Isolation of Actinomycetes and Primary Selection of Anti-Nematode Active Strains Isolation and selection of microorganisms that produce antagonists against harmful soil nematodes were carried out by the following method.

1 g of soil collected from all over the country was suspended in distilled water and allowed to stand for several minutes, and the supernatant was mixed with 3 kinds of antibiotics (C
ycloheximide 25 μg / ml, Nystatin 12.5 μg / ml, Nali
Dixic acid (25 µg / ml) was added to Pridham agar medium, and the mixture was incubated at 27 ° C for 5 to 14 days. The grown colonies were transplanted to Waksman agar medium and incubated at 27 ° C for 5
Actinomycetes were selectively isolated by pure culture for ~ 14 days.

[0017]

The isolated actinomycete strain is treated with TN2 in a large test tube.
A liquid medium (10 ml) was inoculated and cultured at 27 ° C for 5 days with shaking. 10 ml of acetone was added to this culture solution, shaken for 20 minutes, and then centrifuged at 3000 rpm for 10 minutes to separate and remove cells. The supernatant acetone was distilled off with an evaporator,
The residue was used as a test sample.

[0019]

300 μl of test sample and 300 μm of nematode suspension
1 (50 to 60 heads / hole) was mixed on a 24-well plate, left standing in a thermostat at 25 ° C., and the state of nematodes was observed every few hours with a stereomicroscope. At this time, (1) distilled water, (2) TN2 liquid medium, (3) pyrocatechol (final concentration 3
1.25, 62.5, 125, 250, 500, 100
6 stages of 0 μg / ml) were set. 16 methods for determining activity
After 24 hours, the nematode morphology was A (normal: actively moving), B (abnormal: not moving but resembling the shape when moving), C (death: not moving at all. , And the activity index M = A ′ (percentage of A) + B ′ (percentage of B) / 2 was used to calculate the survival rate of nematodes. Finally, the survival rate of the nematodes after 16 and 24 hours was 0% (corresponding to the activity of 1000 μg / ml in the control group (3)), which was used as the primary selection strain.

Example 2 Secondary Selection of Anti-Nematode Active Strains The activity of the primary selected strains was evaluated again while considering the concentration of the test sample. Three levels of concentration (5%, 25%, 5
(0%) and the nematode suspension were mixed on a 24-well plate and allowed to stand in a thermostat at 25 ° C., and the morphology of the nematodes after 16 and 24 hours was examined with a stereoscopic microscope. I observed.
The activity was determined in the same manner as in the case of the primary selection, and a strain having a low motility of nematodes reduced in a short time was selected.

(Example 3) Mycological properties of anti-nematode active strains (1) Culture properties on various agar media Tables 2 to 6 (2) Physiobiochemical properties Table 7 (3) Cell wall components Regarding the diamino acid type in the cell wall of the test strain, all 5 strains were LL bodies (LL-A2pm).

(Example 4) Test of effect of plant culture of anti-nematode active strain on plant Effects of culture supernatant of each strain on growth of vegetables (tomato: Fukuju, Tokage, cucumber: Hayamidori) were investigated. did. TN2 liquid medium (Sakaguchi flask 100 ml)
X 2) at 27 ° C for 5 days with shaking, and centrifugation (300
Supernatant solution after 0 rpm, 10 minutes) in 2 steps (stock solution, 1/1)
The sample diluted with 0) was used as a test sample, and distilled water and TN2 liquid medium were set as control groups. This specimen sample is mixed with the soil (about 6 ml / 3 pots), seeds of the trial product are seeded in the No. 3 pot, and the effects of the trial product on germination, fresh weight, number of leaves, etc. are investigated. did. As a result, there was no tendency to be a problem for plant growth.

(Example 5) Carrier fixation test In order to stabilize the selected strains in soil, the fixation to various carriers was investigated. Each strain was cultivated with shaking in 100 ml of Waksman liquid medium at 27 ° C for 5 days, sprayed on each carrier (vermiculite, zeolite, diatomaceous earth fired grain, paper pulp waste fiber compost, peat moss, granular peat moss) and mixed well, at 27 ° C. It was left standing in a constant temperature room. This was collected periodically and the viable cell count was examined by the dilution plate method. The results are shown in Fig. 1.

As shown in FIG. 1, the number of viable bacteria was high in both organic and inorganic carriers. In addition, the stability of the inorganic carrier was generally high.
In particular, vermiculite has a high stability and a large number of surviving bacteria, so it can be said that it has a high fixability. Although the number of viable bacteria in the organic carrier was small in the initial stage, an increasing tendency was observed after 60 days.

Example 6 Application Effect Test In this example, cucumber (Hayamidori) was tested and the result of application effect of each strain was observed. The results are shown in Tables 8 and 9 . (1) Test scale No. 8 plastic pot (ordinary pot) (2) Application amount of bacteria 200 kg / a equivalent of the following materials was applied. -Material 1: A solution prepared by adding culture fluid to vermiculite so that the density of bacteria in the material is about 1 x 10 ⁶ CFU / g.・ Material 2: The culture broth is added to the base material mainly made of waste pulp and pulp fiber compost, and the density of bacteria in the material is about 1 × 10 ⁶ CFU / g.
Prepared to be (3) Nematode density in contaminated soil High density area (approximately 2.3 heads / dry soil 1 g) Low density area (approximately 0.46 head / dry soil 1 g) (4) Presence or absence of addition of constituent bacteria of test area ( 2) × nematode density (2) × carrier (2) × 2
District system = 16 pots (5) Method for investigating the parasitic degree of root-knot nematodes Goal The degree of engraftment is divided into 5 stages according to the criteria shown in Fig. 2 by looking at the whole root system of cucumber dug at the end of the test. Recorded. (6) Investigation method of root-knot nematode density in contaminated soil The root-knot nematode density in soil pollution was investigated by the Bellman method. Using 50g of contaminated soil before and after this test, 27
Separation was performed at 48 ° C. for 48 hours, and the number was shown per 1 g of dry soil.

[0027]

[Table 8]

[0028]

[Table 9]

[0029]

Industrial Applicability As described above, according to the present invention, it is possible to obtain a soil improving material containing a microorganism having an antagonistic property against harmful soil nematodes and stably growing in soil as an active ingredient. Since it has been found that pests can be controlled without relying on them,
It is possible to provide a farming technique that is safe for the human body and the environment.

[Brief description of drawings]

FIG. 1 is an actual measurement diagram in which the fixability of Streptomyces sp. NA-494 to various carriers is measured.

FIG. 2 is an explanatory view showing the standard of the degree of goal establishment of root system on cucumber.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI C09K 101: 00 (56) Reference JP-A-61-231933 (JP, A) JP-A-4-353593 (JP, A) JP-A-10 -30091 (JP, A) JP-A-9-268089 (JP, A) JP-A-2-108609 (JP, A) JP-A-10-46147 (JP, A) (58) Fields investigated (Int.Cl) . ^7, DB name) C09K 17/32 C09K 101: 00 A01N 63/00 - 63/02 C05F 11/08 C05G 3/02 C12N 1/00 C12R 1: 465 - 1:61

Claims (4)

(57) [Claims] 1. Streptomyces s
p.), and deposit number FERM P-16611,
A soil improvement material containing a strain which is at least one of P-16612, P-16613, P-16614 and P-16627 as an active ingredient. 2. The soil conditioner according to claim 1, wherein the strain has anti-nematode activity against harmful soil nematodes. 3. A soil improving material in which at least one of the microorganisms according to claim 1 is added and stabilized in a concentrated compost carrier or vermiculite carrier containing waste pulp and paper fibers as a main raw material. 4. A soil conditioner in which the amount of the strain according to claim 1 added to the carrier is 10 ³ to 10 ⁸ cells / g.