JP2000217568A - Preparation including live bacteria of genus heteroconium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare the subject preparation capable of keeping bacteria alive for a long term without lowering their survival number and capable of preventing soilborne disease from occurring without polluting soil and subterranean water by dispersing cultivated body cells of bacteria belonging to Heteroconium in liquid. SOLUTION: The objective preparation including live Heteroconium bacteria is prepared by-dispersing cultivated body cells of Heteroconium chaetospira bacteria [e.g. Heteroconium chaetospira bacteria BP-6134 strain (FERM BP-6134) or the like] belonging to Heteroconium in liquid. The preparation can be preserved for a long term without lowering the survival number of bacteria, had an advantage of ease of dilution and can directly be used without being diluted by adjusting its concentration in advance. The preparation is applied to take measures against soilborne disease for the purpose of preventing environmental pollution such as soil pollution and/or subterranean water one.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel viable cell preparation which maintains cells of a bacterium belonging to the genus Heteroconium in a living state.

[0002]

2. Description of the Related Art Continuous cultivation of many cultivated plants causes continuous cropping failure such as poor growth, reduced yield, and poor quality. If the continuous cropping failure becomes severe, it will die. Such continuous cropping disorders are mainly due to soil diseases. As a countermeasure against soil diseases of such cultivated plants, chemical synthetic fungicides are widely used at present. However, the chemical synthetic bactericide may lead to food safety due to residues in cultivated plants, environmental pollution such as soil pollution and groundwater pollution, and its harmfulness is receiving public attention. Under these circumstances, the role of microorganisms is attracting attention as a substitute for chemically synthesized fungicides.

[0003] In recent years, a microorganism called endophyte having the ability to colonize roots and at the same time suppress the crop soil disease has been discovered by the Institute of Biotechnology, Ibaraki Prefectural Agricultural Research Center (WO98 / 42823). Endophyte cannot be established on the root unless it is a viable bacterium. For widespread use of such microorganisms, it is essential to have a technique for stably alive living microorganisms without reducing the number of bacteria.

In general, as a long-term preservation method of microorganisms, a freeze-drying method, a liquid paraffin overlay method, a soil preservation method, an air-drying method and the like have been carried out as general-purpose methods. This preservation method does not basically maintain the viable cell count at the same concentration as at the start of storage, but is based on the idea that the survival of the bacteria should be ensured. Little attention has been paid to bacterial concentration. In other words, the productivity is extremely low as a method for producing a viable bacterial preparation that requires maintaining the initial concentration. In addition, since an appropriate method for preservation greatly differs depending on the type of the target microorganism, a method corresponding to the microorganism must be adopted.However, for heteroconium spp. There was no knowledge about the method for preserving viable bacteria.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above, and an object of the present invention is to provide a preparation which is capable of preserving bacterial cells for a long period of time without reducing the number of viable cells. And

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by suspending a cultured cell of a bacterium belonging to the genus heteroconium in a liquid, The present inventors have found that storage stability is significantly improved, and completed the present invention. That is, the present invention is a viable cell preparation characterized by suspending a cultured cell of a bacterium belonging to the genus Heteronium in a liquid.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The viable bacterial preparation of the present invention is composed of a cultured cell of a bacterium belonging to the genus Heteronium and a liquid. However, the bacterium belonging to the genus Heteronium used in the present invention is not particularly limited.
etospira), Hetroconium citalexili (Heteroco)
nium citharexyli, Heteronium solaninum (He
teroconium solaninum). Among the bacteria belonging to the genus Heteronium, preferred strains include Heteronium ketospira BP-6134 strain (Accession number: FERM BP-61 of the Institute of Biotechnology and Industrial Technology, Faculty of Industrial Science and Technology).
34, deposit date: March 13, 1997). The heteroconium genus has a synonymous name, such as Septonema genus, Septosilindorium (Septosilindorium).
cylindrium). The strain of the present invention (BP-6
134 strains) are listed below. Colonies grow slowly, have a dark green to brown hue, and are powdery. Directly linked spores form on the hypha. Some conidiospores are light brown and have 0 to 3 septa.

When a bacterium belonging to the genus Heteronium is used in the present invention, a cultured bacterium obtained by culturing the bacterium in a medium capable of growing the bacterium belonging to the genus Heteronium may be used. Absent. As a method for culturing a bacterium belonging to the genus Heteronium, the same method as that for culturing an ordinary microorganism can be applied. For example, in the laboratory, a culturing method such as culturing at 25 ° C. for 3 weeks on a potato dextrose agar medium can be mentioned. In the case of mass culture, any of ordinary liquid culture and solid culture using a plant-derived solid component such as bran, a porous body impregnated with a sugar or nitrogen source, or the like is possible.

The viable cell preparation of the present invention can be obtained by suspending a cultured cell of a bacterium belonging to the genus Heteronium in a liquid. There is no particular limitation as long as it can be suspended. Specific examples include water, various solvents (e.g., hydrocarbon, ester, ether, and alcohol). Of these, water is preferred. If necessary, various additives such as surfactants and inorganic salts can be added and used. Since the novel preparation of the present invention is in a liquid state, it can be very easily diluted. Further, by adjusting the concentration, direct use is also possible. According to the present invention, it is possible to provide a viable bacterial preparation which can be easily handled by a general farmer and which has the activity and stability as viable bacteria at the same time.

[0010]

Embodiments of the present invention will be described below. [Example 1] Conidia were collected from the surface of the flora of the heteroconim ketospira BP-6134 strain cultured on PDA for 3 weeks, and the conidia were suspended in sterile distilled water, and the spore concentration was about 1.5 x 10 ⁷ cfu / m.
l of spore suspension was prepared. This suspension was further serially diluted 10 times with sterile distilled water to prepare a dilution series up to 1.5 × 10 ⁵ cfu / ml. Then, spore suspensions of each concentration were dispensed into a sterile tube by a fixed amount, and stored in a dark place at 25 ° C. After that, the number of viable bacteria in each suspension was determined over time by the dilution plate method using PDA. The concentration was measured. Table 1 shows the results.

[0011]

[Table 1]

[0012] As shown in Table 1, the viable cell count in the preparation was stably maintained even after 3 months in the preparations of all spore concentrations. That is, it has been clarified that heteroconim ketospira can be stably stored over a long period of time at all storage concentrations by preparing a preparation based on the preparation method.

[Example 2] As a preculture, the flora of Heteroconim ketospira BP-6134 strain plated on PDA was extracted with a cork borer, and two pieces were placed in a PDB 100ml / 500ml (with baffle) Erlenmeyer flask at 1 week at 25 ° C. The culture was performed with shaking under dark conditions at 120 rpm (preculture). PDB 500ml /
Inoculate 5% to 1000ml mini jar fermenter, 25 ℃, 900
Culture was performed under rpm and 0.5 vvm conditions. After 5 days of culture, the hypha was collected by filtration and resuspended at a level of 10 ⁶ cfu / ml to obtain a liquid preparation. This preparation was stored in each temperature zone of 4, 18, 25, 32, and 40 ° C, applied to PDA by the dilution plate method over time, and the change in the number of viable bacteria was investigated.

Further, the precultured solution is spread on a PDA plate medium, and cultured at 25 ° C. for 4 weeks. The conidia obtained are collected with sterilized distilled water, resuspended at a level of 10 ⁶ cfu / ml, and solid-cultured. A liquid formulation of spores was also made. This formulation is also stored in each temperature zone,
The change in the number of viable bacteria was investigated. For comparison, a section containing only spores was provided, stored in each temperature section, and the change in the number of viable bacteria was investigated. Table 2 shows the results.

[0015]

[Table 2]

As shown in Table 2, when stored only with spores, the results showed that the bacteria rapidly died except at low temperatures. In comparison, the liquid preparation was less affected even at a high temperature of 40 ° C., that is, the temperature resistance was high, and it was shown that the viable cell count can be maintained for a long period of time.
Also, whether derived from liquid culture or solid culture,
Formulation was shown to be possible as well. From the above results, long-term viable cell count maintenance was achieved for both the spores and the hypha at high temperatures.

Comparative Example Conidia of Epicoccorus nematosporus were suspended in distilled water at 1 × 10 ⁵ cfu / ml, stored at 4 ° C., and the number of viable cells was examined over time. . Table 3 shows the results.

[0018]

[Table 3]

As shown in Table 3, the viable cell count of Epicoccosus nematosporus decreased sharply, and no viable cells were confirmed at the 8th week, despite the investigation under refrigerated storage. Therefore, this case shows that the method of maintaining the viable cell count by suspending the cells in the liquid is not applicable to all microorganisms.

[0020]

Industrial Applicability The present invention provides a novel live bacterial preparation of a heteroconium genus. This preparation can be stored for a long period of time without reducing the number of viable bacteria. In addition, there is an advantage that the dilution is easy, and by further adjusting the concentration in advance, it is possible to use directly without dilution.

Claims (2)

[Claims] 1. A viable bacterial preparation characterized by suspending a cultured cell of a bacterium belonging to the genus Heteronium in a liquid. 2. The viable bacterial preparation according to claim 1, wherein the bacterium belonging to the genus heteroconium is a bacterium belonging to heteroconium ketospira.