JPH04353593A - Material for inoculation of microorganism - Google Patents

Abstract

PURPOSE:To provide a material for the inoculation of microorganism, easy to produce and handle, capable of stably keeping the live microorganism during storage and after application to prevent the decrease of the number of living cells and applicable together with a fertilizer before sowing to get an effect comparable to the effect attained by the separate application. CONSTITUTION:The objective material for the inoculation of microorganism is produced by mixing sterilized soil with a water-soluble polymeric substance (preferably polyvinyl alcohol). Microorganisms useful for vegetables are compounded to the material and the obtained mixture is dried at a temperature and means free from adverse effect on the microorganism and mixed to soil preferably after granulating in the form of pellets.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is applicable to the use of airborne nitrogen solid bacteria, antagonistic antibacterial bacteria (microorganisms that have an antagonistic effect on soil pathogens and plant parasitic nematodes, which are the main causes of continuous cropping failure), and diseases during plant cultivation. Resistance-inducing microorganism, PGPR (Plant G
rowth Promoting Rhizobact
The present invention relates to a material for inoculating microorganisms that can sustainably and efficiently supply microorganisms having useful functions such as P. elegans, plant growth-promoting rhizobacteria) into the growing range of plants.

0002

BACKGROUND OF THE INVENTION Microorganisms in soil play a major role in crop production, and if the activity of certain useful microorganisms can be artificially increased, high-level production of crops will be possible. Attempts have been made to artificially introduce microorganisms having these useful functions into the growing range of plants.

[0003] Methods for introducing microorganisms are broadly classified into the following two types. (1) A well-balanced coexistence of microorganisms, each of which has a useful function, is grown in advance on organic matter that serves as nutrients, or a group of microorganisms and organic matter that serves as nutrients are blended, By applying it to the soil, it aims to improve the yield and quality of crops by improving the microflora of the soil, and uses something called compost or soil conditioner. (2) This is a method of introducing specific microorganisms such as rhizobia, VA mycorrhizal fungi, antagonistic antibacterial fungi, and PGPR into the growing range of plants and using microbial materials to express their effects.

[0004] Patents relating to compost and soil improvement materials belonging to (1) are disclosed in, for example, Japanese Patent Laid-Open No. 60-262886 and Japanese Patent Laid-Open No. 61-209981. The disadvantage of these materials is that organic matter accounts for most of the weight of the product. When agricultural waste such as rice straw or livestock manure is used as an organic material, it is becoming difficult to obtain the raw material due to changes in the agricultural structure. It is also possible to use food industry waste (fermentation lees, organic component extraction residue, etc.) and sewage sludge, which are in relatively stable supply, as organic raw materials, but these generally have a high water content and The raw material supply area and the consumption area are often far apart, making it disadvantageous to use it as a raw material in terms of drying costs and transportation costs.

[0005] Furthermore, there are problems in the production process when these organic substances and microorganisms are mixed and fermented.
It takes a long manufacturing period, and as various microorganisms coexist, the microbial flora may undergo a transition due to long-term storage, and in some cases, the efficacy may decrease, making it difficult to supply products with stable quality. There are other problems. Among them, the biggest problem is that even if all of the above problems could be solved, it would be difficult to preferentially propagate the most effective bacterial strain obtained through screening over other microbial groups.

[0006] In order to compensate for these drawbacks, a method has been proposed in which microbial materials are used and specific useful microorganisms are directly inoculated. For example, a method has been developed in which the root-knot fungus is encapsulated in seeds in advance, or a method in which the seeds are coated with powder at the time of sowing. In these methods, even if the microorganism in question has an affinity for plant roots and is motile, such as root-knot fungi, the growth rate of the roots is overwhelmingly high, so the microorganism is It is difficult for the microorganisms to colonize the seeds, and colonization is limited to the root zone around the seeds.

[0007] This drawback is not limited to the application of root-knot bacteria, but becomes even more decisive when, for example, microorganisms that cannot colonize the roots of plants are used. That is, since the survival of the applied microorganisms is inhibited by competition with microorganisms indigenous to the soil, the duration of the effect is limited to a short period immediately after application.

[0008] In order to solve this drawback, the microorganisms to be tested are immobilized, the activity of the microorganisms is maintained according to the growth period of the plant, and the active microorganisms are introduced by applying it in the same manner as fertilizer during the growth period. This method is considered to be effective. As a technology based on the above idea, Japanese Patent Application Publication No. 62-23400
There are microbial preparations for agriculture, forestry and fisheries industries disclosed in Publication No. 5, and a method using sodium alginate and polyvinyl alcohol as immobilization carriers is proposed. Among the above techniques, the gelled product of sodium alginate has a problem in terms of strength. In addition, freeze-drying, gelation using boric acid, etc. are disclosed as methods for preparing polyvinyl alcohol immobilized products. However, these methods involve rapid temperature changes in the presence of viable bacteria, drying steps, or contact with substances harmful to microorganisms such as boric acid, which adversely affect the survival of microorganisms that are sensitive to these conditions. It is believed that the practical implementation of the method is quite limited.

[0009]

[Problems to be Solved by the Invention] The present inventors found that the combination of only polyvinyl alcohol (hereinafter referred to as PVA) and microorganisms has difficulty in stabilizing the number of viable bacteria in the product and has problems in practicality. In view of the fact that when granules are manufactured using only soil and microorganisms, the strength of the granules is insufficient and there are problems with storage and transportation.
We are conducting extensive research to provide a microbial inoculation material that maintains stable viable bacteria throughout the storage period and after application, has no risk of decreasing the number of viable bacteria, and has the same effect when applied at the same time as fertilizer before sowing. This is what I did.

0010

[Means for Solving the Problems] The present invention aims to solve the above-mentioned problems, and is characterized in that a water-soluble polymeric substance, preferably PVA, is added to and mixed with sterilized soil.
Furthermore, it is characterized in that microorganisms useful for plants are blended into this microorganism inoculation material and granulated, preferably into pellets.

[0011] The soil used in the present invention is not particularly limited, but it is preferably fertile soil such as paddy field soil, field soil, and mountain soil. Depending on the nature of the available soil, chemicals such as calcium carbonate and calcium hydroxide are mixed into
H adjustment can also be done. Alternatively, it is also possible to improve the physical and chemical properties of the soil by adding clay minerals such as bentonite and montrollilonite. Furthermore, if necessary, various organic substances can be added to increase the survival stability of microorganisms. Furthermore, it is also possible to add other formulations.

Furthermore, if the soil is passed through a sieve of about 50 to 100 Tyler meshes, subsequent granulation can be carried out smoothly. The soil or the mixture of soil and various additives prepared in this manner must be sterilized before use. Sterilization can be carried out by conventional industrial methods such as high-pressure steam sterilization, ethylene oxide gas sterilization, radiation sterilization, ultraviolet sterilization, chemical sterilization, fumigation sterilization, etc., and high-pressure steam sterilization is particularly preferred. The conditions for autoclaving are 105-135°C for 5-60 minutes, preferably 115-125°C for 15-30 minutes.

[0013] If this operation is omitted, competition will occur between the microorganisms indigenous to the soil and the microorganisms to be inoculated, which will significantly impair the survival of the microorganisms to be inoculated. Sterilized soil or soil mixed with various additives should be kept dry. The drying conditions are either left at room temperature to about 105°C, or dried under ventilation. Preferably, the temperature is 60 to 90°C for 5 to 24 hours.

[0014] PVA used in the present invention can be used either as an aqueous solution or as a solid. Normally, the pH when PVA is dissolved in water is on the acidic side, but the optimum pH for the target microorganism is
Adjust and use. When used as an aqueous solution, one with a viscosity of about 1 to 300 cps is suitable. Viscosity 1cp
If the viscosity is less than 300 cps, the strength of the finished granules will be insufficient, and if the viscosity exceeds 300 cps, the operability during mixing of soil or soil and additives will be poor and unevenness will likely occur, and even if forced to mix, microorganisms will not survive. detract from sexuality. The amount of PVA used is 0.5 to 30 parts by weight, preferably 1 to 10 parts by weight, in terms of PVA base material, based on the soil to be inoculated or the mixture of soil and various additives. When PVA is used in solid form, it is preferably partially saponified and in the form of fine powder from the viewpoint of solubility at room temperature.

[0015] Various types of microorganisms can be used in the present invention, and among them, the following are particularly preferred. For example, Rhizobium spp., Bradyrhizobium spp.
ium), Azotobacter (Azotoba genus)
cter genus), Trichoderma genus (Trich
bacteria of the genus Oderma), bacteria of the genus Gliocladium (Gl
iocladium genus), Fusarium genus (Fu
Sarium spp.), Verticillium spp. (V
erticillium), Enterobacter, Bacillus, Pseudomonas, Serratia
(Serratia genus), Pasteuria genus (Pa
steuria spp.), Streptomyces spp. (S
microorganisms such as treptmyces (genus treptmyces). That is, the present invention allows various microorganisms to survive stably. These microorganisms are cultured in liquid or solid form by conventional methods and used in the form of cells, spores, or the like.

In the present invention, microorganisms are blended into a microorganism inoculation material consisting of soil and PVA and molded. For example, the following method is used industrially. (1) After dispersing the cultured microorganisms in a PVA aqueous solution, granules are produced by spraying onto soil or a soil-based composition using a pan-shaped granulator. If necessary, dry at a temperature that does not damage microorganisms.

(2) After dispersing the cultured microorganisms in a PVA aqueous solution, they are mixed with soil or a soil-based mixture using a kneader or the like, and then extruded into an arbitrary shape using an extruder or the like. Manufacture things. If necessary, dry at a temperature that does not damage microorganisms until the desired hardness is achieved.

(3) After dispersing the cultured microorganisms in a PVA aqueous solution, they are mixed with soil or a soil-based mixture using a kneader or the like, and dried at a temperature that does not damage the microorganisms until a desired hardness is obtained. Then grind.

(4) Partially saponified PVA in the form of fine powder is mixed with soil or a soil-based composition, and a granulated product is produced by spraying a suspension of cultured microorganisms using a pan-shaped granulator. .

(5) Mix partially saponified PVA in the form of fine powder and soil or a soil-based mixture, inject and mix a suspension of cultured microorganisms, and form a molded product according to the methods of (2) and (3). Manufacture.

(6) Mix soil or a soil-based mixture with the PVA solution, granulate it, dry to a desired hardness or slightly harder, and then spray the granulated product with a suspension of cultured microorganisms. Manufacture.

[0022]

[Effect] As mentioned above, with the combination of only PVA and microorganisms, it is difficult to stabilize the number of viable bacteria in the product and there are problems with practicality. The strength of the product was insufficient, and there were problems in terms of storage, transportation, and survival of microorganisms in the soil. The present invention focuses on the appropriate nutritional properties and granulation properties of soil, and has succeeded in providing a material for microbial inoculation that has favorable granulation properties and viable bacteria stability by using PVA. . By using this material, microorganisms that have useful functions for plants are immobilized on the material, and by mixing it with the soil before or after sowing, it is possible to keep a sufficient amount of useful microorganisms alive as the plants grow. This makes it possible to continuously supply these to the roots of plants.

[0023]

[Effect of the invention] The microorganism inoculation material of the present invention has a stable number of viable bacteria even during storage and in the soil, so it can be applied in advance to the soil before cultivating plants, making the work easier. This saves labor and expands the scope of application of various useful bacteria to plant cultivation. In addition, even if large amounts are supplied to the soil, there is no risk of adverse effects on the ecosystem because harmony is achieved with the bacteria in the soil. Furthermore, the product of the present invention is mixed and used in seedling raising soil,
Since the number of viable bacteria in the product is highly stable even when transplanted to the main field along with the seedling soil, the number of viable bacteria in the future will also be stable, resulting in a beneficial effect on the plants.

[0024]

[Examples] The present invention will be specifically explained below using Examples and Comparative Examples. Example 1 Fluorescent P, an isolate capable of colonizing the roots of radish
bacterium of the genus pseudomonas, DK-240 (R
ifanpicin resistant strain) was cultured in a liquid medium at 28°C under aerobic conditions, and granulated using PVA B-17 (manufactured by Denki Kagaku Kogyo Co., Ltd.) and soil within the Research Institute of Denki Kagaku Kogyo Co., Ltd. It became.

DK-240 strain is a bacterium isolated from the roots of radish grown in a radish field in Miura City, Kanagawa Prefecture. This bacterium grows well on broth agar, is polar flagellated, motile, Gram-negative, obligately aerobic rod, and does not produce spores. In addition, catalase activity was positive, and O-F
(High-Leifson) test produces acid aerobically and Pseudomonas agar F
When cultured on (PAF) medium at 28°C for 2 days, a fluorescent dye (fluorescin) is produced.
It was identified as a udomonas bacterium. DK-24
Rifanpicin generated by natural mutation from strain 0
A resistant strain (which can grow at 100 μg/ml of Rifanpicin) was isolated and named strain DK-240.

[0026] The PVA B-17 used was heated in a warm bath (90
℃ or above) to form an aqueous solution, and after cooling, the pH is 6.9 to 7.
．． Adjusted to 1. Add 1 part of the bacterial suspension to 5 ml of this PVA aqueous solution.
．． 2 ml was added to adjust the concentration in the microorganism-containing PVA aqueous solution to 5%.

[0027] This microorganism-containing PVA aqueous solution (viable number of bacteria: 10
After adding 10g of soil to the mixture (containing 12 particles/ml), mix well and extrude using a syringe to obtain a mixture with a diameter of 1mm and a length of 5-6.
The pellets were granulated into a cylindrical shape of mm. The material was dried at room temperature under blowing air so that the moisture content was 30 to 40%, and the desired microorganism-containing material was obtained. There were 2×1010 live bacteria in 1 g of this material. The obtained microbial inoculation materials were subjected to a plant cultivation test using pots.

[0028] Each granulated product was applied at two locations in a pot containing 300 g of soil at a rate of 0.15 g per location. On the day of application and 5 weeks later, radish seeds are sown at the granule application position,
Grown in a greenhouse. The roots of each radish were collected 6 weeks after sowing, and after removing the attached soil in sterile water, they were homogenized in sterile water, and the root suspension of this radish was rifated.
Test bacteria were collected from the root surface by inoculating them onto a PAF medium containing 100 μg/ml of mpicin, and the number of viable bacteria per root weight of the radish was calculated. The results are shown in Table 1.

[Table 1]

Comparative Example 1 DK-240
(Difco)+10 after culturing in 1% glucose medium
0 ml was collected, centrifuged, and the supernatant was discarded. Add 100 ml of a 10% aqueous solution of PVAB-17 to the remaining moist bacterial cells.
were added and mixed to prepare a PVA aqueous solution containing 109 live bacteria. The prepared live bacteria suspension was dropped into a Na2B4O7 saturated aqueous solution (pH 7.0) from a cylinder equipped with a 0.7 m/m injection needle, and spherical live bacteria-containing P
A VA gelling carrier was prepared. The carrier immediately after preparation contained 2 x 108 live bacteria/grain. This carrier was used in a plant cultivation experiment in a pot. The application amount per planting hole was 15 seeds/planting hole in order to equalize the initial amount of viable bacteria, but the procedure was as in Example 1. The results are also listed in Table 1.

Example 2 Bradyrhizobium japonicum
rifam from IFO 15001 by mutation treatment
picin-resistant mutant strain (100 μg rifampi
cin/ml medium) was obtained. The above mutant strain was cultured on a yeast extract/mannite agar medium containing 100 μg/ml of rifampicin at 30° C. for one week and then harvested. The collected mutant strain was treated with PVA adjusted to pH 7.0.
Stir and suspend live bacteria in a 7% aqueous solution of B-17 and add 2 x 101
A suspension of 1 cell/ml was prepared. Add and mix 8 ml of the suspension to 10 g of sterilized soil, extrude through a syringe,
It was dried under blowing air to a moisture content of 25%. 1 x 10 in the carrier
There were 10 live bacteria/g.

1 g of the above carrier was uniformly mixed with 100 g of field soil using a V-type mixer. The field soil containing the carrier was filled into a pot, and incubated at 28° C. in a thermostatic chamber while replenishing water to maintain 60% of the maximum water capacity. The total amount of soil was collected over time, a soil suspension was prepared, and the number of soil test mutants was measured by the dilution plate method on a rifampicin-containing yeast extract/mannite agar medium. The results are shown in Table 2.

[Table 2]

Comparative Example 2 Similar to Example 2, Bradyrhizobium j
A mutant strain of Aponicum IFO 15001 was cultured. The mutant strain was suspended in sterile water and the number of viable bacteria was 5 x 108.
A suspension of viable cells/ml was obtained. 20 ml of this suspension was uniformly dropped onto 100 g of soil using a dropper, and then sufficiently stirred and mixed using a V-type mixer. The number of viable bacteria in the soil was measured according to Example 2 and is also listed in Table 2.

As is clear from Tables 1 and 2, it was found that the microorganism inoculation material of the present invention has the effect of stably allowing specific microorganisms to survive in the soil for a long period of time.

Claims (2)

[Claims] 1. A material for inoculating microorganisms, characterized in that a water-soluble polymer substance is added and mixed with sterilized soil. 2. A material for inoculating microorganisms, comprising microorganisms, sterilized soil, and a water-soluble polymer substance.