JPS62171675A - Production of agricultural field crop inoculum of bacteria - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates generally to a method for producing large amounts of stable, storable, viable microbial material, and in particular to:
The present invention relates to a method for producing stable dry microbial preparations which are particularly advantageous for use as inocula on farm crops to improve productivity.

[Conventional technology]

The use of a biological (i.e., microbial) inoculum on a selected crop species to promote the growth of the nuclide crop or to confer resistance to certain pathogenic organisms in the nuclide crop may be used in agriculture. This has been known and practiced for a long time. Additionally, there are several species of root-colonizing microorganisms that are useful in promoting the growth of flowering plants or acting as antagonists to disease organisms, thus improving crop productivity. There is recent research data that shows this. [Schrot
h) and Hancock, "Disease Suppression Soil and Root-Colonizing Bacteria," Science, 216.1376-1.
381 (1982): ]. The earliest established use of microbial inoculum was the planting of Rhizobium spp.
It is a perfectly normal practice to inoculate soybeans and other legumes with bacterial cultures of the genus um ), so that Rhizobium bacteria colonize the internal root nodules of said soybeans or other legumes; The colony symbiotically fixes nitrogen in the root nodule for the plant as well as the bacteria. Currently, this inoculation is accomplished in several ways, none of which are optimal for all purposes. Current practical methods include applying the seed and dispersing the planted seed or crop, ie, applying a liquid live inoculum to the furrow in which the seed is planted.

Significant efforts have been made in the past to optimize the product for the preparation of bacterial crop inoculum, particularly Rhizobium inoculum. Typical preparation methods for such inocula usually require a fermentation step to grow sufficient quantities of bacteria, and a stabilization or formulation step that involves storing and storing the mature bacteria. The purpose is to formulate the bacteria in the form of active cultures in order to stabilize them in an inert state for transport or to deliver them directly to farms. In the past, the fermentation and formulation steps were typically considered to be quite distinct, requiring one or more handling or processing operations to successfully transfer viable bacteria from the fermentation to the formulation process. becomes. Furthermore, in conventional methods, the usual carrier for viable Rhizobium cultures was beets. Direct culture of bacteria on beets has usually been considered impractical due to microbial contamination of the beets, difficulty in sterilizing the beets, and toxic substances produced in the beets during sterilization. When using beet carriers, a clear separation of the fermentation process from the formulation process was usually necessary.

It has been recognized in the agricultural field that other non-Rhizobium microorganisms can promote the growth of conventional crops. For example, it was known in the prior art that many fungi are found associated with the roots of certain vascular or woody plants. The modes of association between fungi and plants have not been well characterized, and there is no clear consensus among mycologists as to which of these bonds are better characterized as symbiotic, or more appropriately termed pathogenic. There is no understanding or agreement. Said connections produced by plant roots and fungi are often referred to as mycorrhizal connections. However, little is understood about this bond.

It has been particularly reported that certain fungal species or strains have the potential to be antagonistic to certain other plant pathogens. For example, Talaromyces flavus (Talaromyces
Verticillium dahliae (Verticillium dahliae) is a fungus that is used in the cultivation of eggplant (in this case, the original artefact is Verticillium dahliae).
llium dahliae). [Marois et al., [Solanum-Melongena in farms]
Verticillium verticillium (Vert.
Biological control of Icilium Wilt)
, Plant Disease
), 66: 12.1166-1168 (19
82)). Other bacterial species have also been reported to have similar effects. [Papivizas, [
soil and bismuth sativum.

Cultivar Parts Efted - Freezer (Pisu)
m-3ativum Cultivar Perfect
tid-Freezer) and Bean Facella Subal Glass Cultivar Blue Lake (Bean
Phaseolus-VulgarasCultiva
Trichoderma-Harzianum (Blue-1ake)
Phytopatology (Phytopatology)
72: 1.122-125,
(1982)). In U.S. Pat. No. 4,259,317, a formulation for the protection of growing sugar beets against damping-off caused by a parasitic fungus is disclosed, which formulation
ligandrum), which is applied as an inoculum to sugar beet seeds to prevent damage to the plants by other fungal species.

[Problem that the invention seeks to solve]

One problem with using fungi as crop inoculum is that it is difficult to propagate and produce large quantities of vegetative fungal material.

Bacterial culture technology has not yet made much progress. For bacterial species useful as crop inoculants, the bacterial species can be produced in reasonable quantities and the final product of the production process can be adequately and easily handled and commercially useful. It must have a particularly long shelf life. While it is possible to maintain a suitable microbial inoculum alive in a liquid state, liquid cultures present transportation and storage problems.

Therefore, it is clearly preferable that the fungi be able to grow even if they are in a dry powder state when planted in agricultural soil. At least one prior attempt is known to devise a method for producing and preparing grains for use as agricultural inoculum. This method is described in U.S. Department of Agriculture U.S. Pat. No. 4.530.83 by Soper and McCabe.
It is disclosed in No. 4. In this method, the mycelium on the side of the image is cultured in a suitable medium, and then the mycelium is obtained on a certain mesh screen to produce a mycelium mat. The mycelium mat is then treated with a protective agent, preferably until saturated, such as a sugar solution. Furthermore, culturing the mycelium,
Air dry at room temperature. The present invention is intended to be a more convenient method for producing similar dry bacterial products that can be propagated.

[Means for solving problems]

The present invention is summarized in that a method for producing a microbial farm crop inoculum comprises the following steps. That is, granular vermiculite (Vermicelli) was grown in a flexible disposable culture container.
(culite), preparing a medium containing a certain amount of microbial nutrients and water; inoculating the medium in a culture container with a culture of a microbial inoculum; and subjecting said culture container to conditions suitable for the growth of microorganisms. and then growing and maturing the microbial culture in the medium in the incubator.

It is an object of the present invention to provide a method for reliably producing large quantities of stable microbial inoculum for crops.

Another object of the present invention is to provide a method that is convenient and economical to carry out, as it allows the fermentation and formulation processes to be combined in a single step, while still producing a superior product. be.

An advantage of the invention is that the method requires the least possible handling of the microbial culture.

Another advantage of the present invention is that it allows for effective fermentation and formulation of microbial cultures for which no conventional reliable protocols exist.

Other objects, advantages and features of the invention will become more apparent in the following description.

In contrast to most conventional microbial fermentation methods that inherently use liquid media, the method of the present invention contemplates the use of a medium containing a specific carrier moistened with a nutrient-bearing liquid (i.e., water). There is. Rather than growing the microorganisms in a medium and then transferring them to a carrier, the method employs a carrier that is ultimately dispersed as the microbial product at the beginning of the fermentation process. By introducing the carrier at such an early stage, the method reduces the effort of handling, measuring and transporting the culture, making the single-step fermentation and formulation process simple, economical and effective. ing.

The implementation of the production method of the invention begins with the preparation of a medium for the growth and formulation of microorganisms containing carriers. The medium contains a finely divided particulate carrier, preferably vermiculite, nutrients selected for the organism, and water. The medium is placed in a culture container and sterilized in the culture container. The culture container is preferably a flexible disposable container such as a simple polyethylene or polypropylene cup, bucket, tray or bag. The medium is then inoculated with microorganisms in the culture container and the microbial culture is grown. After the growing period, the culture can be used directly as a crop inoculum, and then air-dried or stored in a liquid state for a long period of time. The culture products in culture containers can be stored in these same culture containers and then delivered for use.

The products are stable and easy to use and are highly compatible with applications for uniform seed application.

The first step in carrying out the method of the invention consists of preparing the medium. The majority of the medium consists of finely divided particulate or granular material that is useful as a microbial carrier. It is known that vermiculite is a preferred material as said carrier. Because the price of vermiculite,
This is because they have a combination of advantageous properties such as efficacy, non-toxicity, uniformity and processability. As can be seen from the above method, the vermiculite is not only used as a medium but also as an inoculum carrier. Vermiculite is suitable for both methods because it is naturally exposed to air and has a strong buffering capacity. Flaky vermiculite particularly offers the advantage of large surface area, which is beneficial for microbial growth. Of course, vermiculite is inorganic and easily sterilized. less than 40 messini, usually 40-2 (1
1) Micronized vermiculite pulverized to a particle size of about 100 ml is suitable for the practice of the present invention. The most preferred size of vermiculite is 45
It is in the range of ~80 meters.

Vermiculite provides a substrate for microbial cultures to grow, while
Nutrients are necessary for the growth of microbial inoculum. The appropriate nutrients should be selected for each microbial system produced. For most Rhizobium cultures, yeast extract and mandol nutritional components were known to give good results. For other bacterial cultures, commercially available nutrient broths are often used. For many fungal cultures, and some bacterial cultures, it is surprisingly known that wheat flour and bran flour in general, and in particular conventional unbleached white flour, are advantageous for use as nutrients. ing.

It is known that wheat flour is considered to be particularly advantageous with respect to the stability of the microbial products of many strains produced by this method, for reasons that are not yet fully understood. Although other polysaccharides may be used, especially for fungal products, the resulting products are very stable when grain carriers are used. Generally, the amount of grain carrier is a small proportion to the amount of substrate material, i.e. 1.0 by dry weight.
It is within the range of ~9%. For fungal cultures or other cultures suitable for grain carrier nutrients, 1.
It was known that a dosage of unbleached white flour or bran flour in the proportion of 3% dry weight is very suitable. For Rhizobium cultures and other bacterial cultures, the optimal ratio for appropriate dry nutrients is 1
．． 8% and 1.2%.

The mixture is then moistened to provide adequate water for culture growth. The amount of water added to the granular matrix is variable and left to the choice of those skilled in the art. It is generally known that an amount of water equal to 60% by weight of the granular substrate is most suitable for the growth of the culture. This ratio is 1.5-
It can also be expressed as water/g vermiculite. When using nutrients in solutions such as yeast extract and mandol solution, the nutrient solution is only 1.3rd per gram! is added to vermiculite at a rate of, and as the nutrients are added,
An appropriate amount of water is added to the culture.

In the detailed description of the invention, the following is contemplated. That is, a culture medium formulated for microbial growth as described above is charged to a culture container and preferably also sterilized, and then the microbial culture is introduced. This is done for convenience and ease of handling. Also, if necessary, it is quite possible to prepare a large quantity of medium, sterilize it and then introduce it into a previously sterilized container. A microbial culture is grown in the container. However, it has been found that the process works most quickly when the culture container is a simple, flexible plastic resin container, such as a common polypropylene film bag.

The above mixture is introduced into the container in an appropriate amount, and the culture container filled with the medium is then autoclaved to remove the sterilized medium and the sterilized container in one step. After sterilization, the medium and containers are cooled before use.

A sterilized container containing a suitable medium mixture is then inoculated with the culture to be grown. In the case of bacteria, this inoculum must be a culture of bacteria. In the case of fungi, the inoculum should consist of spores or other viable material.

After introducing the microbial culture, close the container. Polypropylene bags can be easily sealed by heat sealing.

The heat sealing deforms the bag to obtain a sealed container. Alternatively, the bag can also be easily closed mechanically with a zipper or other similar means. These containers are then stored under conditions suitable for the growth of microbial cultures. Typically, these containers are kept at room temperature for a period of time sufficient for the culture to grow on the medium contained within the container.
Simply let it stand. For most fungi, 30
It has been found that a period of at least 45 days, preferably at least 45 days, is required to achieve optimal growth and stability of the culture under room temperature conditions. Regarding bacteria, 7
A period of at least 30 days, preferably at least 30 days, is a satisfactory growth time.

- Once the microbial culture has been grown in the container, the material can be used as an agricultural inoculum directly from the container, or the material can be removed from the container, dried and used as a dry inoculum or as a seed coating. be able to. When the material is used wet from the container, the container itself, preferably a plastic bag, can then be used as a storage and transport container. These microorganisms, especially /
(When Cutilia reaches the limit of solid number density in the container,
Becomes dormant. The wet microbial mixture carried on the upper right side of the heel can be directly introduced from the bag into the furrow or the like separately from the seeds, or can be used by being mixed with the seeds during sowing. For many cultures, and especially fungal cultures, however, dry materials offer attractive benefits in terms of ease of handling and stability.

If a fungal culture is to be dried, it is necessary to choose the harvest period in the container. Generally, for periods of -30 days or less, the shelf life as well as the stability of the dried inoculum are adversely affected. If the culture is aged for a period of more than 30 days and then dried, the dried product becomes extremely stable and shows a high survival rate even after long-term storage.

It has been found that the stability of a dried culture generally does not increase appreciably after growing it for 45 days, and therefore, for the most efficient production processing of dried inoculum, growing the culture for 45 days is preferred. Ta. After growing the culture in the container for a predetermined growth time, the container is simply opened and the culture is allowed to air dry at room temperature.

The culture is left exposed to the outside air at normal room temperature in a room suitable for human life. The standing time is sufficient for the moisture content in the culture to equilibrate with the relative humidity of the room in which the culture is being dried.

It has been found that dried microbial cultures of fungi produced by this method are stable and easy to handle. These cultures have a characteristically granular appearance and are agriculturally versatile and can be easily used by spraying on farms or applying to seeds. The dried culture thus survives for a period of at least two months, with little, if any, loss in fungal survival during that period. Furthermore, the manufacturing process of the present invention is effective to utilize and economical to operate.

Since sterilization and microbial growth can be performed in the same container, handling of the various components of the process and of the microbial culture itself is minimized. Since this method does not require special containers or vessels, the materials required to carry out the method are economically attractive.

And it can be easily done manually. This method thus provides an efficient and economical process, yet results in a highly stable and homogeneous product, while retaining optimal viability.

Example 1 20 g of granular matrix of vermiculite of 40 meshes was
g of flour and 13 mf of water.

This mixture was mixed together in a container and 113.4
g (4 oz) of polypropylene sample force and door pressure.

The specimen cup was sealed and autoclaved to sterilize it, then the cup was cooled.

Once the cup has cooled, it is opened again and the fungus Marasmius oleetes
The offspring of Mius Ore'ates were inoculated. The cup was hermetically sealed again. The cups were then left in the laboratory at room temperature for 45 days.

After 45 days, the cups were opened and the cultures were allowed to air dry in open trays for 2 days.

The dry cultures thus produced were assayed for stability and viability. As a result, the resulting dried culture contained at least 106 viable offspring.

oleates (M, oreates) per gram. After 6 weeks of storage, the viability test was repeated and there was no appreciable decrease in viability of the dried culture.

Example 2 Two gallon autoclaved plastic jars were inoculated with each of the fungal cultures listed in Table 1 below. Each jar was prefilled with 5 (11) g of a growing medium containing vermiculite-flour-water, proportioned as in Example 1. Approximately 0.5 g of fungal culture inoculum was added to each jar and the medium was then thoroughly mixed.

The activity level of the fungi in the medium, also expressed as the number of offspring per gram, was measured 2 to 4 weeks after inoculation. These jars were always stored at room temperature. Additional activity measurements were performed 6 months after inoculation.

Each fungal culture was identified in the attached table to the best of the researcher's knowledge.

Table I 257 0 Lycoderma] '2X10'
2X107 3X107258 0 Ricoderma] 2x105 2x108
2xlO”274 [:) Lycoderma:]
2X10'2X10"
2X108285 [7 Oma (Phoma)
lXIO31XIO62X10'301 [Mucor] 1×1(13 1×106
4×1063 (13 [) Lycoderma]
6X1 (13 6X10'
3X107331C Fully ILt! J7
8X10' 8X105 6X10'
(Alternaria)] 332 [)! J] De + L Ma: l 1x
lO'1xlO'2xlO'
490 [Ascomycetes] 4X1 (134X10'
5X10'491 [Ascomycetes] 3X10'
3X10' lXl07545 [Resobu 2
] lXl0"lXl0'
3X10' (Rhizopus) 546 [:Morcellella] 8X10
'8X10'9X10' Example 3 Cultures of each of the bacteria introduced in Example 2 above were prepared again in a similar manner. The resulting culture was then air-dried. Determination of activity levels was performed on dry basis using standard dilution methods. The dried cultures were then stored at room temperature for 6 weeks, after which culture viability was measured again. The results are summarized in Table ■ below.

144 1 pride 3 ne distortion H...i...ore
ates 189 Trichoderma Trichoderma1
91 Mortierell
a231 Mucor238
Mucorales (edited by Muhei yles257)
Trichoderma Trichoderma 258 Trichoderma Trichoderma 274 Trichoderma Trichoderma 284
Ascomycetes (Muboite 8t°.

285 Phoma301
Mucor 3 (13 Trichoderma 331 Alternaria A 1 ternar 1a332 Trichoderma 474
Alternaria Alternaria475
Cladosporium Cladosporium
490 children') 6 bacteria frequent
es491 Ascomycetes (e?boy5e
Te.

545 Rhizopus Rh1zopus
546 Mortierel
la■Table II゛1〕ゝLevel 6°・mouth゛゛2 Main level 3 X 10'
2 x 10"3 x 10'
2 x 1062 x
10' 7 x 10'
2 x 10' I
X 1 (15) X 10'
2 x 1062 x 10b
2 x 10'I x 1
0” I X 1011
8 x 10' I
X 10"2 X 10'
9 x 10'4 x 10'
2. X 10S2 X 1(
15) X 1063X10'
IX'lO'6 X 10'
5 x 1053 x 10'
2 x 10'5
X 10' 6 X
10'2 x 10'
9 x 1061 x 10'
3 x 1066 x 106
2 X 10'2X10. '
6X10'2 X 1
05 6 X 10'2
X 10' 3 X
106 Example 4 A portion of the dried formulation of each culture prepared in Example 3 above was isolated for cold storage viability testing.

Samples of each dried culture were cooled at 4°C for 5 months.

Activity levels in each culture were determined 3 months and 5 months after the start of storage.
Measurements were taken after several months had passed. The results are shown in Table ■ below.

1-1- Bacterial culture Activity level at the time of manufacture: 144 Marasmius (Ma
rasmius 3 x 10
"Oreates" 189 Trichoderma
3 x l Q ”191%Atf
sshi5 (Mortierella)
2x IQ'231 Mucor
(Mucor) 2X
10'238 Mucorales
ales) l x l Q
'257 Trichodel? (Trichoder
ma) 2 X 10@258 Trichodel? (Trichoderma)
I X 10.274 Trichodel?
(Trichoderma)
3 x IQ'284 Ascomycetes
(Ascomycetes) 2
X 10 '285 F# -7 (Phoma)
4 X 10'301
Mucor 2
X 10″3 (13 Trichodel? (T
richoderma) 3
10'331 Alternaria (Alter)
naria) 6 x 10'
332 Trichodel? (Trichoderm
a) 3 X 10'474
Alternaria
5 X 10'475 Cladosporium
2 X 10 '490 Ascomycetes (7) Fungi
(Ascomycetes) I
X 10 '491 Ascomycetes (7) Fungi
(Ascomycetes) 6 X
10 '545 Rhizopus
2 X 10'546 Mortierella
2 X 1 Activity level after 53 months Activity level after 5 months 3 X 10
' I X 10' I X
10” 9 X 10@
5 X 10' 4 X
10'3 x 10'' 9
X 10'2 X 10"
8 x 10"5 x 10'
2 x 10'2 x 10'
7 x 10'5 x
10" 5 x 10'4
X 10' 3 X 1
0'5 x 10' 2
X 10@2 X 10'
7 X 1G"2 X to'
2 x 10'I x 10'
2 x 10・4 x 1
0'4 x 10'6
X to' I X 1
0@I X 10@9 X 10'I X
10@I X 10'I X 10@I
X 10'8 X 10'
2 x 10'3 x 10'
6 x 10'9 x 10'
5 x 10' Example 5 Standard commercially available vermiculite
) Milled to a nominal 40 mesh. This crushed vermiculite was sieved to obtain a fraction of 45 to 80 mesh,
This was packed into a flexible polypropylene bag used as a culture container. The vermiculite in this bag was autoclaved for 45 minutes.

Rhizobium jabonicum isolated in a soybean field in Iowa.
um) was grown to stationary phase in yeast extract-mannitol broth. The stationary phase state of this inoculated culture was determined by optical density measurements. This optical density remains at a constant value when the stationary phase is reached. A sample was removed from the above inoculum culture and diluted with fresh yeast extract-mannitol broth so that the bacterial density in the bacteria-nutrient solution was slightly less than 1 (slightly less than 14 bacteria) per ml of solution. This bacteria
The nutrient solution was added to the vermiculite in the bag at a rate of 1 ml of solution per gram of vermiculite. The liquid and vermiculite were then thoroughly mixed and the bag was sealed. These bags were then stored at room temperature to allow bacteria to grow. Table 2 below summarizes the growth status of the sample cultures.

Table ■ Bacteria density (per 1g) Sample No. Initial (t=o) 7
Day 30 Day 1 1.4x
lO' 1.7x108 1.3x1 (19)
2 1.4xlo'3.1xlO"
1.3xlOg3 1.4xl(1'
3.2xlO81, 6xlo94 1.
4X10' 3.2X108 1.0X10'
Thus, approximately 1 (11). (11) 0x growth was achieved in 30 days. The resulting densely grown bacterial culture was granular and at a density suitable for direct application to agricultural fields. In this field, 1 bacterium per 1 g
Since it is normal to achieve a density of 105 bacteria per seed by coating the seeds with inoculum at a density of 0.8 bacteria, even if there is an order of magnitude reduction in viability, this is achieved here. The density obtained would still achieve sufficient bacterial density for use. Thus, Rh, izobeium in the bag.
) cultures were suitable for use in agricultural fields without further treatment. This is because it already contained a suitable carrier and a high density living culture.

Example 6 The method of the present invention was applied to other Rhizobium
) strain, we applied the above culture method to four other Rhizobium japonicum (Rhizobium japonicum) strains.
inmJapon icum) strain, Louisiana (Lou
three mutant strains of wild-type bacteria isolated from soybean plants in Minnesota (14inn) and
It was repeated for one culture isolated from a soybean plant of A. esota). The harvesting method was the same, and the logarithmic growth phase curve obtained gave the same pattern as that obtained in Example 5 above and shown below.

Table 7 Displacement strain 1 3.6xlO5&QxlO8//
2 3.9X105 1.5X1 (19) //
3 1.9XIO66,0X108 These results demonstrate that the method of the invention is not strain dependent.

Example 7 The method of the present invention was applied to a wide range of Rhizobium
In order to further confirm that it is suitable for bacteria other than m), the culture method of Example 5 was repeated for six types of soil bacteria specimens. These six specimen cultures are soybean root-associated cultures that do not nodulate soybean roots and differ from Rhizobium in growth rate and cell and colony morphology. These cultures have not been classified taxonomically. These cultures were treated in the same manner as Rhizobium. A commercially available nutritional broth was used instead of the yeast extract-mannitol broth. The results are summarized in Table ■ below.

Nα1 8.0xlO'3.0xlO'N
o, 2 8. OXl 0' 5. IXl
0'No, 3 2.7 X 1
(19) No, 4 2.5X1
(19) No, 5 L I
X 101ONo, 6 9.6
X108 These results demonstrate that the method of the invention is also suitable for a wide range of soil-dwelling or root-associated bacteria that may be of agricultural importance. Although the logarithmic growth curves varied from species to species, significantly higher growth rates were observed for these cultures. Because of the effectiveness afforded by the method of the present invention, it can be used against most known and unknown soil-associated microorganisms.

Claims (31)

[Claims] (1) (a) introducing a medium containing a finely ground granular carrier, a certain amount of nutrients and water into a culture container; (b) inoculating the medium in the container with a culture of a microbial inoculum; ) manufacturing a microbial farm crop inoculum, comprising the steps of: storing said container under conditions suitable for the growth of microorganisms, and growing and ripening said microbial culture on a medium in said container; Method. (2) The method according to claim (1), wherein the nutrient is a grain. (3) The method according to claim (2), wherein the grain flour is unbleached white wheat flour. (4) The dry weight ratio of the above grain flour to the granular carrier is 1
．． Claim No. 3 characterized in that it is 3 to 9% (
The method described in section 2). (5) Claim No. 4, characterized in that the dry weight ratio of the grain flour to the granular carrier is about 1.3%.
) Method described in section. (6) The method according to claim (4), characterized in that the weight ratio of the water to the granular carrier is about 60%. (7) The method according to claim (1), wherein the granular carrier is vermiculite. 8. The method of claim 7, wherein the vermiculite is ground to a particle size generally less than 40 mesh. (9) The method according to claim (1), further comprising the step of sterilizing the culture container containing the medium before the inoculation step. (10) The method according to claim (9), wherein the sterilization is performed by autoclaving. (11) After the storage step, open the container,
The method according to claim 1, further comprising the step of air-drying the culture at room temperature. (12) The method according to claim (11), wherein the preservation step is for at least 30 days. (13) An agricultural inoculum of dried microorganisms obtained by the method according to claim (11). (14) The method according to claim (1), wherein the microbial inoculum is a fungus. (15) The method according to claim (1), wherein the microbial inoculum is bacteria. (16) The above bacterium is a species of the genus Rhizobium (Rhi).
zobium) strain according to claim (15). (17) The method according to claim (1), wherein the culture container is a plastic bag. (18) An agricultural inoculum of microorganisms obtained by the method according to claim (1). (19) (a) Place a certain amount of crushed vermiculite in a plastic bag, (b) Add a medium containing water and microbial nutrients to the vermiculite in the bag, and (c) Add microorganisms to the contents of the bag. inoculating the culture, (d) hermetically sealing the bag, and (e) storing the bag at room temperature and growing the microbial culture in the medium within the bag. Method for producing microbial farm crop inoculum. (20) The method according to claim (19), wherein the vermiculite is crushed to a size smaller than 40 mesh. (21) The method according to claim (19), further comprising the step of sterilizing the bag containing the vermiculite before the step of adding the medium. (22) The step of adding the medium and inoculating the contents of the bag comprises diluting a certain amount of the microbial culture with a solution of nutrients, and adding the microbial culture and the nutrient solution together into the bag. The method according to claim 19, characterized in that the method is introduced into a method. 23. The method of claim 19, further comprising the step of dispensing said microbial culture into said bag for use as an inoculum. (24) The method according to claim (19), wherein the microbial inoculum is bacteria. (25) The above bacterium is a species of the genus Rhizobium (Rhi).
zobium) strain according to claim (24). (26) An agricultural inoculum of microorganisms obtained by the method according to claim (19). (27) A harvesting medium for microbial cultures comprising micronized vermiculite, water, and an amount of grain flour equal to about 1.0-9% of vermiculite by dry weight. (28) The culture medium according to claim (27), wherein the vermiculite is crushed to a finer particle than 40 mesh. (29) The culture medium according to claim (27), wherein the amount of water is about 60% by weight of the vermiculite. (30) The culture medium according to claim (27), wherein the grain flour is white unbleached wheat flour. (31) The amount of the above grain flour is approximately 1.3 of vermiculite by dry weight.
% of claim (27).