JPH0655068B2 - Crop cultivation methods - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for cultivating agricultural crops.
More specifically, it relates to a method for cultivating agricultural crops using novel microorganisms having plant growth-promoting activity.

[0002]

BACKGROUND ART A wide variety of microorganisms inhabit the rhizosphere or root surfaces of plants, and have a major impact on the growth of the organisms, the occurrence of diseases, etc. Therefore, attempts have been made to isolate industrially useful microorganisms from these rhizosphere microorganisms and to utilize them to improve agricultural productivity, and many research reports have been published.

For example, nitrogen-fixing bacteria fix nitrogen from the air and supply plants with nitrogen, one of the three major nutrients. It is also known that certain mycorrhizal fungi increase the efficiency of phosphorus utilization in the soil and promote plant growth by supplying plants with phosphorus, an essential element for plants.
In addition, a wide variety of plant pathogens live in the soil, but it is also known that there are microorganisms that have antagonistic effects against these pathogens. For example, bacteria belonging to the genus Pseudomonas have been isolated as antagonistic microorganisms, and research is being conducted on their use.

However, since mycorrhizal fungi are parasitic fungi, the plant itself is required for their cultivation, making it difficult to cultivate them in large quantities industrially.
It has not yet been put to practical use. Furthermore, even if nitrogen-fixing bacteria can be mass-cultured industrially, when they are sprayed on soil, the number of bacteria in the soil decreases over time. As a result, the amount of nitrogen fixed decreases, which is considered to be problematic in terms of economic efficiency compared to using commercially available nitrogen fertilizer. Furthermore, since many antagonistic bacteria produce substances (antibiotics) that are antagonistic to the growth of plant pathogens, they may also have a more or less inhibitory effect on plant growth.

[0005]

[Means for solving the problem] The object of the present invention is to isolate useful microorganisms from plant root fungi that are easy to mass-cultivate, have good adhesion to the plant rhizosphere and root surface, and have the effect of promoting plant growth, and to establish a method for mass-cultivating these microorganisms and then applying them to improve the cultivation efficiency of agricultural crops, etc.

In order to achieve the above-mentioned object, the present inventors have widely searched for microorganisms that have plant growth-promoting properties among plant root fungi, and have found that a novel microorganism belonging to the genus Enterobacter, Enterobacter cloacae, isolated from cucumber root fungal soil, meets the object of the present invention by promoting the growth of a variety of useful agricultural crops, including cucumber, and have thus completed the present invention.

[0007] That is, the first aspect of the present invention relates to a method for cultivating agricultural crops, which comprises directly applying a microorganism belonging to the genus Enterobacter and having a plant growth-promoting effect to seeds, etc., and then sowing the seeds in soil, or alternatively mixing the microorganism into soil and then sowing the seeds, etc. The second aspect of the present invention relates to a method for cultivating agricultural crops, which comprises cultivating seedlings or crops in a hydroponics fertilizer solution to which a microorganism belonging to the genus Enterobacter and having a plant growth-promoting effect has been added.

The novel microorganism used in the present invention, Enterobacter cloacae, was isolated by the present inventors from cucumber rhizosphere soil,
It has the following mycological properties.

1. Morphology (1) Cell shape and size: Bacillus, 0.8-1.0 x 1.5-
3.0 microns (2) Cell pleomorphism: Absent (3) Motility: Present (movement by peritrichous flagella) (4) Spores: Absent (5) Gram stain: Negative (6) Acid-fastness: Negative

2. Growth state in each medium (1) Nutrition agar plate culture: The cells do not produce significant pigments.
It grows with a pale yellow, creamy appearance. It grows vigorously. (2) Meat broth agar slant culture: Same as (1) above. (3) Meat broth liquid culture: It grows while becoming cloudy overall and does not form a mycelium. (4) Meat broth gelatin stab culture: It liquefies very slowly. (5) Milk culture: No significant changes such as liquefaction, coagulation, or pH fluctuations are observed.

3. Physiological properties (1) Nitrate reduction: positive (2) Denitrification: negative (3) MR test: negative (4) VP test: positive (5) Indole production: negative (6) Hydrogen sulfide production: negative (7) Starch hydrolysis: negative (8) Citric acid utilization: positive (9) Inorganic nitrogen source: Utilizes nitrate and ammonium salts as nitrogen sources. (10) Pigment production: No significant production of soluble or insoluble pigments was observed. (11) Urease: positive (12) Oxidase: negative (13) Catalase: positive (14) Growth range: Grows in the temperature range of 15 to 45°C.
The optimum temperature is 28-37°C. The pH for growth is near neutral. (15) Attitude towards oxygen: Facultative anaerobic (16) O-F test: F type (17) Production of acid and gas from sugars

[0012]

[Table 1]

4. Other Properties (1) DNase Production: Negative (2) Tryptophan Deaminase Production: Negative (3) β-Galactosidase Production: Positive (4) Arginine Decomposition Test: Positive (5) Lysine Decarboxylation: Negative (6) Ornithine Decarboxylation: Positive (7) Esculin Decomposition: Negative

As described above, this strain is a Gram-negative, facultatively anaerobic bacillus, does not produce spores, and has the morphological characteristics of moving by peritrichous flagella. It also exhibits physiological properties of being oxidase negative and nitrate reducing activity positive, and therefore it is judged to belong to the family Enterobacteriaceae. Furthermore, based on various physiological properties, it is also judged to belong to the family Enterobacteriaceae.
It would be most appropriate to place them in the ter cloacae.
This strain has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology under the accession number FERM BP-1529.

Next, the first and second aspects of the present invention will be explained. Plant growth can be promoted by applying a plant-growth-promoting microorganism belonging to the genus Enterobacter, such as Enterobacter cloacae, at a rate of ¹⁰ to ^{10 cells} per plant seed and then directly applying the microorganism to the soil, or by mixing the microorganism into soil at a rate of 10 to ¹⁰ cells per gram of soil and then sowing seeds in this soil. In the case of hydroponics, plant growth can be promoted by mixing the microorganism into the nutrient solution at a rate of ¹⁰ to ¹⁰ cells/ml and adding the microorganism as needed during the cultivation period.

In the cultivation of agricultural crops, the effect of inoculating the above microorganisms is firstly manifested as a growth promotion effect on roots, and then the growth of stems and leaves is promoted.

The present invention is applicable to any type of plant, but is particularly suitable for agricultural crops. Here, agricultural crops refer to all agricultural crops, including husks, vegetables, flowers, and fruit trees, as well as the harvested products thereof. Plants also include seedlings, such as seedlings of vegetables such as cucumbers, pumpkins, eggplants, tomatoes, melons, and watermelons, seedlings of flowers, and seedlings of grains such as rice, as well as seedlings of all useful plants.
In the present invention, the term "seeds, etc." includes not only seeds but also seed potatoes, etc., in the case of tuberous roots such as potatoes. Furthermore, the term "hydroponics" refers to all hydroponics, including hydroponics, sand culture, gravel culture, and rock wool culture.

[0018]

EXAMPLES The present invention will now be described in more detail with reference to the following examples. Reference Example 1 (1) Enterobacter cloaca
e) Large-scale culture: glucose 1.0%, peptone 0.5%, KH ₂ PO ₄ 0.1%,
400 ml of a liquid medium containing 0.05% MgSO ₄ ·7H ₂ O (hereinafter referred to as M medium) was added to a 1-liter Erlenmeyer flask, sterilized at 120°C for 30 minutes, cooled, and inoculated with one platinum loop of Enterobacter cloacae (FERM BP-1529).
The seed culture solution is prepared by cultivating the medium at 30°C and 240 rpm for 24 hours. 20 liters of M medium is placed in a 30-liter jar fermenter, sterilized at 120°C for 30 minutes, and then
After cooling to 0°C, the seed culture solution was inoculated and cultured at a temperature of 30°C.
The culture was carried out at 0°C, stirring at 200 rpm, and aeration at 100 vvm for 24 hours. The number of Enterobacter cloacae bacteria in the culture medium was 1 x 10 ¹⁰ cells/ml.

Example 1 (1) Cucumber seedling raising: Soil for raising seedlings was added to a seedling raising tray (30 cm x 50 cm x 3 cm), and 10 cucumber seeds (variety: Kifujin) were planted therein.
After cultivating at 20-30°C for one week, the seeds were transplanted into 3-inch pots and raised for another two weeks. The experimental plots were as follows: Control plot: The soil for raising seedlings was used as is. Soil-added plot: Enterobacter cloacae (FERM BP-15
29) (obtained in the reference example, the same applies below) at 1 x ¹⁰⁷ /
Seed application area: Seeds (variety: Kifujin) were added to a culture solution of Enterobacter cloacae (number of bacteria: 1.0 x 10 ¹⁰ cells/ml) and grown.
The seeds were immersed in the solution, and then sown in soil to which the bacteria had not been added and grown as seedlings.

The results are shown in Table 1. In the control plot, the height above ground was 1
In the soil-added area, the proportion of S-size seedlings was 6%, and in the seed-applied area, the proportion of S-size seedlings was 18%, which was lower than in the control area.

As described above, by treating the seeds with Enterobacter cloacae, large, healthy seedlings could be grown.

[0022]

[Table 2]

(2) Cultivation of Cucumbers The seedlings obtained as described above were planted in greenhouse soil at 80 cm intervals, and the cucumbers were cultivated for three months in the greenhouse under natural temperature and natural light conditions. Inorganic fertilizer (Kasei No. 14) was added to the roots as needed. From the second week of cultivation, Dysyston (trade name) was applied as needed to control aphids. The test results are shown in Table 2.

As is clear from the table, the above-ground parts of both the soil-added plot and the seed-applied plot were larger than those of the control plot, and the cucumber yield also increased by 8 to 12% compared to the control plot.

[0025]

[Table 3]

(3) Colonization of Enterobacter cloacae on the root surface During the cultivation period, roots were sampled at regular intervals and the number of Enterobacter cloacae growing on the root surface was measured. The measurement of root surface microorganisms was carried out in accordance with the method described on page 380 of Soil Microorganism Experimental Methods (edited by the Soil Microorganism Research Society, Yokendo Publishing). Enterobacter cloacae was grown in Martin medium (1% glucose, 0.5% peptone, _0.1 % _KH2PO4 ,
MgSO ₄ · 7H ₂ O 0.05%, Rose Bengal 0.0033
When cultured at 30°C for 24 to 48 hours on a 2.0% agar solution (pH 6.8), the colonies exhibited characteristic polysaccharides, color, and shape, and these properties were used as indicators to count the number of colonies.
The results are shown in Table 3.

[0027]

[Table 4]

Example 2 Two sunny lettuce seeds were sown on a 4 cm square urethane mat, and immersed in a liquid fertilizer containing 0.15% of Otsuka House Fertilizer No. 1 and 0.1% of Otsuka House Fertilizer No. 2 (both trade names).
After cultivating for 10 days under conditions of 8000 lux, the plants were transplanted into a hydroponic cultivation device and cultivated for 3 days under conditions of 8000 lux and 24°C.
The plants were cultivated for 5 days. The experimental plots are as follows:

Control group: Otsuka House fertilizer group without microorganisms. Added group: Otsuka House fertilizer was added with Enterobacter cloacae at a rate of 1 x ¹⁰⁵ cells/ml to 1 x 109 cells/ml. Enterobacter cloacae was added at a rate of 1 x 105 cells/ml to 1 x ¹⁰⁹ cells/ml once a week.
Enterobacter cloacae was cultured in the same manner as in Reference Example 1 and subjected to the experiment.
The test results are shown in Table 4.

[0030]

[Table 5]

As is clear from the table, the addition of Enterobacter cloacae increased the yield and root weight, with the effect being particularly notable at 1 x 10 ⁶ to 1 x 10 ⁸ cells/ml.

Example 3 40 g of rice seeds (variety: Akinishiki) were sown in a nursery box (50 cm x 15 cm x 20 cm) filled with black soil, covered with soil 3 to 4 mm thick, and then allowed to germinate at 30 to 32°C for 2 days, followed by cultivation at 25°C for 17 days. The experimental plots are as follows:

Control group: Soil (black soil) to which no bacteria were added was used. Added group: Black soil to which 1.0 x ¹⁰⁷ Enterobacter cloacae/g of soil was added and the covering soil was used.

Enterobacter cloacae was prepared in the same manner as in Reference Example 1. The average stem and leaf length of rice seedlings was 16.3 cm in the control group and 18.4 cm in the added group, showing a growth of 113% compared to the control group.

Example 4 200 seeds of spinach (variety: Jiromaru) were sown in a pot (50 cm x 15 cm x 20 cm) and incubated at 20°C, 300°C.
The plants were cultivated for 40 days under conditions of 100 lux.

Control group: Soil (black soil) to which no bacteria were added was used. Added group: Enterobacter cloacae was added at 1.0 x 10 ⁸ cells/g of soil.

Enterobacter cloacae was prepared in the same manner as in Reference Example 1. The results are shown in Table 5.

[0038]

[Table 6]

As is clear from the table, the average weight of the added group was 135% compared to the control group.

Example 5 One corn seed was sown in a 3-inch pot containing black soil and cultivated for 20 days under conditions of 25° C. and 5,000 lux. The experimental plots are as follows:

Control group: Cultivated in black soil without adding Enterobacter cloacae. Addition group: Cultivated in black soil with adding Enterobacter cloacae at 1 g per 1 g of soil.
The plants were cultivated by adding ^1x104 cells/ml to ^1x109 cells/ml.

Enterobacter cloacae was cultured and subjected to the experiment in the same manner as in Reference Example 1. The test results are shown in Table 6.

[0043]

[Table 7]

As is clear from the table, Enterobacter
Addition of Cloaca increased the length of stems and leaves.
In the plots to which 1×10 ⁵ cells/g soil to 1×10 ⁸ cells/g soil were added, growth promotion of 114 to 137% was observed compared to the control.

[0045]

According to the present invention, by using novel microorganisms having plant growth promoting activity in the cultivation of agricultural crops and the like, it is possible to efficiently cultivate useful agricultural crops.

──────────────────────────────────────────────────── Continued from the front page (72) Inventor: Shinji Miyamichi 760 Shioka-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture, Meiji Seika Kaisha, Ltd. Pharmaceutical Research Institute (72) Inventor: Hidemasa Hidaka 580 Horikawa-cho, Saiwai-ku, Kawasaki, Kanagawa Prefecture, Meiji Seika Kaisha, Ltd. Biological Science Research Institute (56) References: CANADIAN JOURNAL OF MICROBIOLOGY 28 (5), 1982, pp. 462-467 CANADIAN JOURNAL OF MICROBIOLOGY 29 (10), 1983, pp. 1301-1308

Claims (4)

[Claims] [Claim 1] A method for cultivating agricultural crops, comprising applying a microorganism belonging to the genus Enterobacter and having plant growth-promoting properties directly to seeds, etc., and then sowing the seeds in soil, or mixing the microorganism into soil and then sowing the seeds in the soil. 2. The method according to claim 1, wherein the microorganism belonging to the genus Enterobacter and having plant growth-promoting activity is Enterobacter cloacae. 3. A method for cultivating agricultural crops, comprising hydroponically cultivating seedlings or crops using a fertilizer solution containing a microorganism belonging to the genus Enterobacter and having a plant growth-promoting effect. 4. The method according to claim 3, wherein the microorganism belonging to the genus Enterobacter and having plant growth-promoting activity is Enterobacter cloacae.