JP2000191421A - Plant growth promoter - Google Patents

Abstract

(57) [Abstract] (Solution) Pseudomonas marginalis (Pseudom
onas marginalis) or a mutant thereof. [Effect] Since it can be produced at low cost and exhibits an excellent plant growth promoting effect, the number of times of treating the plant may be small.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a plant growth promoter, and more particularly, to a novel plant growth promoter having the effects of improving plant yield, promoting flowering and extending the growth period by promoting plant growth. For accelerators.

[0002]

2. Description of the Related Art It is important to promote plant growth and increase its yield, and many plant growth promoters have been studied in the past. Of these, plant hormones mostly act on a part of plant organs, but do not act on whole plants and increase yield.

[0003] On the other hand, N-
Photosynthetic ability improving drug such as allyl-N-methylglycine,
Although plant hormones and the like have been developed, their effects are small and not sufficiently satisfactory.

[0004]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a plant growth promoter which acts on the whole plant and improves the yield of the plant.

[0005]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies and as a result, have found that microorganisms belonging to Pseudomonas marginalis or mutants thereof have an extremely small but excellent plant growth effect, The present invention has been completed.

That is, the present invention provides a plant growth promoter comprising as an active ingredient a microorganism belonging to Pseudomonas marginalis or a mutant thereof.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Pseudomonas marginalis or a mutant thereof, which is an active ingredient of the plant growth promoter of the present invention, is obtained, for example, in soil obtained by adding lime to peat moss, peat, etc., heating and pressurizing, and mixing humus with soil. The aqueous solution of 5,6-dimethyluracil is allowed to act for about one year for a long period, and can be collected from the soil.

[0008] Pseudomonas marginalis or a mutant thereof can be grown in a conventional nutrient medium. For example, after preculture in a normal nutrient medium containing organic substances or salts, simple liquid fertilizers other than organic substances, for example, If the main culture is performed using an aqueous solution containing inorganic salts of nitrogen, phosphoric acid, and potassium as a medium, about 10 ⁶ to 10 ⁷ / mL of the number of cells can be obtained. It can be used as a growth promoter. In addition, Pseudomonas fluorescens is known as an antagonistic microorganism and is also known to have a growth promoting effect [Disease control by antagonistic microorganisms (Agricultural and mountain fishing village culture association), Phytochemical regulation society (30th Annual Meeting B- 30, 58P)]. However, it is not known that Pseudomonas marginalis can be an active ingredient of a plant growth promoter.

The Pseudomonas marginalis or a mutant thereof used in the present invention is not particularly limited, and examples thereof include strain M-1 having the following taxonomic properties.

(1) Method 1) GC% measurement of DNA [FEMS Microbiol. Lett., 67:12]
7-130 (1990)] After decomposing the DNA extracted from the cells into bases, the phosphate groups were further removed, and the molar ratio of each base was calculated as a nucleoside by liquid chromatography to determine GC%. 2) Biochemical properties test As shown in Table 1, the morphology, culture properties, and various physiological properties were tested. The physiological property test was performed by a test tube method according to the method described in New Bacterial Culture Studies Course <Second Edition>, Kindai Shuppan, 1998. 3) Testing using a simple identification kit API 20NE (BioMérieux Vitec Co., Ltd.)
Identified by

(2) Results 1) Measurement of GC% of DNA The GC% of the M strain was 56.7% (Table 1). The GC% of Pseudomonas aeruginosa measured at the same time was 64.4%. 2) Biochemical property test Table 1 shows the results of each property test. 3) The test results using the simple identification kit are shown in Table 2.

[0012]

[Table 1]

[0013]

[Table 2]

The M-1 strain tested as described above was a Gram-negative aerobic bacillus, which was oxidase-positive and catalase-positive by oxidizing glucose, and was considered to be a strain belonging to Pseudomonas. However, since the species cannot be identified by these tests alone, the 5S of 16S rRNA is
The M-1 strain was identified from the base sequence of the terminal 500 bp.

<Method> (1) Extraction of Nucleic Acid After extracting nucleic acid using "Sepa Gene" kit (manufactured by Sanko Junyaku Co., Ltd.), nucleic acid was recovered by ethanol precipitation. (2) PCR About 1.5 kbp using two universal primers specific to 16S rRNA (5'-end and 3'-end)
Was amplified. (3) Purification of PCR amplification product After completion of amplification, purification was performed using a spin column (Pharmacia). (4) Analysis of base sequence of 16S rRNA The purified PCR amplification product was subjected to base sequence analysis of 500 bp at the 5 ′ end side using an Applied Biosystems 377 DNA sequencer (manufactured by Perkin Elmer). (5) Homology analysis by 16S rRNA database search A search was performed online from Genebank database.

<Results> According to the results, unlike the Pseudomonas fluorescens, the M-1 strain had a Pseudomonas marginalis (Pseudomonas marginalis) with 99.6% homology.
as marginalis).

However, since the taxonomic properties of the M-1 strain do not match any of the known strains of the genus Pseudomonas, they were determined to be new strains and the FERM P-
16437 was deposited with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry. The nucleotide sequence of the M-1 strain is shown in the sequence listing below (SEQ ID NO: 1).

The culture of the M-1 strain of the present invention may be carried out using an organic substance or a simple liquid fertilizer as a medium in the same manner as described above.

As the Pseudomonas marginalis or a mutant thereof used in the present invention, the culture obtained by the above method may be used as it is, or may be used after being purified by a conventional method.

[0020] The plant growth promoter of the present invention is sufficiently effective only with Pseudomonas marginalis or a mutant thereof, but in addition thereto, other plant growth regulators;
Sugars; inorganic salts including nitrogen, phosphoric acid and potassium; urea;
Amino acids, organic acids, alcohols, vitamins, other minerals and the like can be added. Further, other pesticides, fertilizers and the like may be used as a mixture as long as the effects of the present invention are not impaired.

The dosage form of the plant growth promoter of the present invention includes:
In addition to liquids, powders, granules and the like can be mentioned, and these can be produced according to a conventional method.

The following methods are exemplified for applying the plant growth promoter of the present invention to plants. (A.) A method in which seeds are immersed in the plant growth promoter solution of the present invention and then seeded. The number of cells in this solution is preferably 10 ⁴ to 10 ⁸ / mL, and particularly preferably 10 ⁶ to 10 ⁷ / mL. Is preferred. The immersion is preferably for about 1 to 3 days. (B.) Method of irrigation into soil A solution having the same number of cells as in (a.) Is sterilized at 60 ° C. for 24 hours before use. The time of irrigation may be appropriately determined depending on the plant, but the time when the true leaves are about 3 to 6 cm long (12 hours after sowing)
To 16 days) is preferred. The amount of irrigation was determined by inoculating M-1 bacteria into an inorganic salt medium [medium obtained by dissolving 5 g of ammonium nitrate and 5 g of dipotassium hydrogen phosphate in 1 L of purified water, adjusting the pH to 7.0, and then autoclaving] per 10 ares. It is preferably about 2 kg to 4 kg after culturing at 10 ° C. for 10 days and evaporating water. (C.) Spraying method once using the liquid of (b.) Once (1-7 days after sowing) when the length of the true leaves is 1-2 cm, and then spraying once or twice every 10 days thereafter I do.

In either of the above methods (a.) And (b.), The growth of (c.) Is remarkably improved by one or two treatments, and the growth in the middle and late stages is remarkable. And at the time of collection,
The yield is increased by 1.5 to 2.5 times as compared with the control not using the product of the present invention. Further, the plant growth promoter of the present invention also has a flowering promoting effect and a flowering period extending effect.

However, if the treatment period is too long or the cell concentration is too high, the growth may be suppressed. Therefore, it is desirable to determine the treatment period by testing each crop. In addition, when the concentration is slightly higher, the growth is temporarily suppressed, but then recovers, and finally, the sales start to increase.

Plants to which the plant growth promoter of the present invention can be used include, for example, komatsuna, spinach, large eastern vegetables, nozawana, Hiroshimana, Chinese cabbage, radish, cabbage,
Vegetables such as turnips, pumpkins, peppers, tomatoes; rice,
Cereals such as barley, wheat, barley, corn, millet; fruit trees such as tangerines, apples, oysters, plums, pears, grapes, and peaches; Trees such as Satsuki, Kunugi, Sugi, Hinoki, Japanese oak, Beech; Beans such as adzuki bean, soybean, peanut, faba bean, peas; Lawns such as black mulberry, bentgrass, wild grass, potato, sweet potato, sweet potato, yam ,
Potatoes such as taro; leek such as leek, onion, raccoon; and grasses such as alfalfa, clover, vetch and the like.

Among them, flowers can also have a flowering promoting effect.

[0027]

Industrial Applicability Pseudomonas marginalis or a mutant thereof, which is an active ingredient of the plant growth promoter of the present invention, can be cultured in a large amount in a simple medium and is economical. In addition, the plant growth promoter of the present invention is excellent in growth promotion effect, so that it can be applied to plants as a fertilizer, the amount of use is extremely small, and a large amount of plants can be treated. In addition, the treatment has the advantage that one or two treatments are sufficient for crops with a harvest period of 30 to 40 days, and no labor is required. In addition, since cultivation can be easily performed even in nitrogen-containing substances such as urea as well as inorganic salts containing three elements of fertilizer, the efficacy of chemical fertilizers, liquid fertilizers, slow-release fertilizers, and the like can be increased. Of course, the fertilizer effect of organic fertilizer can also be increased.

[0028]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

Example 1 1 L of 5,6-dimethyluracil 1000 PPM aqueous solution was allowed to act for 1 year on soil mixed with 5 L of humus and 5 L of improved material obtained by adding lime to peat moss and peat and heating and pressurizing. Pseudomonas sp. M-1 was obtained from among bacteria collected from this soil.

Example 2 The M-1 strain obtained in Example 1 was cultured at 28 ° C. for 2 days in a broth liquid medium (tryptosome bouillon medium: manufactured by Nippon Suisan) or a 100% molasses aqueous medium. Next, this culture 1
Each mL was inoculated into 1 L of the following inorganic salt medium A or 1 L of 100% aqueous solution of Hyponex liquid fertilizer (manufactured by Hyponex Japan), and cultured at 20 ° C. for 10 days. The inorganic salt medium A is made of an inorganic salt (ammonium nitrate 5 g, hydrogen phosphate 2).
(5 g of potassium) dissolved in 1 L of purified water and adjusted to pH 7.0. The number of bacteria after culture was about 7
× 10 ⁶ / mL. This viable or sterilized solution was used below.

The plant growth promoter of the present invention thus obtained was applied to plants under the following conditions.

1. Soil 1) Takii planter soil (Takii seedlings KK), granular power soil (Kureha Chemical KK) and commercial culture soil. 2) A mixture of equal amounts of black clay and Akadama clay (small grains) (IB
No. 48 or ordinary chemical fertilizer, 1 per 500g of pot soil
g). Both 1) and 2) were given 0.5 g to 1 g per pot 20 days after sowing of the fertilizer as an additional fertilizer. Put 500 g of the soil of 1) and 2) above into one pot (12
20 pots in the control section and 20 pots in the treatment section, respectively.

2. Test crops Komatsuna (Goku Rakuten), spinach (Okame), large eastern vegetables, Nozawana, Hiroshimana, cosmos.

3. Treatment method (a) Seed treatment (same size) Each of the above seeds was put into a petri dish, and the viable cells cultured in the Hyponex liquid fertilizer 100 times aqueous medium were put into each dish.
The immersion treatment is carried out at 0 ° C. for 1 to 2 days (1 day, large mountain eastern vegetables, nozawana, Hiroshima vegetables; 1.5 to 2 days, spinach and komatsuna). As a control, a 100-fold aqueous solution of Hyponex liquid fertilizer was used. 10 seeds are seeded in one pot, and four leaves are left when the true leaves appear for about 7 to 10 days after sowing, and the others are removed.

Growing state and results From about 14 to 20 days after sowing, the difference between the control and the growth gradually appears. At the time of harvest (40 to 50 days after sowing), about 1.5 to
A difference of about 2.0 times (live weight) was observed (especially in late stage, growth is remarkable).

When cosmos was treated for 3 days and then sown, growth of foliage and promotion of flowering were observed.

The same experiment was carried out using live bacteria cultured in the above-mentioned inorganic salt medium A, and almost the same results were obtained (one day of immersion, three days of komatsuna / spinach, three days cosmos;
2x diluent 1 day, large Yamato greens, Hiroshima greens).

(B) Soil irrigation treatment solution 1) A solution obtained by sterilizing bacteria cultured in the inorganic salt medium A at 60 ° C. for 24 hours (the sterilizing solution of Example 2). 2) A solution obtained by sterilizing the bacteria cultured in the Hyponex liquid fertilizer 100 times solution at 60 ° C. for 24 hours (the sterilizing solution of Example 2). 3) 1 L of a 1% aqueous solution of urea was sterilized, and 1 mL of a liquid obtained by culturing the M-1 strain at 28 ° C. for 2 days in the broth medium (tryptosoy bouillon medium) or a 100-fold aqueous solution of molasses was added thereto.
After culturing at 0 ° C. for 10 days, the solution was sterilized at 60 ° C. for 24 hours (the number of cells was about 7 × 10 ⁶ ).

Test crops: Large-sized Shandong, Komatsuna, Hiroshima greens.

All three liquids 1), 2) and 3) were used as treatment liquids. Make 30 pots of each test crop, plant 10 seeds of the same size of each test crop, 3 to
When it reached 6 cm, four of the same growth were left and the others were removed. This is assumed to be 15 pots each for the treatment section and the non-treatment section. Apply 1), 2), 3) solutions 10 to
A 20-fold solution is used, and 20 to 30 mL per pot is irrigated, and a control solution to which a sterile solution of 1), 2) and 3), in which no bacteria are cultured, is diluted in the same manner is injected. In each case, only one injection was performed. As a result, the growth is generally gradually promoted from about 7 to 14 days after the treatment, and the medium to late period (25 to 3 days)
0)) and gradually became different from the control, and from 40 days to 5 days after irrigation.
On day 0, sales increased 1.5 to 2 times. In the case of Torenia, 4 pots each having a height of about 10 cm are used in the same pot, and the treatment solution of 3) is used. When 20 mL of a 10-fold solution is added to each pot, the growth is promoted after 20 to 30 days, and the flowering period is promoted and the flowering period is prolonged. It was observed.

(C) Foliar spraying treatment solution: 5-fold diluted sterile culture solution of 1), 2), 3) same as in the case of soil irrigation.

Test crops Nozawana, Komatsuna.

10 days after germination, when the true leaves reach 1 to 2 cm, once for four individuals of the same growth type, and once for 20 days after growth,
Each time, ³ to 6 mL was sprayed per 100 cm ² of leaf area. Scatter 4
Harvesting 0 days later, the fresh weight was measured, and as a result, a 1.5 to 2-fold increase in yield was observed as compared to the control (a sterile solution in which each microorganism was not cultured was sprayed at the same concentration).

[0044]

[Sequence List] SEQUENCE LISTING <110> MATSUDAIRA, OSAMU <120> Growth promoter for plants <130> P04241110 <160> 1 <210> 1 <211> 500 <212> DNA <213> Pseudomonas sp M-1 <400> 1 cggcaggcct aacacatgca agtcgagcgg tagagagaag cttgcttctc ttgagagcgg 60 cggacgggtg agtaatgcct aggaatctgc ctggtagtgg gggataacgt tcggaaacga 120 acgctaatac cgcatacgtc ctacgggaga aagcagggga ccttcgggcc ttgcgctatc 180 agatgagcct aggtcggatt agctagttgg tgaggtaatg gctcaccaag gcgacgatcc 240 gtaactggtc tgagaggatg atcagtcaca ctggaactga gacacggtcc agactcctac 300 gggaggcagc agtggggaat attggacaat gggcgaaagc ctgatccagc catgccgcgt 360 gtgtgaagaa ggtcttcgga ttgtaaagca ctttaagttg ggaggaaggg ccattaccta 420 atacgtgatg gttttgacgt taccgacaga ataagcaccg gctaactctg tgccagcagc 480 cgcggtaata cagagggtgc 500

Claims (3)

[Claims] [Claim 1] Pseudomonas marginalis (Pseudom
onas marginalis) or a mutant thereof. 2. The plant growth promoter according to claim 1, wherein the microorganism belonging to Pseudomonas marginalis is a microorganism named Pseudomonas sp M-1 and deposited as FERM P-16437. 3. Pseudomonas sp.
sp) A microorganism named M-1 and deposited as FERM P-16437.