JPH0265775A - Novel microorganism and production of gibberellins - Google Patents

Abstract

NEW MATERIAL:Pheospheria sp. L-487(FERM P-10137) having producibility of gibberellins. USE:Production of gibberellins useful as a growth hormone of plants. PREPARATION:Ascocarp of the above-mentioned microorganism adherent to gramineous plant body is collected.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a novel microorganism that produces gibberellins, which are known as one of the plant hormones that directly control plant growth, and a method for culturing the microorganism. The present invention relates to a method for producing gibberellins.

(Prior Art) To date, 74 types of gibberellins of microbial and plant origin are known [Journal of the Japanese Society of Agricultural Chemistry, Earthworks.

1526 (1938), Arch, Bioche
m, Biophys,.

54, 240 (1955)).

The most remarkable physiological effect of gibberellin on plants is its growth-promoting effect on intact plants, which is particularly noticeable in young plants and dwarf plants. It is also known to have the effect of breaking dormancy in certain plants, and to induce parthenocarpy in tomatoes, cucumbers, grapes, etc. In particular, seedless production of grape varieties such as Praware has been put into practical use.

[Problem that the invention seeks to solve]

Traditionally, gibberellins have been used exclusively for the rice bacterium Gibber.
ella fujikuroi (Fusarium
moniliforme), but in recent years, the pathogenic bacterium Sphaceloma m that parasitizes challah mackerel
GA was isolated from B. anihoticola (Bio
chem, Biophys, Res, Comm,,
91+35 (1979) ), Neuros
G A x was isolated from pora crassa (
Agric, Biol, Chem, + 47 (71+
1693 (1983)), which is G, fuj 1
This is the first example showing that a mold other than Kuroi produces gibberellin.

However, the number of bacteria that produce gibberellins is still small. Therefore, the inventor of the present invention
As a result of intensive studies to provide a gibberellin-producing bacterium other than erella fujikuroi, we found that bacteria of the genus Phaeosphaeria produce gibberellin A4 (OA4'') and gibberellin A9 C.
GAq) was discovered, and the present invention was completed.

(Structure of the Invention) That is, the gist of the present invention is to obtain Phaesphaeria sp.
eosphaeria sp, L-487).

Hereinafter, to explain the present invention, Phaesphaeria sp. L-487 according to the present invention belongs to the genus Phaeosphere, which is a locula ascomycete, and its mycological properties are as shown below.

(1) Morphological characteristics (see Figure 2) Ascocarps are scattered on the host plant and are initially buried under the plant epidermis. Later, the epidermis is ruptured and the papillary neck protrudes. The shape of the ascocarp is spherical to subglobular or conical, and the size is 15 mm in diameter.
It is 0 to 280 μm. The shell wall consists of two to three thin layers, the cells of which are polygonal and relatively flexible. The neck is papillary, and the inner surface of the cervical foramen lacks a velophyllus.

Asci occur in large numbers, their shape is cylindrical to seed rod-shaped, tapering towards the base, rounded apex, thick membrane, double-walled structure, octospores, abundant pseudolateral filaments. , filamentous, with septa. The size is 95-130 x 10-12 μm. Ascospores are arranged in two rows within the ascus, with a long oval to long cylindrical shape and a size of 20-50 x 4-4.7μ.
m, is yellowish brown in color and has 3 to 8 septa (usually has 7 septa). Each cell of the ascospore lacks prominent ampullary cells, also lacks constricted cells, and is occasionally surrounded by a gelatinous sheath.

(2) Culture characteristics on various media (a) Potato glucose agar (PDA), 27°C23
Colonies cultured for a week are velvety, yellow-brown in the center and periphery, and sand-colored on the underside. Aerial hyphae width 2.5-3.7
It has septa, is branched, and has a pale yellow appearance. The basal hyphae extend radially and branch;
Width 14. It reaches 0μm. It also has many partition walls and has a yellowish brown appearance. After 3 weeks of culture on PDA, no formation of complete or incomplete reproductive organs was observed.

(rl) Colonies cultured on malt agar medium (MA) at 27°C for 3 weeks are fluffy, pale yellowish brown in the center, yellowish brown in the periphery, and brownish gray on the underside. Aerial mycelium has a width of 2.0~
It is made of 3.8μ steel, has partition walls, and has a pale yellow appearance. The basal hyphae are radially elongated and branched, with a width of 5.7
-6.0μ■. It also has many partition walls and has a yellowish brown appearance. After 3 weeks of culture on MA, no formation of complete or incomplete reproductive organs was observed.

(c) Colonies cultured on oatmilk agar (OA) at 27° C. for 3 weeks spread like fluff, exhibiting a dark yellow-brown color from the center to the periphery, and a dark yellow-brown color on the underside.

The aerial hyphae are branched and reach a width of 3.7 μm. It also has partition walls and has a pale yellow appearance. The basal hyphae are radially elongated and branched, reaching a radius of 4.7-5.0 μm.

It also has many partition walls and has a yellowish brown appearance. During 3 weeks of culture on OA, no formation of complete or incomplete reproductive organs was observed.

(d) Leonian's agar medium
Colonies cultured on gar) at 27°C for 3 weeks spread out in a velvety manner, with a pale yellow-brown center, a dark yellow-brown periphery, and a brown-gray underside.

Aerial hyphae are branched and up to 3.0 μm wide. It also has partition walls and has a pale yellow appearance. The basal hyphae are radially elongated and branched, reaching a width of 7.5-8.0 μm.

It also has many partition walls and has a yellowish brown appearance. Leo
During 3 weeks of culture on Njan's agar, no formation of complete or incomplete reproductive organs was observed.

(3) Physiological properties (a) Optimal growth conditions Optimal pH near ~ 8 (LCA liquid medium (Miura, Koo,
Journal of the Dutch Society of Japan, Volume 1).

1) 6-1) 8 pages, 1970) Medium, 14-day culture) Optimal temperature: 20-27°C (PDA agar, above ground 4
Day-long culture) (II) Growth range pH: 3 to 9 (LCA liquid medium [Miura, Engyo, Journal of the Japanese Dutch Society, Vol. 1).

1) 6-1) 8 pages, 1970] Temperature: 5-30°C (cultured on PDA agar for 4 days above ground) (4) Taxonomic considerations (a) Higher taxonomic considerations Location This fungal strain (L-487) grows epiphytically on grass plants and forms frass-shaped and cup-shaped ascocarps. has a double-walled structure.Ascospores have septa and are multilocular, so 1.Ho1m+Sy
mb, Botan, Upsal, 14 (3H188
(1957); E. S. Lutterell, Loc.
uloascomycetes, The Fungi.

Vol, 4 A (ed, G, C, Ainswor
th eL al, ) + 135-219 (19
73); J, 8, von Arx & E,
Miller.

5tud, Mycol, 9. 1 159 (197
5) Loculoascomycetes (Loculoascomycetes), etc.
es) - Pleosporales
- Belongs to the family Pleosporaceae.

(Tl) Identification at the genus level This strain (L-487) has 1) ascocarps that are spherical to subglobular or conical in shape; 2) the ascocarp wall is covered with abundant brown filamentous hyphae and has setae. 3) The septum consists of 2-3 thin layers, the cells are polygonal and flexible, 4) Ascospores have 3
It consists of ~8 septa (usually has 7 septa), is yellowish brown in color, and is characterized by the fact that the spores are covered with a gelatin sheath. Regarding these properties, L, Ho1n+Symb, B
otan, [Ipsal.

14 (3); 1 188; E, S, Lutt
erell.

Loculoascomycetes, The Fu
ngi, Vow, 4 A (ed, G, C.

AinsworLhetal, ), 135-219
(1973); J. A. von Arx & E.
, MLiller, 5tud, Mycol, 9
A search using the taxonomic literature on the Bleosvoraceae described in 1) 59 (1975) revealed that this strain (L-487) belongs to the genus Phaeosphaeria.

(c) Species level identification, Ho1m, Symb, Botan, Upsal, +
14 (3); 1-188 (1957) and G,
A, Hedjaroude, Sydowia22:5
7-107 (1968) of the genus Pheosphelia (Ph
According to the taxonomic literature on genus aeosphaeria, 21 species have been described in this species. Each of these species has different characteristics of the ascospores, namely, spore shape, size,
They are identified by the number of septa, the location of ampullary cells, the presence or absence of gelatin sheaths, and the color of the spores.

This strain (L-487) has: 1) Ascospores are long oval-shaped.
Cylindrical, size 20-50 x 4-4.7μm, 2)3
It has the following characteristics: it has ~8 septa, 3) it lacks ampulla cells, 4) it is yellowish brown in color, and 5) it is sometimes covered with a gelatin sheath.

When searching the above-mentioned literature regarding these properties, we found that
No species matching this strain (L-487) was found.

Therefore, this strain (L-487) is presumed to be a new species of the genus Phaeosphaeria, but the formal naming of the species will wait for future taxonomic studies and at this stage it is called Phaeosphaeria. Phae
osphaerta sp.) L-487.

Strain L-487 was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 10237 (FERM P-10237).
It has been deposited as.

Generally, Phaeosphaeria sp. L-48
7 is susceptible to changes in its properties, as seen in the case of other fungi.

For example, strain L-487, or a mutant strain (naturally occurring or induced) derived from this strain, a trait zygote, or a genetically recombinant strain, has the ability to produce gibberellin A4 and gibberellin A9. All can be used in the method of the invention.

In the present invention, the above bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As a source of nutrients,
Glucose, starch syrup, dextrin, sucrose, starch, molasses, animal/vegetable oils, etc. can be used. Further, as a nitrogen source, soybean flour, wheat germ, cornstarch liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used. others,
If necessary, it is effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions.

As a culture method, a culture method under aerobic conditions is suitable. The appropriate temperature for culturing is 20 to 30''G, but in most cases it is cultured at around 20 to 27''C.

Production of gibberellin varies depending on the medium and culture conditions, but its accumulation usually reaches its optimum within 3 to 10 days in both shaking culture and tank culture. When the amount of gibberellin accumulated in the culture reaches its maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

Since the gibberellin of the present invention is a fat-soluble substance, its properties can be utilized to isolate and purify gibberellin from a culture. That is, solvent extraction using ethyl acetate, chloroform, etc.; column chromatography using silica gel, alumina, etc.; furthermore, preparative thin layer chromatography using silica gel alone are effective.

By the above methods or by appropriately combining these methods, gibberellin having the above-mentioned physicochemical properties and good purity can be obtained.

In addition, for gibberellin assay in each purification step, Chinese cabbage seeds were placed in a petri dish containing the test solution for 25 min.
After 4 days of growth at °C, the length of the hypocotyl of the Chinese cabbage was measured and compared with the control.

〔Effect of the invention〕

Pheospherea sp. 487 (P
haeosphaeria sp, L-487)
is a novel microorganism that produces gibberellins, which are useful as plant growth hormones.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Cultured starch syrup 4.0%, soybean oil 0.3%, soybean flour 2.0%, cottonseed meal 1.0%, sungrain 0.5%, CaCO50.3
%, P13SO4・7Hz00. OO1%, CoCf
fz ・61)zOO, OOO1% and NaC1z
' Seed medium containing 6HzO0,0001% (ρ
)16.0) was dispensed into 10 Erlenmeyer flasks of 2Of)n+ffi at 40raR each, and autoclaved at 121'C for 20 minutes.

Next, 1 Phaesphaeria sp. Shi-487
Platinum loopfuls were inoculated and cultured with shaking at 210 rpm for 4 days at 26°C to prepare a seed culture solution.

Separately, starch syrup 2.0%, soybean oil 0.15%, soybean flour 1.0
%, cottonseed meal 0.5%, sungrain 0.25%, CaC
03Q, 1%, Fe5Oa ・7Hz00. OO
O5%, CoCP2・6HzOO, OOOO5% and
NaC (!

Main fermentation medium (p
l(6,0) and its 801) IP in 500ml
l! The mixture was dispensed into 100 Erlenmeyer flasks and autoclaved at 121°C for 20 minutes.

This main fermentation medium was inoculated with 4 + 12 of the above seed culture solution, and cultured with shaking at 210 rpm for 5 days at 26°C. The obtained culture was centrifuged to obtain a culture supernatant and cultured cells.

Purification method 20 liters of the above culture filtrate was acidified (pH=3.0) and extracted with 15 liters of ethyl acetate.

After concentrating the ethyl acetate layer under reduced pressure, a benzene-acetone (10:i) soluble portion was prepared from 3.5 g of the extract, and it was subjected to low-pressure liquid chromatography (CQ3 silica gel). When fractionated using acetone (8:1) mixed solvent, one strongly active area and one weakly active area were found (Figure 1).Of the strongly active areas, the one that was used first was the active area! (91 mg), the latter part is active area ff (1
41 mg), and 1 blue was prepared by repeating low pressure liquid chromatography and preparative TLC.

As a result, 1 part of the active substance was found in the active area, 3 parts of colorless columnar crystals.
1 mg (1) 98-199°C1 crystal solvent: hexane-ethyl acetate), and active substance 2 from the active area
Colorless acicular crystals 6.6 mg (mp 210-21)'C5
Crystal solvent: hexane-ethyl acetate) was obtained.

NMR, MS, and IR spectral data for active substances 1 and 2 are shown below.

[On active substance] El-MS Engineering/Engineering: 316 (M”)
298.272, 229, 203, 183, 159.1
29.91.79. IRνmax(KBr)cm-':
3100.2940.2800~2400.1770°1735.1653,880°'H-NMR (CDCffi,) δ: 1.10 <
3! (,S,)2.47 and 2.71 (each L H,A
Bq, J=1. l I(z).

4, 83 (I II, br, S,), 4.94
(18, br, S,) [Active substance-2) (a
) o 10' (cm0.18.Meoll
).

El-MS m/z: 332 (M”), 314
,296286.270,256,242,241,2
14゜203.183,157,105,91,79゜IRνmax(KBr)cm-': 3500.336
0°2930.2700~2400,1734.171
B.

1653, 870° According to the above (7) NMR, MS, and IR spectral data, the active substances 1 and 2 have the following structural formulas (1) and (
They were identified as gibberellin A, (GA, ) and gibberellin A, (GA4) shown in II) (see Table 1).

[Brief explanation of the drawing]

FIG. 1 shows the elution pattern of an ethyl acetate extract of Phaesphaeria sp. L-487 culture subjected to low pressure liquid chromatography. The horizontal axis shows the fraction number, and the vertical axis shows the length (mm) of the hypocotyl of Chinese cabbage. Figure 2 shows a schematic diagram of the morphology of Phaesphaeria sp. A, Cross-sectional view of the ascocarp buried under the host epidermis B, Enlarged view of the shell wall showing 2-3 layers of polygonal cells C0 Immature ascus double-walled structure; 8 Vigorous ascus E containing spores. Enlarged view of the ascus tip. A clear double-walled structure F. Three to eight ascospores are separated by septa, some of which are covered with gelatin sheaths. Applicant Mitsubishi Kasei Corporation Agent Patent Attorney Hase - 1 other person; C scold note 21 A 100 people

Claims (2)

[Claims] (1) Pheospherea that has the ability to produce gibberellins
SP L-487 (Phaeosphaeriasp
．． L-487). (2) A method for producing gibberellins, which comprises culturing a gibberellin-producing bacterium belonging to the genus Phaesphaeria and collecting gibberellins from the culture.