JPH0951734A - Bluegrass and introduction of symbiotic microorganism into bluegrass - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain bluegrass which has improved characters, particularly excellent in insect and infection resistances, and is useful for lawn grass and pasture by efficiently introducing a specific type artificially proliferated symbiotic microorganisms into bluegrass by inoculating and infecting the microorganisms artificially into the bluegrass. SOLUTION: Symbiotic microorganisms composed of Hyphomycetes, such as Acremonium sp. Po-001, Acremonium sp. Po-023 or Epichloe sp. Po-048 (respectively FERM P-14798, FERM P-15029 or FERM P-15030) are separated and artificially proliferated. The artificially proliferated symbiotic microorganisms are artificially inoculated into bluegrass to infect the bluegrass with the symbiotic microorganisms, and introduce the symbiotic microorganisms into the bluegrass.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bluegrass obtained by introducing a symbiotic bacterium and a method for introducing the symbiotic bacterium into the bluegrass. The bluegrass is a plant belonging to the genus Strawberry Alliaceae in the family Gramineae, belonging to the family Strawberry Allele.

[0002]

2. Description of the Related Art Conventionally, as bluegrass, Kentucky Bluegrass, Canadian Bluegrass, Poa annua, Rough Meadowgrass, Bullbow Meadowgrass, Alpen Meadowgrass, Wave Meadowgrass, Wood Meadowgrass, Balfour Meadowgrass, Swamp Meadowglass Plants such as grass, broadleaf meadowgrass, narrow leaf meadowgrass, smooth meadowgrass, spreading meadowgrass, and flattened meadowgrass are known.

These plants are included in the Gramineae family and belong to the family Strawberry Rhododendron in the subfamily Strawberry Rhododendron and are plants belonging to the genus Strawberry Rhododendron in the family Strawberry Rhododendron.
For example, Kentucky Bluegrass has the scientific name Poa pra
It is called tensis. Poa plate
nsis means Clayton et al. (Genera Gr.
aminium, Grass of the Wor
ld) 1986).

Bluegrass of the grass family represented by Kentucky bluegrass is important for humans among many bluegrass, and is widely used for grass and grass.

However, blue grass has a wide range of uses,
Moreover, because of its vast area of use, damage due to diseases and insect damage is great, and the Bluegrass website
The damage caused by (Japanese name Shibatatsuga) is so great that the larvae immediately after hatching are in a place where the chemical spray cannot reach them, and the damage is so great that the grass disappears overnight.

[0006] By the way, conventional breeding and cultivation methods for gramineous plants include an artificial crossing method, a selection method, a mutation method, a cell fusion method, a gene introduction method and the like. Recent advances in biotechnology have reduced the breeding period, which has taken over 10 years, to several years. In particular, the gene transfer method, which is a transformation technique, is a method using Agrobacterium, electroporation,
There is a particle gun method and the like, and application to many crops is being implemented.

[0007] However, it has been pointed out that the above-mentioned gene transfer method is extremely low in the efficiency of the above-mentioned gene transfer method in plants of the Poaceae family.
In the Agrobacterium method, gene transfer is very difficult because of the difficulty in infecting gramineous plants with Agrobacterium. In addition, in the electroporation method, it is essential to develop a regeneration system from protoplasts of gramineous plants, and even if regeneration is possible, it is known that the characteristics of the original plant are impaired due to culture mutation and the like. I have.

In the particle gun method, it is known that a given plant often becomes a chimera because a gene is randomly introduced into a plant or a culture.

[0009] Even in bluegrass represented by Kentucky bluegrass, a cell breeding method including cell fusion and gene transfer requires a complicated operation and its efficiency is low, so that it has been successfully developed at a practical level. Is very few.

By the way, among wild plants existing in nature, there are filamentous fungi which are endosymbiotic bacteria, that is, plants in which endophytes coexist. In the tissues of the plant,
In particular, such symbiotic bacteria grow in gaps between cells called cell gaps.

Such an endosymbiotic bacterium, ie, endophyte, is a symbiotic filamentous fungus that not only has no adverse effect on the host plant, but also provides a useful substance to the host plant, and has an environmental stress. It contributes to imparting a property having resistance to the plant.

That is, endophyte is an insect resistance of symbiotic plants (Siegel et al., 1987 Ann. R).
ev. Phytopathol, 25: 293-31
5), disease resistance (Gwinn and Gavin, 19)
92 Plant Disease 76: 911-9
14), environmental stress (dryness, etc.) resistance (Arachev)
alta et al., 1989 Agron. J. 81: 8
3-90), growth promotion (Latch et al., 1985 N.P.
Z. J. Agric. Res. 28: 165
-168) is known to be improved. Particularly in perennial ryegrass infected with endophyte, it is known that insect resistance is improved by repellent substances and alkaloids produced by this endophyte.

Also, New Zealand lychees (Latc
h) et al. have collected and investigated the endophyte of perennial ryegrass and tried to find an endfight called end safe which is less toxic to livestock and has excellent insect resistance.

However, many plants in which such endophytes coexist are wild plants with poor utility to humans, and it is necessary to introduce the efficacy of endophytes into useful gramineae plants. Has occurred. Therefore, attempts have been made to introduce endfight into perennial ryegrass, a major grass. The techniques are roughly divided into artificial hybridization and artificial inoculation.

The artificial mating method is a method of introducing a useful trait by pollen using a plant infected with endophyte as a mother, but in the conventional method, it is limited to introduction between species and qualities capable of mating. ing. In the artificial inoculation method, a plant body or a cultured tissue is inoculated and infected with endophyte isolated and cultured.

Compared with the artificial mating method, the artificial inoculation method is
Although the range of introduction was expanded, it was limited to perennial ryegrass due to technical problems such as the cultivation method of endophyte, inoculation conditions, and plant conditions. In addition, a method has been reported in which callus is used in tissues of a plant to be inoculated to improve the infection efficiency. According to this method, development of a system for regenerating a plant from callus is required, and thus, the method has only been introduced into perennial ryegrass.

[0017]

In the conventional cell breeding method, complicated operations and skill are required. On the other hand, according to the transformation or the cell fusion, the transduced trait or the transfected trait may be lost due to culture mutation or the like. Practical application has been difficult due to the influence on the traits.

Further, in the gene introduction method, when the gene affecting the plant trait or morphology was not elucidated at the time of gene introduction, it was impossible to introduce these traits.

In particular, traits related to complicated factors such as environmental stress could not be introduced by a method such as a gene introduction method. Further, in many cases, a decrease in seed fertility has been observed in plants produced by the cell breeding method.
This is a fatal defect in gramineous plants because reduced seed fertility leads to reduced yield.

In view of the above situation, the breeding method using endophyte or the improvement of traits is a completely new method for solving the above problems.

In the case of artificial inoculation of endophyte, it is limited to perennial ryegrass because of the search for endophyte, examination of culture system, etc., and application to other useful gramineous plants such as Kentucky bluegrass is recommended. Not done at all. Furthermore, in the callus inoculation method, it is essential to develop a regeneration system for the introduced plant. At the time of inoculation, no inoculation conditions have been developed to improve the infection efficiency of endophyte.

Among the types of endophytes, currently useful endophytes have been searched only from perennial ryegrass, tall fescue, and meadow fescue, which is a major obstacle to introduction. In particular, the host plants of these endophytes are exotic species, and no endophytes adapted to the Japanese environment have been found from plants native to Japan.

[0023]

An object of the present invention is to provide a bluegrass artificially introduced with endophyte and a method for artificially introducing endophyte into bluegrass not infected with endophyte.

[0024]

The present invention relates to a symbiotic bacterium consisting of a filamentous fungus, that is, bluegrass obtained by artificially introducing endophyte into bluegrass not infected with endophyte. Here, bluegrass is a gramineous plant, and is a plant of the genus Strawberry, which is included in the family Strawberry Rhododendron in the subfamily Rosaceae, Kentucky bluegrass, Canadian bluegrass, bluegrass, rough meadow grass, bullbows Meadow Glass, Alpine Meadow Glass, Wavy Meadow Glass,
The plants include plants such as wood meadow glass, balforce meadow glass, swamp meadow glass, broadleaf meadow glass, narrow leaf meadow glass, smooth meadow glass, spreading meadow glass, and flattened meadow glass.

Introducing such endophytes into bluegrass involves searching for and discovering endophytes that coexist in plants that grow in nature, and separating endophytes from plants and artificially culturing them. It is artificially introduced into the bluegrass. It is known that a plant that grows in the natural world and has endophyte exists in the cyperaceae, rush family and the like in the natural world.

For example, the following endophytes that have been discovered and cultivated by the present inventors and have been cultured, and the following endophytes may be used.

Acremonium sp. Po-0
01 (Deposited Bacteria, FERM P-14798) Acremonium sp. Po-023 (Seikokuken deposit FERM P-15029) Epichloё sp. Po-048 (Seikokuken deposit FERMP-15030) The bluegrass into which such an endophyte is introduced also includes the progeny thereof.

Next, a method of introducing a symbiotic bacterium consisting of a filamentous fungus into bluegrass, that is, an endophyte will be described. This method separates endophytes coexisting with plants existing in the natural world to perform artificial growth. ,
Bluegrass is artificially inoculated with artificially grown endophyte. And by infecting bluegrass with artificially inoculated endophyte and coexisting,
Introducing End Fight to Bluegrass.

In the step of artificially inoculating the endophyte, conidia of the endophyte may be used. In addition, any of the above endophytes deposited at the Institute of Industrial Science may be artificially inoculated. Further, the introduction of the endophyte into the bluegrass is not necessarily one type of the endophyte, and two or more types of the endophytes may be introduced together.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION The introduction of endophyte into bluegrass according to the present invention will be described in detail according to the procedure thereof.

Step 1 Detection step of presence / absence of endophyte and separation step (1) Detection step of endophyt The epidermis of leaves and leaf sheaths of the plant collected by search and the like is peeled off, and put in an aniline blue dyeing solution for staining. Endophytes in the tissue are detected under an optical microscope to confirm the presence or absence of endophytes.

(2) Separation / Culturing Step of Endophyte After sterilizing a plant piece in which endophyt is confirmed, it is placed on an endophyte separation medium and cultured for several months.

(3) Endophite classification process The separated endophytes are classified according to the host. Alternatively, cultivation is performed in a state where environmental conditions are changed using a plate culture method, and the cells are classified by morphological part. In addition, liquid culture is performed, and the cells are classified by morphological part. In addition, slide culture is performed, and classification is performed based on morphological parts.

Step 2 Alkaloid Analysis Step Alkaloids produced from endophyte are analyzed under symbiosis with the fungus alone or with a plant to confirm the insect resistance. At this time, it does not prevent the test for disease resistance, environmental stress resistance, growth promoting property, and the like.

Step 3 Introduction Step of Endophite The separated endophyte is artificially introduced into a target or target bluegrass. Methods for introducing endophyte include a method of directly inoculating a plant and a method of once inoculating a plant into undifferentiated cells such as callus and then regenerating the plant from the callus. These may be arbitrarily selected according to the type of plant to which endophyte is introduced.

Step 4 Introduction Confirmation Step In the individual into which the endophyte was introduced, the explants were stained with a staining solution, observed under a light microscope, and examined for the presence of endophyte or infection by enzyme immunoassay. Detect the presence or absence.

Step 5 Endophyte-introduced plant assay step (1) Insect resistance assay Using an endophyte-introduced symbiotic plant and an endophyte-free plant, an insect to be damaged by insects is bred. , Artificially perform feeding damage test.

(2) Disease resistance test step Using the bluegrass into which endophyte was introduced and the same type of bluegrass without endophyte, and using the pathogenic fungus to be the disease target, The pathogen is inoculated artificially, and the disease resistance test is performed according to the degree of disease.

(3) Assay using progeny After seeds in which endophyte is present are collected and germinated to confirm the presence of endophyte, each assay described above is performed.

[0040]

【Example】

(1) Detection of Endophyte Endophyte was detected from a plant belonging to the genus Poa, which is a wild grass family plant, by the following method.

The epidermis of the leaves and leaf sheaths of the above plants were peeled off, and the presence or absence of endophytes in the tissues was confirmed under an optical microscope. For this confirmation, several drops of a staining solution of 5 ml of lactic acid, 10 ml of glycerin, 5 ml of water and 0.02 g of an aqueous aniline blue solution are dropped on a slide glass. Then, the leaf sheath was peeled off, and the back surface epidermis was peeled off with tweezers in the vein direction. The peeled epidermis was placed on a slide glass, covered with a cover glass, boiled with a gas burner flame, and the tissue was observed under an optical microscope. If endophyte is present under these conditions, the mycelium will be colored blue, thereby detecting endophyte.

As a result of the detection, endophytes were detected in three kinds of Poa plants. And from the life cycle of the fungus, two of them are Acremonium type endophytes that do not go out of the plant only for the asexual generation, and the other one has a sexual generation and chokes at the heading time of the plant. Epichlo forming the mass of the called Constellation
It was a ё type end fight.

(2) Classification of end fights Three types of Po whose end fights were confirmed in the above item (1)
Endophyte was separated from the plant a genus by the following method.

The endophyte was separated from the plant body by washing the leaves and the sheath portion with water and then using 1% aqueous 70% ethanol solution.
After immersion for 0 second and then for 10 minutes in a 2.5% aqueous sodium hypochlorite solution, the plate was washed three times with sterile water, placed on an endophyte separation medium, and cultured at 25 ° C. in the dark. .

The separation medium is PDA adjusted to pH 5.6.
(Potato dextrose agar) Medium at 121 ° C.
After sterilization for 15 minutes, penicillin and streptomycin were added to a concentration of 100 mg / l each,
20 ml each was dispensed into a 9 cm-diameter plastic petri dish and used.

Approximately 3 to 8 weeks after the placement, the hyphae were separated from the explants, and the formed colonies were taken out with a cork borer having a diameter of 5 mm and transplanted to the same PDA medium for growth.

(3) Classification and identification of endophytes in the flora by the plate culture method The mycelia transplanted to the PDA medium were cultured under dark conditions at 25 ° C, and the flora formed was investigated. As a result of the investigation, the flora on the medium was all white and flocculent, the growth rate was very slow, and the colonies grew only to a radius of about 2 cm in one month.

Of these, three kinds of separated end fights were deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology. The display of the bacteria and the deposit number are as shown below.

Acremonium sp. Po-0
01 FERM P-14798 Acremonium sp. Po-023 FER
MP-15029 Epicloё sp. Po-048 FERM
P-15030 (4) State of hyphae by slide culture A PDA medium having a thickness of 2 to 3 mm was placed on a slide glass, and hyphae were proliferated thereon to investigate the morphology of hyphae and the formation of conidia. This culture was performed under dark conditions at 25 ° C.

As a result of the examination, all the hyphae were colorless, and the partition walls were observed with a width of 1 to 2 μm. Conidia could be easily formed with all endophytes.

Conidia were formed at the tip of hyphae or the tip of monophyllic phialide standing upright from the side, and most of them were conidia.

All the conidia were colorless and the cells were unicellular. Most of the conidia had a kidney shape, and the size was 3 to 8 × 1 to 3 μm. Further, the formed phialides were all cylindrical, thinned toward the tip, and separated from the mycelium by partition walls.

(5) Analysis of alkaloids from plants FERM P-14798, FERM P-1502
9, alkaloids were analyzed from each host plant of FERM P-15030 by the following method.

After freeze-drying the leaves and leaf sheaths of each plant,
100 mg of a sample was put in a mortar and ground, and 1.5 ml each of methanol and chloroform was added and mixed, and collected in a centrifuge tube. Slowly mix at 18 ° C for 30 minutes, then add 3 m each of n-hexane and water.
l and stirred for 30 minutes. Then 2000r.
p. m. For 10 minutes. Thereby, the organic layer and water were separated.

The water layer portion collected was 3 ml of BioRad.
AG2 × 8 and Analychem Blond
After purifying with an Elut CBA column, concentrating and adding 100 μl of 80% methanol, 20 μl of each sample was dropped on a thin-layer plate using Merck silica gel 60, and chloroform: methanol: acetic acid: water was used as a developing catalyst. The mixture was developed at a ratio of 10: 10: 1, developed, and analyzed by TLC (thin layer chromatography).

For the organic layer portion, 2 ml of the sample was placed in an Eppendorf tube, 500 μl of each sample, and the solvent was completely evaporated with a centrifugal evaporator, and finally diluted with chloroform to 200 μl. After dilution, Merck silica gel 60 was used for the thin layer plate, 3 μl of each sample was dropped, and chloroform: ethanol mixed at a ratio of 9: 1 was used as a developing solvent, and analyzed by TLC. Done.

After the extraction of the aqueous layer and the organic layer, U
V, the color reaction and Rf were measured using Ehrlich reagent (1.0 g of p-dimethylbenzaldehyde dissolved in 96% ethanol) and nitrosonaphthol reagent.
And measured.

As a result, FERM P-14798, F
Aqueous layer extracts from ERM P-15029 and FERMP-15030 host plants were confirmed to have blue-violet spots when the Ehrlich reagent was used, and these were indole alkaloids based on the color reaction with the reagent and the Rf value. I confirmed that there is.

FERM P-14798, FERM
The organic layer extract from each host plant of P-15029 and FERM P-15030 has a blue-violet spot from all the plants using the Ehrlich reagent, and when the nitrosonaphthol reagent was used, the organic layer extract of FERM P-15030 was obtained. A reddish purple spot was confirmed at the same position only from the host plant, and it was confirmed from the color reaction by the reagent that these were indole alkaloids.

(6) Liquid growth medium FERM P-14798, FERM P-1502
9. After culturing FERM P-15030 in PDA medium under dark conditions at 25 ° C. for 2 months respectively, colonies were taken out with a cork borer having a diameter of 5 mm, and 100 ml of PD (potato dextrose broth) medium was put into a shake flask of 300 ml. Put one in each flask in sterilized at 121 ℃ for 15 minutes, 150 ℃ at 25 ℃
r. p. m. Shaking was performed.

As a result, all the bacteria grew in a flask in one month.

(7) Artificial inoculation using plants Endophyte FERM P-14798 isolated,
FERM P-15029, FERM P-15030
Was used to artificially inoculate Kentucky bluegrass in bluegrass.

The target plants used here are as follows. I mean Kentucky Bluegrass,
The varieties of Baron, Merion, Kenblue and Troy were used.

The above endophyte was used after being grown by the method of item (6), transplanted to a fresh PD medium and cultured under the same conditions for 5 to 12 days. For inoculation, germination was performed in MS basic medium in which 3.0% shoe cloth and 0.7% gellan gum were added to MS (Murashige-Skoog) medium, and 1 week after germination,
The plant was incised with a scalpel, and 10 μl of the mycelium that had been liquid-cultured in item (6) was dropped.

After culturing these under dark conditions of 25 ° C. and 30 ° C. for several weeks, they were transferred to 16 hours of illumination or immediately transferred to 16 hours of illumination and cultured, and individuals that had grown to green had the same composition. The cells were transferred to a medium and cultured for 1 month.

As a result of confirming the introduction of the endophyte in these individuals using the method of (1), FERM
P-14798, FERM P-15029, FER
It was confirmed that all of MP-15030 were introduced into plants. The infection efficiency was clearly higher at 30 ° C than at 25 ° C.

(8) Artificial inoculation using callus Callus induction of Kentucky bluegrass was performed as a sample for artificial inoculation. The materials used were Baron, Merion, Kenblue and Troy varieties in Kentucky Bluegrass, and 2.0 mg / l PIC (picloram) and 0.2 mg / l BAP (6-benzylaminopurine) were added to MS basic medium. ) Was added to give a callus induction medium.

Seeds immediately after germination in MS medium were transplanted to a callus induction medium and cultured under dark conditions at 25 ° C. for 2 months to obtain callus having redifferentiation ability.

FERM P-14798, FERM P
-15029 and FERM P-15030 were used for artificial inoculation using callus of Kentucky bluegrass. Each callus was induced in the above-mentioned induction medium, and the obtained calli were transplanted to an MS basic medium without a plant hormone.

Immediately after the transplantation, the callus was cut with a scalpel, and the mycelia grown in the item (6) were added in an amount of 50 μl per callus.
It was dropped.

Callus was cultivated under dark conditions of 25 ° C. and 30 ° C. for several weeks, and then either transferred under illumination for 16 hours or immediately transferred under illumination for 16 hours for culturing, and regenerated individuals were kept fresh. It was transplanted to MS medium and cultured for 1 month. As a result of confirming the introduction of the endophyte using the method of the item (1), FERM P-1479
8, FERM P-15029, FERM P-150
The introduction of 30 was confirmed in all varieties.

Further, using the same method as the item (8), FER
Two types of bacteria, MP-14798 and FERM P-15030, were introduced into Kentucky bluegrass variety Baron. As a result of separating the mycelium from the introduced plant and culturing, two types of bacteria with slightly different flora were separated and detected.

As a result of analyzing these using the PCR method,
FERM P-14798 and FERM P-1503
A band pattern similar to that of 0 was shown, and symbiosis of two types of endophytes was confirmed from Kentucky bluegrass into which two types of endophytes were introduced.

(9) Mass production of conidia FERM P-1 confirmed to form conidia in a liquid medium
4798 was used for mass production of conidia. FER
MP-14798 was cultured by the same method as in (6), transplanted to a fresh PD medium, and 5 to 12 days after the conidial formation ability reached its peak, 20 ml of the culture solution was taken out, and a 20 μm mesh was used to Unnecessary hyphae are removed with a stack of layers, and the filtrate is put into a 10 ml centrifuge tube for 1000
r. p. m. For 10 minutes. The supernatant after centrifugation was discarded, and 1 ml of PD medium was added to obtain a conidium suspension.

(10) Inoculation method using conidia FERM P-14798 conidial suspension was cultivated in Kentucky bluegrass using the callus inoculation method described in (8) to produce varieties Baron, Merrion, Kenblue and Troy. Each was artificially inoculated. As a result of assaying each obtained individual by the method of (1), endophite-introduced individuals were confirmed at high frequency, and even if compared with the method of (8) which is an inoculation method using hyphae. A significant difference was recognized.

(11) Detection of endophyte by enzyme-linked immunosorbent assay (ELISA) From leaf pieces of a plant in which endophyte was confirmed by the artificial inoculation method described in (7), (8), and (9), The end fight was detected by the following method.

A buffer solution was added to 0.5 g of the fresh weight of the explant of the plant and ground in a mortar to obtain an extract. Add 50 μl of the extract to the wells of a microplate and
The adsorption was performed for 0 minutes. Then, the unbound antigen was washed.

The inside of the well was filled with a blocking solution (3% skim milk powder solution) and washed 30 minutes later. Further, anti-endophyte rabbit antiserum (primary antibody) was added to the wells and reacted at room temperature for 60 minutes. Then, the unbound antibody was washed.

A diluted secondary antibody (goat anti-rabbit IgG antibody labeled with alkaline phosphatase) was added to the wells,
The reaction was performed at room temperature for 60 minutes. Unbound antibody was washed out.
Thereafter, wells were added to the substrate solution to cause an alkaline phosphatase reaction. Then, the reaction was stopped with 0.5N NaOH, and the absorbance was measured at a wavelength of 405 nm.

As a result, a color reaction was confirmed from all of the endophyte-introduced individuals produced in (7), (8), and (9), and the introduction of these endophytes into Kentucky bluegrass was proved. Was done.

(12) Resistance test with shibatatoga Endophite FERM P-1479 according to item (8).
Kentucky Bluegrass with 8 introduced (variety Baron)
(Hereinafter referred to as KB-Baron 14798),
A resistance test against grasshopper was carried out.

KB-Baron 14798 was 2 after inoculation
Baron seeds that had not been inoculated as a control were soaked in 70% ethanol for 10 seconds, then immersed in 2.5% sodium hypochlorite aqueous solution for 10 minutes, and then sterilized water. The cells were washed 3 times with the above, air-dried, transplanted to MS basic medium, and cultured for 2 months after germination, which was acclimated and used.

Each of these materials was placed in a plant box of 20 × 30 × 10 cm with a culture soil up to a height of 8 cm, and 20 individuals of each material were planted and managed in a greenhouse for 120 days. After 120 days, the eggs of the grasshopper were soaked in 70% ethanol for 10 seconds, washed with sterilized water three times, and air-dried, and about 200 seeds were sown in each plant box.

As a result, the eggs hatched in 1 week and feeding damage started. The larvae of Shibattoga started from KB-Baron 1
Both 4798 and control were eaten, but KB-Baron 14798 with endophyte stopped eating and the control was completely eaten in 3 to 4 days.
KB-Baron 14798 was the only early feeding damage.

(13) Assay for resistance to leaf blight Drechslera sp., A pathogen isolated from leaf blight occurring in Kentucky bluegrass , Curv
ularia sp. , And Bipolaris
sp. Was cultured on a PDA medium for 2 weeks, conidia formed on the surface of the microflora were scraped off with a needle, and suspended in sterile water to adjust the concentration to 5,000 to 10,000 cells / ml.

KB-Baron 14798 was 2 after inoculation
In the control group, the non-inoculated baron seeds were soaked in 70% ethanol for 10 seconds, and then in 2.5% sodium hypochlorite aqueous solution for 10 minutes. After washing 3 times, air-drying, transplanting to MS basic medium, and culturing for 2 months after germination, 6 × 6 × 10
Sterilized culture soil was placed in a cm plant box, 10 materials were planted each, and the culture was performed for 2 weeks.

The pathogen was inoculated by spraying or coating on the entire surface of the plant and culturing at 28 ° C. for 16 hours with a photoperiod of 1 month.

As a result, in the control group in which all the bacteria did not contain endophyte, leaf rot started from the tip of the leaf 1 week after the inoculation and the plant completely died within 3 weeks, whereas
KB-Baron 1479 containing Endophyte
In No. 8, a black spot with a diameter of about 2 mm in the early stage of infection and a leaf withering of about 1 cm at the tip of the leaf were detected, but there was almost no spread of lesions after that, and Drech
slera sp. , Curvularias
p. And Bipolaris sp. There was a clear significant difference in the occurrence of leaf blight caused by the disease.

(14) Assay for resistance to rust The spores formed on the leaves of Kentucky bluegrass infected with black rust were adhered to a 5 × 5 mm damp paper. A paper filter with summer spores was attached to leaves of KB-Baron 14798 and control plants prepared in the same manner as in (12), and inoculated. The inoculated plants were incubated for 12 hours in a humid, 20 ° C., dark condition, then transferred to light for 16 hours, and cultured for 3 weeks.

As a result, 10 days after the inoculation, KB-Baro was used.
n. spores were formed on the surface of the leaves of both n 14798 and the control. After that, in the control section, the whole plant body was covered with a large amount of summer spore stacks formed secondarily in 2 weeks, whereas in KB-Baron 14798, the newly formed summer spore stacks were very polar. A small amount did not cover the whole plant with summer spore deposits, and there was a significant difference in the occurrence of symptom.

[0091]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a symbiotic bacterium consisting of a filamentous fungus, that is, a bluegrass obtained by artificially introducing endophyte into a bluegrass in which endophyte does not exist, and an artificial bluegrass It is about how to introduce End Fight.

Therefore, according to the present invention, at least insect resistance and disease resistance can be imparted by introducing the endophyte into the bluegrass and causing the endophyte to coexist in the bluegrass. Therefore, the traits of bluegrass can be improved, and useful bluegrass having excellent traits can be provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 645)

Claims (6)

[Claims] 1. A bluegrass obtained by artificially introducing a symbiotic bacterium comprising a filamentous fungus into an individual in which a symbiotic bacterium comprising a filamentous fungus does not exist. 2. The symbiotic bacterium introduced into the bluegrass is A
cremonium sp. Po-001, Acre
monium sp. Po-023, or Epic
hloё sp. Po-048 (Deposited Bacteria of RIETI FE
RM P-14798, FERM P-15029, or FERM P-15030), The bluegrass according to claim 1. 3. A step of isolating and artificially growing a symbiotic bacterium comprising a filamentous fungus that is symbiotic with a plant, a step of artificially inoculating bluegrass with the artificially symbiotic bacterium, and the artificially inoculated symbiotic bacterium. A method of introducing symbiotic bacteria into bluegrass, which comprises the steps of coexisting with bluegrass to infect. 4. The method of introducing symbiotic bacteria into bluegrass according to claim 3, wherein conidia of the symbiotic bacteria are artificially inoculated in the inoculating step. 5. A symbiotic bacterium artificially inoculated into the bluegrass is Acremonium sp. Po-001, Acr
emonium sp. Po-023, or Epi
chloё sp. Po-048 (Strain deposited by the Institute of Industrial Science and Technology F
ERM P-14798, FERM P-15029 or FERM P-15030), The method for introducing a symbiotic bacterium into bluegrass according to claim 3, wherein 6. The method for introducing a symbiotic bacterium into bluegrass according to claim 3, wherein one symbiotic bacterium or two or more symbiotic bacterium is artificially introduced.