JPH0937667A - Plant with symbiotic endophyte and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide lawn and glassland reduced in both insect injury and disease injury in administering evergreen lawn grass, while enabling reduced use of insecticides, germicides and herbicides. SOLUTION: This plant with symbiotic endophyte belongs to Agrostis clavata, Poaanua L. var. reptans Haussk., or Festocaparvigluma. Lawn glass and lawn having the above-mentioned advantages are provided by using this plant.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plant in which endophytes coexist, and turfgrass and lawn using the same.

[0002]

2. Description of the Related Art Among turfgrass currently used as grasslands and lawns, cold-type turfgrass, which exhibits evergreenness, prefers a cool climate. Therefore, in a hot and humid climate like Japan, a great deal of management is required. It requires labor. For example, bentgrass and Kentucky bluegrass, which are very often used as cold-season turfgrass, suffer from diseases and insect damage in the summer, and therefore the use of pesticides is indispensable to maintain and manage them. In Bentgrass, diseases such as take-all patch, leaf blight, brown patch, psium bright, snow rot, anthracnose, dollar spot, etc. are mentioned as the disease, and death occurs unless an appropriate fungicide is applied. Further, as insect damage, insects such as Shibattoga, Sugiyoteto, Tamanaga moth and Japanese beetle are eaten, and in some cases, the turfgrass is completely killed. The use of insecticides is essential to control this.

[0003] By the way, it has been known that gramineous fungi called endophytes coexist in grasses (Hironori Koga, “Plant Protection”, Vol. 47, No. 7, 1993, Hironori Koga, “Lawn grass research”). Vol. 22, No. 2, 1993). Unlike other pathogenic bacteria, this endophyte is known not only to show no pathogenicity but also to improve plant insect resistance and disease resistance. It is considered that alkaloids and antibacterial substances have an influence on pests and pathogenic bacteria among the physiologically active substances produced by endophyte. For example, peramine, an indole alkaloid produced by endophyte, is known to have repellent effects against pests (Siegel et al. Ann. Rev. Phytopatho
l. 25 p.293-315 (1987)). In addition, as a result of the insect resistance test in perennial ryegrass (Lolium perenne L.), which is the main grass, clearly symbiotic individuals show resistance to insect pests such as grasshopper (Koga et al.,
Plant Protection No.47, 7 p.13-16 (1993)). Similarly, in the grass grass timothy (Phleum pratense L.), it is known that the endophyte symbiotic individuals show resistance to the disease, black rust (Puccinia graminis Pers.). (Tamiya et al., Hokkaido Agricultural Experiment Station Report N
o.34, p.8-11 (1990)). As described above, in grasses, it is known that symbiosis of endophytes greatly improves insect resistance and disease resistance, but in turf grass,
There is no example. For example, in bentgrass and Kentucky bluegrass, which are the main turfgrass, since endophytes do not coexist, improvement in insect resistance and disease resistance due to endophytes could not be expected.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel plant which enables management of turfgrass without relying on the use of agricultural chemicals due to disease or insect damage.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, found a plant in which endophyte coexists among plants having properties suitable as turfgrass. The present invention has been completed. That is, the present invention belongs to Agrosticus clavata (Japanese name: Yamanukabo), Poa Anua Linne Variety Reptans Hausku (Japanese name: Tsurusuzumenokatabira), or Festka parvigurma (Japanese name: Tobosigara), and End It is a plant in which fights live together.

Further, the present invention is turfgrass using the above-mentioned plants. Furthermore, the present invention is a lawn using the plant described above. Hereinafter, the present invention will be described in detail. Examples of plants to which the present invention is applied include Agrostis clavata, Poa anua linnae,
Variety Reptans Hausuk (Poaannua L. va
r. reptans Haussk.) and Festaca parvigluma. This is because the Poa Anua Linne Variety Reptans Hausuk is a perennial plant with a stalk of stem, so it does not die completely in the summer like the Poa Anua Linne, so it is an evergreen lawn. It can be used as The features of each plant will be described below.

[0007] Agrostis clavata, which belongs to the genus Agrostis to which bentgrass belongs, is a grass weed distributed in the temperate zone of the Eurasian continent, and has not been used for turfgrass or the like. This plant is a perennial plant that grows naturally in remote forests and lowland grasslands. The culm has a height of 30 to 70 cm, the leaf blades have a length of 7 to 15 cm, and the leaf width is 1.5 to 5.0.
mm, leaf sheath smooth, split to base, leaf tongue height 1.5-3.0
mm, height of panicles is 10 to 20 cm, branches are sparse, and lower nodes are 3 to 5 and half rings. At the time of flowers, a type that opens diagonally,
There is a pattern in which the ears are densely attached. Spikelets are 1.9 to 2.5 in length
mm, pale green, with approximately the same length of bursa, with a dorsal carina on the back along the central vein. Inner stem is about 0.2 mm, anther is 0.3 to 0.5 mm, chromosome is 2n = 42, and flowering period is May to August.

Poa Anua Linne Variety Reptans Hausku is a Poa genus to which Kentucky bluegrass belongs, but it is a grass weed distributed all over the world. Not used In addition, this plant is a perennial that grows naturally in remote mountains and lowlands, and is considered to be a variety of Poa Anua Linnaeus. Pore Anua Linnaeus is a 1-2-year-old plant and exhibits a stock type, whereas Poa Anua Linnaeus
Variety Reptans Hausuk is a perennial plant with stalks. The culm is a bunch, the height is 8 to 25 cm, the leaf blades are bright green and hairless, the length is 5 to 12 cm, the leaf width is 2.0 to 3.5 mm, and the leaves are folded in two along the central vein and inside the leaf tips. Has a boat shape. Leaf tongue is 0.5 to 2.5 mm, white and almost invisible. The inflorescences are 6 to 13 cm in length, and the bursa with a pale green spikelet on the top has lengths of 1.5 mm and 2.0 mm with short hairs in the marginal veins.
Anthers have a length of 0.6 to 1.0 mm, a flowering period of 4 to 7, and chromosomes are 2n = 28, but there are variations.

[0009] Festoca parvigurma is a genus of Nostoc commune to which tall fescue and meadow fescue belong, and is a grass weed distributed in Japan, and has not been used for turfgrass or the like. This plant is a plant of the genus Festoka that grows naturally in Japan, grows in the hot and humid environment of summer in Japan, and is a plant suitable for the environment of Japan. In particular, it has strong cold resistance and retains a green color all year long in winter. In addition, the negative resistance is also good, and the growth in shaded areas is excellent. In addition, this plant grows naturally in forests and grasslands and is a perennial plant. The culm is fasciculated, the height is 30 to 60 cm, and thin rhizomes are seen. Leaf blades are 10 to 25 cm long and 1.5 to 3.0 mm wide,
Smooth and shiny with leaf sheath. Leaf tongue is 0.2-1.0 in height
mm. The panicles are 8 to 15 cm long and singly grown from each node, and sparse spikes are attached to the upper half. Spikelets are green, 7-9m long
It consists of florets measuring 3 to 5 mm. Inclusion is 1mm in length and 1.5m
m, the awn is 6-9 mm. Anther is 0.5-0.7mm, green body is 2
n = 28, the flowering period is May to July.

A feature of the plant of the present invention is that endophytes coexist. The endophyte to be the subject of the present invention may be any endophyte as long as it can coexist with the above-mentioned three kinds of gramineous plants. The endophytes identified in the examples produce indole alkaloids, but those that do not produce such alkaloids are also subject of the invention. Further, the endophyte confirmed in the example imparts traits such as insect resistance, drought resistance, grass resistance, and disease resistance to plants, but imparts only some of these traits. Those that give other traits,
It is the subject of the present invention.

The plant of the present invention can be obtained by collecting the above-mentioned three types of plants from the natural world and selecting an individual living with endophyte from them. Since it is difficult to visually identify whether endophytes are symbiotic, it is a method of observing the mycelium in plant tissues such as seeds and leaf sheaths with a staining solution such as aniline blue, and observing with an optical microscope or an electron microscope. It is possible to know whether endophytes are symbiotic by a method of detecting endophytes in a plant extract using an enzyme-labeled antibody, a method of separating and culturing endophytes on a medium, and the like. The above-mentioned three types of plants are all weeds that grow naturally in Japan and can be easily obtained.

When the plant of the present invention is used as turfgrass or lawn, it can be managed in the same manner as conventionally known grasses and lawns of Poaceae. However, as described above, the plant of the present invention exhibits properties such as insect resistance and disease resistance, so that the amount of pesticide and the like can be reduced.

[0013]

【Example】

(Example 1) Agrostimus clavatta was collected from the wilderness of Fujinomiya City, Shizuoka Prefecture. The epidermis of the leaves and leaf sheaths of this Agrostis clavata was peeled off using tweezers or the like, and the presence or absence of endophyte in this tissue was detected by an optical microscope. Specifically, peel off the tissue of the leaf sheath,
Carefully peel off the back skin using tweezers, put the skin on the slide glass on which the aniline blue or lactophenol blue solution has been dropped, cover with a cover glass, and then boil with a flame such as a gas burner to boil the reagent. After cooling, the structure was observed with an optical microscope after cooling. × 150
At ~ × 300, it was observed that the endophyte hyphae were colored blue.

This operation was repeated several times, and the plants in which the endophytes were detected were designated as endophyte symbiotic individuals, and the plants in which the endophytes were not detected were designated as endophyte non-symbiotic individuals. Since endophyte is seed-transmittable, endophyte is present in seeds of symbiotic individuals. Utilizing this characteristic, seeds of symbiotic individuals were collected. (Example 2) Pore Anua from forest in Fujinomiya city, Shizuoka prefecture
Linne Variety Reptans Hausk was collected. Seeds of endophyte symbiotic individuals were collected from this Poa Anua Linne Variety Reptans Hausuk by the same method as in Example 1. (Example 3) Festka parvigurma was collected from a forest in Fujinomiya City, Shizuoka Prefecture. Seeds of endophyte symbiotic individuals were collected from this Festoca parvigurma by the same method as in Example 1. (Example 4) The seeds of Agrostis clavata obtained in Example 1 were sown at a density of 20 g / m ^{2 in} a well-horizontal horizontal field, and then covered with 0.5 cm to 1.0 cm of soil. It was Seeding of seeds was possible for a period of 3 to 10 months. One treatment section was 1 m ^{2 and} was repeated 3 times.

After seeding, germination started 7 to 10 days, and the first leaves developed. Then, within 10 to 20 days, 2.0 to 3.0 cm from the ground.
At the time of growing, the mowing was performed with a mower at a height of 20 mm. After that, mowing was performed at intervals of once / day to once / 3 days to form turf. Fertilization is carried out annually with nitrogen (60kg / 10a), phosphoric acid (20kg / 10a), potassium (45kg).
kg / 10a) was carried out regularly.

Further, the sandy soil was uniformly applied about once a month so as to be about 2 to 3 mm above the ground. In addition to this treatment area, creeping bentgrass (Agrostis
stolo-nifera L.) variety Pencros, colonial bentgrass (Agrostis tenuis Sibth.) variety Highland were established and managed in the same manner.

No fungicides, insecticides or herbicides were applied. The above turfgrass management was carried out throughout the year. As a result, from June to July, Shibattoga (Dediasia teter)
rellus Zincken), Japanese beetle (Popillia japonice Ne)
wman), Swordweed (Spodoptera depravata Butle)
r) larvae were found in each treatment area.
The treated area of Clavata was scarcely damaged by feeding, whereas the treated areas of Penncloth and Highland were particularly damaged by Shibatga, and most of the treated area became bare land.

From August to September, the dry condition is 4
After 7 days, the target areas, Penncross and Highland,
Withering was noticeable, but the treated area of Agrostis clavata continued to retain a green color. Also, from September to October,
Since weeds such as Poa annua invaded the bare land, many weeds were seen in the target areas of Penncross and Highland, which had died and were in bare land conditions, but weeds were invaded in the treated area of Agrostis clavata. Obviously few.

In the winter months from January to February, after the environment below freezing continued, the green color of the pen cloth decreased, and then the highland also decreased. However, Agrostis
The treated area of Clavata continued to retain the green color. (Example 5) 45 g of Poa anua linne Variety Reptans Hausk seeds obtained in Example 2
Sown in a well-consolidated field at a density of m ² , then about 1.
0 cm of soil was covered.

Seeding of seeds was possible for a period of 2 to 11 months, preferably 3 to 5 months and 9 to 10 months. One treatment area was 1 m ² and the treatment was repeated 3 times. Germination started about 10 days after sowing, and the first leaves developed about 14 days later. Then about 20
It grew to 2.0-4.0 cm above the ground in a day. At this time, height
Pruning was performed with a mower at 20 mm. After that, once /
A mowing operation was performed at intervals of once a day / 3 days to form a turf. As the rolling pressure increased, the stalks of the plants grew, and turf was formed in a short period of time.

Fertilization was regularly carried out annually using nitrogen (50 kg / 10a), phosphoric acid (25 kg / 10a) and potassium (25 kg / 10a) as a guide. Grain soil is about 2
It was uniformly performed about once a month so that the size was about 3 mm. In addition to this treatment area, we set the target area as Kentauki bluegrass (Poa pratensis L.) variety Baron variety Kenblue and performed the same management.

No fungicides, insecticides or herbicides were sprayed at all. The above management was implemented all year round. As a result, from June to July, larvae of Spodoptera litura occurred in each treatment group. The outbreak situation is that the plants died because the larvae of Spodoptera litura were found on the rhizome of turfgrass, which had died above ground, and the roots were damaged.

In Baron and Kenblue, which are the target areas, the above-ground part of turfgrass was withered and left bare due to the feeding damage of striated Ytoto, whereas the treatment area of Poa Anua Linne Variety Reptans Husk was , Was not damaged at all. In addition, after the dry condition continued in the summer, the target areas, Baron and Kenblue, were prominently dead, but the treated area of Poa Anua Linne Variety Reptans Hausque, which has drought resistance, is in a green state. While holding, continued to grow.

[0024] In autumn, weeds such as Poa annua and C. serrata rapidly invaded the surface that was bare in summer. Especially, Baron and Kenblue, which are the target areas,
Due to the large amount of bare land in summer, weeds were heavily damaged. Further, in the winter months from January to February, water stress was caused under the freezing temperature environment, the green color was decreased in each treatment section, and a part became dark purple.

However, the treated area of Poa Anua Linne Variety Reptans Hausuk continued to grow while maintaining a green color. (Example 6) The seeds of Festoca parvigurma obtained in Example 3 were uniformly sown at a density of 30 g / m ^{2 in} a well-compressed field, and then covered with about 1.0 cm of soil. Seeding was possible from February to November. 1 treatment area is 1m ²
As a unit, it was repeated 3 times.

[0026] Approximately 8 days after sowing, germination started, and the first leaves developed approximately 12 days later. After that, when it grew to a height of 30 mm or more, it was cut to 30 mm using a mower. After that, mowing was performed at intervals of once / 3 days to once / 7 days to form turf. Also, the grained soil should be 3-4 mm thick once a month
This was done twice a month so that the plant was completely covered with sand.

Fertilization was carried out using nitrogen (50 kg / 10a), phosphoric acid (30 kg / 10a), potassium (30 kg / 10a) as a guide year. In addition to this treatment area, tall fescue (Festuca arundinace Schreb.) Cultivar Yamanami cultivar Hokuryo was set as the target area and the same management was performed.

No fungicides, insecticides or herbicides were sprayed at all. The above management was implemented all year round. The situation is described below. From June to August, larvae of Spodoptera litura and Scutellaria japonica developed in each treatment group. The target areas, Yamanami and Hokuryo, were damaged by feeding and the above-ground parts of the grass were withered, and the grass was partially discolored to brown. When the turf grass was peeled off, a large number of larvae such as scorpion grass and bean beetle were found 3 to 4 cm underground. The average number of individuals in each treatment area was 68 Japanese beetle larvae
There were 79 and 36 Festka Parvigurma.

In addition, after being dried in August, Yamanami and Hokuryo, which are the target areas, died due to the damage caused by the Japanese white squirrel and the Japanese beetle, almost all of which turned brown. However, the processing zone of Festka Parvi Gurma is
Although some yellowing was observed, it did not die.
Furthermore, in September, the part that died in the summer became bare land,
From there, weeds such as Poa annua, Rhizopus, and Aquinoe foxtail invaded. Yamanami and Hokuryo in the target area, which had a lot of bare land in the summer, were particularly damaged.

In the winter months from January to February, the temperature became below freezing, and the water color changed from green to dark purple. In the shaded area of Festka Parvigurma, green and vigorous growth continued throughout the year, but the target areas, Yamanami and Hokuryo, are not only slow-growing in the shaded area, but also have many dead plants. I was seen. In the target area of the shaded area, 60% (area ratio) of the whole area was bare land. (Example 7) The disease resistance test of Agrosticus clavata against four kinds of diseases including take-all patch, dollar spot, brown patch, and leaf blight was carried out. The disease resistance test was performed by a spontaneous method. Evaluation of disease resistance is A: strong, B:
The test was performed in four stages of moderately strong, C: slightly weak, and D: weak.
In addition, the pathogens were similarly inoculated to Pencloth and Highland to serve as controls. The results are shown below. Take all patch duller spot braun patch leaf blight Pencross C C B B Highland B C C B A.clavata B A A A (Example 8) Black rust, powdery mildew, and leaf blight Pore Anua Linne Variety Reptans Hausque was tested for disease resistance. The disease resistance test was performed by a spontaneous method. Evaluation of disease resistance is the same as in Example 7. Baron and Kenblue were similarly inoculated with pathogenic bacteria and used as a control. The results are shown below. Black rust Powdery mildew Leaf blight Baron CBC Kenblue CBB P.annua L.reptans A A A (Example 9) Festka against 3 types of diseases, rust, powdery mildew and leaf blight Parvigurma was tested for disease resistance. The disease resistance test was performed by a spontaneous method. Evaluation of disease resistance is the same as in Example 7. In addition, pathogens were similarly inoculated to Yamanami and Hokuryo as a control. The results are shown below. Rust Disease Powdery mildew Leaf blight Yamanami B C B Hokuryo B C B F.parvigluma A B A (Example 10) After lyophilizing and drying 100 mg of Agrostis clavata plant tissue, it was ground and ground. , 1.5 ml each of methanol and chloroform were added, and the mixture was placed in a glass centrifuge tube and mixed for 30 minutes to 1 hour. This allowed the solvent to extract the alkaloid component from the tissue. The organic solvent layer was recovered, 3 ml of n-hexane and 3 ml of water were mixed and stirred for 30 minutes to 1 hour. After centrifugation at 5000 rpm to 8000 rpm, the organic layer and the aqueous layer were separated.

The aqueous layer was purified with an ion exchange column (Biorad AG2 × 8), dried and dissolved in an 80% aqueous methanol solution. Using this sample, TLC
After development and separation by (thin layer chromatography), four color reagents of Dragendorff reagent, p-dimethylbenzaldehyde reagent, nitrosonaphthol reagent and 1,2-naphthoquinone-4-sulfonic acid reagent were developed. make use of,
Alkaloids were detected.

Specifically, silica gel 60 manufactured by Merck was used as the thin layer plate, and chloroform was used as a developing solvent.
Methanol: acetic acid: water = 20: 10: 1: 1 was used. P-Dimethylbenzaldehyde was used as a color reagent for alkaloids. As a result, the Rf value is 0.1 to 0.8
At, several blue-violet spots were observed with the p-dimethylbenzaldehyde reagent. From the above results, it was clarified that Agrostys clavata produces indole alkaloids. Dragendorff reagent, nitrosonaphthol reagent, 1,2-
Alkaloids were detected in the same manner as above using a naphthoquinone-4-sulfonic acid reagent. As a result, all reagents were positive.

The colors of each reagent and the target substances are as shown in Table 1. ─────────────────────────────────── Reagent Coloring target substance ─────────── ───────────────────────── Dragendolph reagent Orange amine, alkaloid p-Dimethylbenzaldebit reagent Reagent Blue-purple indole alkaloid Nitrosonnaphthol reagent Blue indole 1,2 -Naphthoquinone-4-sulfonic acid reagent Red-purple aromatic amine ─────────────────────────────────── (Implementation Example 11) Pore Anua Linne Variety
The alkaloid component was extracted from the tissue of Leptance husk in a solvent in the same manner as in Example 10, and the alkaloid was detected using four types of color reagents. As a result, Pore
The tissue extract of Anua Linne Variety Reptans Hausku was positive for all four color reagents. (Example 12) Alkaloid components were extracted from the tissue of Festoca parvigurma into a solvent in the same manner as in Example 10, and the alkaloids were detected using four kinds of coloring reagents. As a result, the Festoca parvigurma tissue extract was positive for all of the four coloring reagents.

[0034]

INDUSTRIAL APPLICABILITY The present invention provides a lawn and a grassland which can reduce insect damage, disease and insecticides, fungicides and herbicides while controlling evergreen turfgrass.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Naoya Hiruma 2971 Hinohira Hinohira, Fujinomiya City, Shizuoka Prefecture (72) Inventor Fumiyoshi Akaike 26-1 Yamamiya, Fujinomiya City, Shizuoka Prefecture Inoue Munehiro Fujinomiya City, Shizuoka Prefecture Kitayama 5018-1 (72) Inventor Satoshi Shinozaki 723 Izumimachi, Fujinomiya City, Shizuoka Prefecture Izumi Palace No. 103

Claims (4)

[Claims] Claims: 1. Agrostis Clavata, Poa Anua Linne Variety Reptans Husk,
Or a plant that belongs to Festka parvigurma and coexists with endophyte. 2. The plant according to claim 1, wherein the endophyte is an endophyte that produces an indole alkaloid. 3. Turfgrass using the plant according to claim 1 or 2. 4. A lawn using the plant according to claim 1.