JP3479183B2 - How to control turf grass snow rot - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for controlling turfgrass snow rot. More specifically, the present invention relates to an antagonistic bacterium against pathogens of turfgrass snow rot and a method for controlling turfgrass snow rot by applying sodium alginate to turfgrass to enhance the antagonistic force of the antagonistic bacterium. According to the present invention, it is possible to effectively control snow rot, which is a disease of turf, by using an antagonistic bacterium having an antagonistic force and a material used in combination therewith.

[0002]

2. Description of the Related Art In the snow areas of Hokkaido and the Tohoku region, wheat and grass are heavily damaged by snow rot under root snow. Most of the major pathogens of snow rot that occur in turf have been identified, and in Japan, there are 1. Typhula ishikariensis , 2.
( Typhula incarnata) , 3. Red snow rot ( Monographella n
ivalis [Incomplete Generation: Microdochium (Fusarium) nival
e ]), 4. Snow rot large grain sclerotia ( Sclerotinia borealis ), 5.
It is classified as brown snow rot ( Pythium spp. ). Among them, snow rot caused by Typhula spp. Mainly occurs in golf courses in areas where the amount of snow is large in winter, the ground temperature under snow is relatively high, and the soil freeze period is short. The symptom is the diameter when the snow melts
It forms a light gray circular affected area of 5 to 20 cm, and in severe cases, the affected areas fuse and become an irregular affected area with a diameter of 30 cm to 60 cm. Light white hyphae and black or brown sclerotia are often found on diseased leaves and on the crown (on the ground).

The host range of Typhula spp. Is wide, and it affects dicotyledonous plants (alfalfa, red clover, rapeseed) and monocotyledonous plants (autumn wheat, orchardgrass, timothy). As for the host range of turf, in addition to creeping bentgrass, colonial bentgrass and velvet bentgrass as well as other species such as bluegrass, Kentucky bluegrass, trefoil fescue, tall fescue, and perennial ryegrass are invaded. At the golf course in Hokkaido, creeping bentgrass is mainly used on the green and Kentucky bluegrass and perennial ryegrass are used on the fairway due to its characteristics as a sports turf, but these are all susceptible to snow rot caused by Typhula spp. is there.

As a method for controlling such a snow rot of the Typhula genus, in addition to the cultivated methods such as removal of thatch, aeration, and appropriate fertilization, preventive control by spraying chemicals is performed. As the drug, an organic copper agent using 8-hydroxyquinoline copper or the like as an active ingredient, a mepronil agent containing 3'-isopropoxy-2-methylbenzanilide as a main component, and 3-hydroxy-5-methylisoxazole are used. A hydroxyisoxazole agent, which is the main component, is used.

A control method using an antagonistic bacterium is disclosed in Japanese Patent Laid-Open No. 7-
This is made clear by Japanese Patent No. 289242. However, these methods have a drawback that the colonization of the antagonistic bacteria is poor and the effect is insufficient depending on the conditions of the spraying field. Further, Japanese Patent Laid-Open No. 4-164008 has devised a method of adsorbing an antagonistic microorganism on beads of calcium alginate. To use this method, the microorganism is once dissolved in a sodium alginate solution, which is then dissolved in calcium chloride or the like. It is necessary to add it dropwise to the aqueous solution. This method has a drawback that the steps are complicated and that it takes time and labor to manufacture the beads of calcium alginate. Also,
When this method is applied to grass, there is a problem that effective effects cannot be obtained unless a large amount of beads are scattered on the grass.

[0006]

Problems to be Solved by the Invention Since snow rot has no manifestation of symptoms before root snow, it is inevitable that it will be scattered all over the place where it is expected to occur, which requires a great deal of labor and expense.
Further, in the chemical control, there is a problem of emergence of resistant bacteria if the chemicals are selected incorrectly and they are continuously used. In addition, preventive spraying uses a large amount of pesticides in a wide range, which may cause environmental pollution. Concerning damage to pesticides at golf courses, there is concern about pollution of drinking water due to environmental pollution, death of fish in rivers and ponds, and poor health of caddies and golfers. Therefore, the use of pesticides at golf courses has been strongly criticized by the public, and the government has made it necessary for the prefectures to prevent the construction of new golf courses and to operate golf courses without pesticides. .

Pesticides are ranked for their toxicity to fish based on the Agricultural Chemicals Control Law. Previously, organocopper agents (fish toxicity B) were mainly used as control measures against snow rot on golf courses. In recent years, golf courses have tended to voluntarily refrain from using pesticides of fish toxicity class B in consideration of environmental pollution. in this way,
The disease control of golf courses is in the direction of reducing pesticides, and various efforts are being made at each golf course by combining agricultural control and pesticide control. At present, it is difficult to deal with all diseases without using pesticides at all, but a biological control method using an antagonistic microorganism has been established,
If it can be combined with chemical control, it can be expected to contribute to the reduction of pesticides on golf courses. Therefore, an object of the present invention is to provide a method for controlling turfgrass snow rot using microorganisms.

[0008]

Means for Solving the Problems The present inventor has succeeded in obtaining a bacterium having an antagonistic activity against the snow rot fungus from the sclerotium of the snow rot fungus. As a result of further research aimed at developing a method and a control material for snow rot using this antagonistic bacterium, this antagonistic bacterium is applied to turfgrass together with a highly viscous and highly hydrophilic substance such as sodium alginate. As a result, they have found that turfgrass snow rot can be controlled extremely effectively, and completed the present invention. Further, this method was able to obtain a stronger control effect by spraying the same amount of antagonistic bacteria as compared with the conventional method using calcium alginate beads. Therefore, the gist of the present invention is a biological control method of turfgrass snow rot, which controls snow rot by using an antagonistic bacterium against the pathogen of turfgrass snow rot in combination with sodium alginate as an auxiliary agent.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The antagonistic bacterium against the pathogen of turfgrass snow rot used in the present invention may be any bacterium having an antagonistic power, and is not particularly limited to a specific bacterium. The antagonistic bacteria can usually be obtained from the sclerotia of snow rot fungi. For example,
Snow rot Black small-grain sclerotium disease, snow rot large-grain sclerotium disease, snow rot brown small-grain sclerotium disease, etc. After sterilization, it can be obtained from growing microorganisms by culturing on agar medium. Further, it can be obtained in the same manner from the leaves of dead grass after snow melting, or can be obtained from the soil of turfgrass in which snow rot has occurred using the dilution plate method. As a test method for the antagonistic force when obtaining the antagonistic bacteria, an assay by confrontation culture, an assay using turfgrass foliage, an assay by a sod cup test, etc. can be adopted. For the acquisition of these antagonistic bacteria and the assay of their antagonistic activity, see Turfgrass Research, Vol. 22,
No. 1, October 1993, pages 21-27 and Japanese Utility Model Publication No. 5-236939.

Representative of the antagonistic bacteria include bacteria belonging to the genus Pseudomonas which are separated from the sclerotium of snow-corrosion small grain sclerotia. More specifically, Pseudomonas fluore
scens , especially Pseudomonas fluorescens A11 strain (FERM P-14269). In order to apply the antagonistic bacterium to turfgrass, a method of culturing the antagonistic bacterium by tank culture, shaking culture, etc., and spraying a culture solution as it is or by appropriately diluting the solution is adopted. For example, A11
The strain is tank-cultured in MYPG medium (3.0 g of yeast extract, 3.0 g of malt extract, 5.0 g of polypeptone, 10.0 g of glucose in 1 liter), and this culture solution is sprayed. As the culture medium for this tank culture, generally all culture media for culturing bacteria can be used. Application rate of antagonistic fungi, typically the broth is cell content ^{10 8 ~10 9 cfu / ml,} 10~3000ml / m 2, preferably applied in an amount of 300 ~3000ml / m ^2.

In the present invention, as an adjuvant together with antagonistic bacteria
Apply sodium alginate. Sodium alginate
As for the grass, the powder of sodium alginate is scattered on the grass as it is.
May be clothed or added to a diluent such as water
You may spray in the state. Application rate of sodium alginate
Is usually 5-100g / m²And preferably 5 to 50 g / m
²And more preferably 20-40 g / m²Is. Argi
Sodium nitrate helps the growth of antagonistic bacteria, but
Becomes a highly viscous liquid, so if it is sprayed in a large amount, breathing problems will occur.
Is not preferable. Sodium alginate is an antagonist
It may be applied after spraying, or before spraying antagonistic bacteria.
You may apply. For example, golf where disease is expected to occur
First, add 20 to 40 g / m of sodium alginate on the field grass.²Scattered
Those who apply the cloth and spray the tank-cultured antagonistic bacteria immediately after that.
Method or after spraying antagonistic bacteria, sodium alginate
The method of spraying the mu can be mentioned. The application time is
Antagonist is, for example, 2 months before root snow, 1 month before root snow,
It may be sprayed three times or once before root snow. this
In this case, the auxiliary agent sodium alginate should be sprayed every time.
Alternatively, the first spraying may be applied. Also, culture of antagonistic bacteria
To the solution, add sodium alginate powder,
It can also be applied to turfgrass together. Natri alginate
When spraying um in 3 times, the total amount of 3 times is 100g
/ m²It is preferable not to exceed 50 g / m²Below
Preferably.

[0012]

INDUSTRIAL APPLICABILITY According to the present invention, snow rot of turfgrass can be effectively controlled by using sodium alginate as an adjuvant together with an antagonistic bacterium against the pathogen of snow rot. The method of the present invention can be easily applied to golf courses and the like, and is extremely significant as a control method without environmental pollution.

[0013]

EXAMPLES The present invention will now be described in more detail with reference to examples. Example 1 (1) Acquisition of antagonistic bacteria against snow rot black small grain sclerotia and
Measurement of antagonistic force i) 2000 strains of psychrophilic microorganisms were screened from the sclerotia of turfgrass and snow rot black small grain sclerotia in soils all over Japan and Hokkaido golf courses. With respect to these 2000 strains, 30 strains of antagonistic bacteria were obtained as shown in Table 1 below by the in-face culture assay and the turfgrass medium assay method described in JP-A-5-236939. Specifically, in the in-face culture assay, a potato dextrose agar medium was poured into a sterilized petri dish, one of the petri dishes was inoculated with a test microorganism, and the other side was inoculated with a black rot black small grain sclerotium bacterium, and after 1 month at 4 ° C, the naked eye was examined. Alternatively, the antagonistic force was determined by observation with an optical microscope. In the turfgrass medium assay, cut bentgrass, cut to a length of 10 to 20 mm, and drain the dish as it is, pack it in an appropriate amount on a petri dish after sterilization at 121 ° C for 20 minutes so that the test microorganism contains 10 ⁸ cfu / ml. It was prepared and sprayed on a petri dish so that the spraying amount was 500 ml per 1 m ² . Immediately thereafter, the flora of the snow rot black small grain sclerotium pre-cultured on potato dextrose agar was
It was cut with a mm cork borer and planted in the center of a petri dish. Culturing was performed at 4 ° C, and after 1 month, antagonistic bacteria in which the development of the flora was completely suppressed were screened. The results are shown in Table 1.

[0014]

[Table 1] Table 1 Screening for antagonistic bacteria of snow spores of small black rot fungus Antagonistic anti-culture test Turfgrass medium assay After selection by number of test strains of bacteria After selection, number of strains of isolated source (%) * Number of strains (%) * Soil 1500 60 (4.0) 10 (16.7) Turfgrass 200 17 (8.5) 8 (47.1) Sclerotium 300 33 (11.0) 12 (36.4) Total 2000 108 (5.4) 30 (27.8) ───── ──────────────────────── * Selection rate (%) = (Number of strains after selection / Number of strains before selection) x 100

Ii) For the 30 strains shown in Table 1 selected by counterculture and turfgrass medium assay,
Antagonism was measured by the assay method by the sod cup test described in 6-327493. Commercial bentgrass sod [variety: pen cloth; sand specification; pH 6.9;
(Yes) Nasu turf glass (Tochigi Prefecture)] was cut into an appropriate size and packed in a rectangular container (108x259x85mm ³ ). Sterile Fusuma Vermiculite Medium (Fusuma 1000ml, Vermiculite) was punched out from 4 to 5 discs from the bacterial flora on MYPG agar medium that had been previously cultivated at 9 ° C for 14 days.
1000 ml, glucose 14 g, distilled water 700 ml) and transplanted to about 500 ml. After static culture at 9 ° C for 1 month, the obtained bacterial cells were stirred and used as an inoculum source. 1.0 g of the inoculum was packed in a cylindrical plastic container (inoculum container) having an inner diameter of 22 mm and a depth of 9 mm, and placed so that the medium surface was in contact with the center of the shiba in the rectangular container. Thirty candidate antagonistic strains selected in the turfgrass medium assay were cultured in MYPG medium for 3 days at 25 ° C, and the culture solution was sprayed as it was onto a bentgrass sod at an amount corresponding to 1000 ml / m ² .

A cloth of fine synthetic fibers is laid so as to cover the entire surface of the shiba, and absorbent cotton that has absorbed water is further placed on it.
The rectangular container was closed with a lid. This container was subjected to low temperature treatment at 0-1 ° C, which is almost the same temperature as under snow. After processing starts 1
By day 4, hyphae grew from the inoculum container and came into contact with bentgrass foliage. This day was counted as the day of inoculation, and the major and minor diameters of the flora diameter of this pathogen that developed between the day of inoculation and the 21st day of inoculation were measured, and the inoculation container diameter of 24 mm was subtracted from the average value of both. The value was used as the amount of bacterial lawn development (Fig. 1). From this, the rate of bacterial flora development per day was calculated. The significant difference in the measurement results was determined by t-test. As a result, 5 more effective strains were obtained from the 30 strains shown in Table 1. The results obtained for these 5 strains are shown in Table 2.

[0017]

[Table 2] Table 2 Assay for inhibition of bacterial flora development of snow rot black small grain sclerotium by spraying with antagonistic bacterial suspension Strain source of bacterial strain antagonistic bacteria Progress of flora (mm / day) A11 strain Snow rot black small grain sclerotium Disease Sclerotium 0.18 ± 0.06 a B58 strain Snow rot black small grain sclerotia Sclerotium 0.20 ± 0.05 a G10 strain Snow rot black small grain sclerotium Sclerotium 0.20 ± 0.06 a G59 strain Diseased part Turfgrass 0.20 ± 0.05 a H62 strain Snow rot Black small grain sclerotium sclerotia 0.20 ± 0.07 a Control (water) 0.25 ± 0.06 b Control (medium) 0.26 ± 0.06 b ──────────────────────── ───────────── a and b are significantly different with P <0.05 by t test.

Iii) The A11 strain shown in Table 2 grows in a basal medium of bacteria such as malt extract / yeast extract / broth medium such as peptone medium as shown below. Medium name: Malt extract, yeast extract, peptone medium (MYPG agar medium) Medium composition: 0.3% Malt extract 0.3% Yeast extract 0.5% Polypeptone 1.0% Glucose medium pH: pH 6.8 Medium sterilization condition: 121 ° C 20 minutes incubation Temperature: 25 ° C Culture period: 3 days

The oxygen requirement of strain A11 is aerobic and can be stored by the freeze-drying method. A11 strain is Pseudomonas fl
belongs to uorescens , Pseudomonas fluoresc
ens A11 strain was deposited at the Institute of Biotechnology, Institute of Industrial Science on April 8, 1994, and the deposit number was FERMP-14269.
Is given. Table 3 shows the detailed characteristics of the A11 stock.
I wrote it in.

[0020]

[Table 3] Table 3 Properties of Antagonistic Bacterial A11 Strain Item Test Result Morphology Bacillus Gram stainability-spores-motility + flagellum hyperproliferative oxidase + catalase + OFO PHB accumulation-fluorescent dye production + arginine dihydrase + Growth at 41 ℃ -Denitrification-Nitrate reduction-Indole production-Urea decomposition-Gelatin liquefaction + Starch decomposition-Assimilation glucose + trehalose + 2-ketogluconate + meso-inositol + geraniol-L-valine + β- Alanine + DL- Arginine + Sorbitol + Adonitol + Quinone Q-9 ────────────────────────────

Iv ) The effect of controlling Pseudomonas fluorescens A11 strain and a sodium alginate auxiliary agent against the snow rot black small grain sclerotium bacterium was evaluated in a petri dish. The test microorganism was transplanted to one end of the minimum medium or MYPG agar medium and cultured at 9 ° C for 10 days. At this time A11
The growth status of the strain was expressed as 0 (bad) to 3 (good). Sodium alginate was used by directly adding it to the minimal medium or MYPG agar medium. After that, T. ishikariensis lawn discs were transplanted onto the medium. At this time, place four flora discs linearly at intervals of about 15 mm from the boundary of the test microorganism,
After culturing at 9 ° C for 10 days, it was observed from which disc the hyphae developed. If the flora develops from the disc closest to the test microorganism, the antagonistic force is 0, and if it develops from the 2nd and 3rd or later, the antagonistic force is 1, and if it develops only from the 2nd and 4th, the antagonistic force is 3 Expressed to determine relative antagonistic force. Also, use the minimum medium or MYPG agar
Transplanted only T. ishikariensis flora discs, 1 at 9 ℃
The diameter of the flora was measured after culturing for 5 days. Each experiment was repeated twice. The results are shown in Table 4.

[0022]

[Table 4] Table 4 Sodium alginate addition concentration and A11 strain growth, antagonism, flora diameter of snow rot black small grain sclerotium fungus diameter Sodium alginate A11 strain growth antagonizing snow rot black small grain sclerotium addition concentration Disease flora diameter (mm) (g / liter) Minimum medium MYPG medium Minimum medium MYPG medium Minimum medium MYPG medium 0 0 1 0 1 35 56 0.5 0 1 0 1 38 56 1 0 1 0 1 40 56 2 0 1 0 1 44 53 5 0 2 0 2 42 52 10 0 3 0 2 42 51 20 1 3 2 3 35 44 50 1 3 2 3 24 36 ───────────────────── ────────────────

As is clear from Table 4, the higher the concentration of sodium alginate added, the more the growth of the A11 strain was promoted, the stronger the antagonistic power, and the smaller the diameter of the flora of the snow rot black small grain sclerotium bacterium. . It is considered that the sprayed antagonistic bacteria acted synergistically with sodium alginate.

Example 2 The same experiment as in iv) of Example 1 was carried out to measure the antagonistic activity of the A11 strain when sodium alginate was added by the assay method by the sod cup test described in JP-A-6-327493. did. Bentgrass sod on the market [Category: Pencloth; Sand specification; pH 6.9; (Now) Nasu Turfgrass (Tochigi Prefecture)]
8x259x85mm ³ ). Sterile 4 to 5 discs were punched out from the bacterial flora on MYPG agar that had been cultivated at 9 ° C for 14 days in advance.Sterile fusuma vermiculite medium (fusuma 1000ml, vermiculite 1000ml, glucose 1
4 g, 700 ml of distilled water) and transplanted to about 500 ml. After static culture for 1 month at 9 ° C, the obtained bacterial cells were stirred and used as an inoculum source.
1.0 g of the inoculum was packed in a cylindrical plastic container (inoculum container) having an inner diameter of 22 mm and a depth of 9 mm and placed so that the medium surface was in contact with the center of the shiba in the rectangular container. Sodium alginate powder 0, 0.5, 1, 2, 5, 1 in grass sod in the container
0,20, 50 g / m ² Evenly spread, immediately after that A11 strain
A culture solution (cell content 1 × 10 ⁸ cfu / ml), which had been cultured in MYPG medium at 25 ° C. for 3 days, was sprayed at 1000 ml / m ² .

A fine synthetic fiber cloth is laid so as to cover the entire surface of the shiba, and absorbent cotton that has absorbed water is further placed on it.
The rectangular container was closed with a lid. This container was subjected to low temperature treatment at 0 to 1 ° C, which is almost the same temperature as under snow. After processing starts 1
By day 4, hyphae grew from the inoculum container and came into contact with bentgrass foliage. This day was counted as the day of inoculation, and the major and minor diameters of the flora diameter of this pathogen that developed between the day of inoculation and the 21st day of inoculation were measured, and the value obtained by subtracting the inoculation container diameter of 24 mm from the average value of both It was defined as the amount of bacterial lawn development.
The significant difference in the measurement results was judged by t-test. The results are shown in Table 5.
It was shown to.

[0026]

[Table 5] Table 5 Effect of Sodium Alginate Addition Concentration Used in A11 Strain Culture Solution on the Development of Snow Rot Fungus Flora Alginic Acid Amount Added Snow Rot Inhibition Rate (g / liter) (g / m ² ) Flora Progress (mm) (%) 0 0 40 ± 12 35 0.5 0.5 39 ± 11 37 1 1 39 ± 12 37 2 2 36 ± 11 42 5 5 32 ± 9 * 48 10 10 30 ± 7 * 52 20 20 30 ± 7 * 52 50 50 30 ± 7 * 52 No cell spray 62 ± 15 0 ───────────────────────────────── * Indicates that the suppression rate was significantly enhanced by the t-test compared to the 0 g / m ² addition (P <0.05)

When sodium alginate was used at 10, 20, 50 g / m ² , a significant control enhancing effect was obtained, which was 75% when only the antagonistic bacteria were used, and when no antagonistic bacteria were added.
Up to 48%, it suppressed the spread of snow rot bacterial flora.

Example 3 The same test as the previous example was conducted in an actual golf field field. The test was conducted in 10 sq. Ft. Of 1 golf course at ^two golf courses in Hokkaido where snow rot is found every year. Sodium alginate as a powder was evenly applied to turfgrass at 0, 0.5, 1,2,5,10,20,50 g / m ² and immediately after that, the A11 strain was grown in MYPG medium at 25 ° C 3
The culture solution (cell content 1 x 10 ⁹ cfu / ml) that was cultivated for a day
1000 ml / m ^{2 was} sprayed. Division was performed in the same manner as in Example 2. At the same time, the culture solution of the A11 strain was made into calcium alginate beads by the method disclosed in JP-A-4-164008 and sprayed as a control. The results are shown in Table 6. Increasing the amount of sodium alginate added tended to suppress the snow rot susceptibility. Moreover, when the amount of sodium alginate added was 5 g / m ² or more, the snow morbidity rate was significantly suppressed. On the other hand, the method using calcium alginate beads was effective only when the addition amount was 20 g / m ² or more. Further, the effect of the method according to the present invention was stronger when the amount added was the same. Therefore, by using sodium alginate according to the present method, the sprayed antagonistic bacteria settle on the turf, and by enhancing its effect,
It is considered that the morbidity of snow rot fungi was suppressed.

[0029]

[Table 6] Table 6 Effect of sodium alginate concentration on snow rot morbidity Sodium alginate snow rot morbidity (%) Addition amount (g / m ² ) Method of the present invention Addition of bead method antagonist 0 90 90 0.5 88 88 1 86 86 2 75 84 5 62 * 81 10 58 * 71 20 45 * 62 * 50 45 * 55 * No antagonist 100 100 ──────────────────── ─────────────── * indicates that the suppression rate was significantly enhanced by the t-test compared to the group with an addition amount of 0 g / m ² (P <0.05).

[Brief description of drawings]

FIG. 1 is a schematic diagram illustrating a method for measuring a microbial antagonistic force by a sod cup test.

Claims (3)

(57) [Claims] 1. A lawn planting area, characterized in that an antagonistic bacterium against a pathogen of turfgrass snow rot and powder of sodium alginate as an auxiliary agent are sprayed as they are in an amount of 5 to 100 g / m ² . How to control turfgrass snow rot. 2. The antagonistic bacterium is Pseudomonasflu
The method of controlling turfgrass snow rot according to claim 1, which is Orescens. 3. The antagonistic bacterium is Pseudomonasflu
orescens A11 strain (FERMP-14269)
The method for controlling turfgrass snow rot according to claim 1 or 2.