JPS6322005A - Control of soil blight of solanaceae family plant - Google Patents

Abstract

PURPOSE:To control soil blight, by applying an immobilized material produced by immobilizing living cells of nonpathogenic Pseudomonas solanacearum M4S strain in combination with a bacteriophage capable of lysing said strain and pathogenic Pseudomonas solanacearum. CONSTITUTION:(A) Living cells of Pseudomonas solanacearum M4S strain (FERM P-7370) which is a nonpathogenic bacterium and (B) a bacteriophage capable of lysing said strain and pathogenic Pseudomonas solanacearum are immobilized e.g. with a polymeric substance or its monomer and the obtained immobilized product is applied to soil to control bacterial wilt of tobacco and Solanaceae vegetables. The present method is characterized by the application of immobilized M4S cell and phage to soil before the transplantation of seedling and exhibits long-acting controlling effect.

Description

[Detailed Description of the Invention] [Industrial Application Fields Tobacco damping off and bacterial wilt of Solanaceae plants, which cause great damage as soil-borne diseases of Solanaceae plants, have the scientific name of Pseudomonas solanacearum (Pseudomonas solanaceae).
This is a difficult-to-control disease caused by the infection and parasitism of G5T3 (as solanacearum) into the plant body.

The present invention relates to a method for controlling tobacco damping-off and bacterial wilt of solanaceous plants other than tobacco, such as eggplants, tomatoes, potatoes, and green peppers.

[Prior Art] Soil sterilization using soil disinfectants such as chloropicrin and methyl bromide has been widely practiced as a method for controlling tobacco damping-off and solanaceous plant bacterial wilt. However, such soil sterilization methods using pesticides have the disadvantage that they indiscriminately sterilize even the beneficial microorganisms that live in the soil, turning the cultivation soil into "dead soil." Furthermore, the use of these pesticides may have harmful effects on the natural environment, such as the accumulation of harmful halides in the soil.

Therefore, the present inventors first developed a method for controlling soil-borne diseases of Solanaceae plants in place of the above-mentioned soil sterilizing and disinfecting agents. Method of inoculating live bacteria of a non-pathogenic Pseudomonas solanacearum M4S concave strain into the roots of a Solanaceae plant (International application published under the Patent Cooperation Treaty, International Publication No. wO8). 4 Inoculate the roots of the plants, and then add a bacteriophage growth solution that lyses the original bacteria and pathogenic Pseudomonas solanacearum.
Method of spraying on the roots of Solanaceae plants (Patent application 1986-2)
No. 61354〉 ■ Inventions regarding methods for improving the above (■), such as a method of immobilizing living bacteria of the same strain using a polymeric substance and applying the resulting immobilized product to soil (Japanese Patent Application No. 60-288013). We have just completed the process and filed a patent application.

[Problems to be solved by the invention] The method described in the above-mentioned prior art
-1 The method of inoculating the roots of plants is not highly effective in suppressing the onset of disease in the field, and there are problems with its sustainability in particular. Following the root inoculation of live bacteria, a bacteriophage propagation solution that dissolves both the original bacteria and pathogenic Pseudomonas solanacearum is sprayed onto the roots, and the method described in (1) above involves inoculating the live bacteria with a polymeric substance. In order for any of the methods of immobilizing the product and applying the obtained immobilized product to the soil to be put to practical use in a wide range of cultivation fields, it is necessary to further enhance the control effect 3.

[Means for Solving the Problems] The present invention is an improvement on the prior art method described above ((ii)), and its purpose is to control tobacco damping-off disease and An object of the present invention is to provide a method for controlling bacterial wilt of solanaceous plants.That is, the present invention provides a method for controlling bacterial wilt of solanaceous plants.That is, the present invention provides a method for controlling bacterial wilt of solanaceous plants.
This is a method for controlling tobacco damping-off and solanaceous plant bacterial wilt, which is characterized by applying to soil an immobilized product obtained by immobilizing a bacteriophage that lyses both Solanaceaeum.

The Pseudomonas solanacearum M4S strain (hereinafter abbreviated as M4S seedlings), which is a non-pathogenic seedling used in the method of the present invention, is published in 1988 as FAIKEN BIKIYO No. 7370.
It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on December 14, 2003. This was obtained by sudden transition from the Pseudomonas solanacearum u-7 strain, which is known as the tobacco blight-causing bacterium, and grows well in competition with pathogenic homologous bacteria. inhibits
Furthermore, it is a known strain that is completely non-pathogenic to eggplant f4 seeds.

The method for culturing M4S bacteria is, for example, using a known CPG medium (1 g of Casamino H, 10 g of glucose, 10 g of peptone,
The culture was inoculated into 1,000 ml of water and cultured at 28-30°C with shaking around 48 o'clock.The culture solution thus obtained was centrifuged and collected using a vessel to obtain wet bacterial cells.

In addition, the cteriophages (hereinafter abbreviated as phages) used in this invention are used in the method described in Microbiology Experimental Methods (edited by the Microbial Research Methods Council, published by Kodansha Scientific in 1975). can be separated by applying That is, for example, a tobacco stem stained with tobacco damping-off disease is cut into pieces of 1 cm or less on each side, and the Lo
g was suspended in Looml bactericidal solution and shaken for 1 hour. Thereafter, the supernatant was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was filtered through a Millipore filter (0.45 μm).
Send one at

Next, 0.1 mt of the fermentation liquid was inoculated into SOml of a known CPG medium containing pathogenic Pseudomonas solanacearum, and cultured with shaking for 48 hours. The obtained culture P solution was centrifuged, and the supernatant was filtered through a Millipore filter, serially diluted, and 0.4 ml of the diluted solution was added to 3 CPG agar plates containing pathogenic Pseudomonas solanacearum bacteria. Pour into a petri dish with a diameter of 9 cm, and heat at 30°C.
There will be a 24-hour recuperation. Phage are isolated from lytic plaques formed on a petri dish.

The isolated phage strain was suspended in 3 liters of CPG agar medium containing M4S bacteria, poured into a petri dish with a diameter of 9 cm, and cultured at 30°C for 24 hours. Aliquot the phages again. The isolated phage has the property of lysing both M2S seedlings and pathogenic Pseudomonas solanacearum bacteria.

Mass culture of phages is similar to the culture of M4S bacteria.

That is, in a liquid medium inoculated with M4S bacteria and phages at the same time, the cells are cultured with shaking at 28 to 30°C for 36 to 72 hours.

The obtained culture solution is high 3! ! (For example, 15,000 rotations/
The heart is separated at a rotational speed of 1 minute, and the supernatant is used as a phage solution. In actual immobilization described below, the number of phages is set to Lm! The number is adjusted to 1011 to 109 and used.

Next, the method of immobilizing ocahedrons and phage, in which both the wet cells of M4S bacteria and the phage liquid are immobilized using a polymeric substance or its monomer, is a method known as the entrapment method (
"Enzyme Engineering", Fukui Mibe et al., ed., p. 157-202), i.e. natural substances such as starch and its derivatives, konjac flour and its purified products, alginic acid and its salts, gelatin, or algae-derived materials such as carrageenan. It is sufficient to make a polymeric substance such as a polysaccharide substance into a sol and gel it by adding wet bacterial cells of M4S bacteria and phage solution.Alternatively, monomers such as polyacrylamide or acrylamide acid copolymer can be mixed with wet bacterial cells of M4S bacteria. It is also possible to add a prepolymer such as polyvinyl alcohol or a photocurable resin to the phage solution and gel it to immobilize it.

Per particle of the obtained immobilized product (average particle size: 2 to 3 mm
) The number of live M4SN bacteria contained in
The number of living phages needs to be 1 x 104 or more, preferably I x 10' or more.

By applying the thus obtained immobilized M4S bacteria and phage to soil, it exhibits an excellent control effect against soil diseases of Solanaceae plants.

The application method is not particularly limited, but from the day when basal fertilizer is applied in the home country or the day when furrows are formed, transplanted to the field (during this period, approximately 2 to
4 weeks), mix well with the soil and apply at a rate of about 21 ml per 10 hols. It can be mixed with compost and other fertilizers.

[Operation of the Invention] The mechanism by which the method of the present invention controls soil diseases of Solanaceae, namely tobacco damping-off and bacterial wilt of Solanaceae plants other than tobacco, is considered as follows.

That is, the M4S bacteria contained in the immobilized material applied to the soil before seedling transplantation invades the plant body, and the action of the M4S bacteria induces the plant's own defense response. Next, M
4 The phages immobilized with the S bacteria migrate into the plant body while lysing the M4S bacteria as the M4S bacteria proliferate (phages have mycoparasitic properties, and it has been experimentally proven that phages migrate into the plant body). (confirmed), this phage inhibits the growth of pathogenic Pseudomonas solanacearum that invades later, suppressing the onset of disease.

The method of the present invention is characterized in that an immobilized product of M4S bacteria and phages is mixed into soil before transplanting seedlings. Living M4S bacteria in the soil not only invades the newly elongated roots after transplantation and exerts a control effect, but also λff43.
As the M4S bacteria proliferate, the immobilized phages can also be sequentially transferred and distributed within the plant body.
Demonstrates a sustained pest control effect. Thus, by the method of the present invention, the effect of controlling soil diseases of Solanaceae plants is sustainable, and the control effect in the field can be further enhanced.

Next, examples of production of immobilized products of M 4 S':3 and phages used in carrying out the method of the present invention and examples of field tests will be described.

[Production Example] Production Example 1 M4S bacteria was inoculated into a liquid medium containing 0.1% casamino acid, 1% glucose, and 1% peptone, and cultured at 30°C for 40 hours. This culture solution was collected using a centrifuge to obtain wet bacterial cells. In addition, casamino acid 0-1%, glucose 1%,
M4S bacteria and phage were simultaneously inoculated into a liquid medium containing 1% peptone and cultured at 30"C for 60 hours. This culture solution was centrifuged, and the supernatant was filtered through a Millipore filter (0
, 45 μm)? A phage solution was obtained.

A bacterial cell suspension obtained by suspending wet bacterial cell Log in 11 demineralized water and 31 ml of a 3% sodium alginate aqueous solution was mixed with 0.21 phage solution to prepare a sodium alginate suspension. . By dropping this into a 145% calcium chloride aqueous solution, a spherical M4S bacterium and phage immobilized with calcium alginate with a diameter of 2 to 3 mm was obtained.

The obtained immobilized product contained approximately 1×106 M4S particles/grain.
and contained 1×104 phages/grain.

Production example 2 M in a liquid medium containing 1% yeast extract and 1.5% glucose
Inoculate the 4S bacteria and incubate at 30°C for 48 hours.

The culture solution was collected using a centrifuge to obtain wet bacterial cells. Furthermore, M4S bacteria and phage were simultaneously inoculated into a liquid medium containing 1% yeast extract and 1.5% glucose, and cultured at 30°C for 66 hours. The culture solution was centrifuged, and a phage solution was obtained as the supernatant.

A 150 mt bacterial cell suspension was prepared using sterilized water per 1 g of wet bacterial cells. On the other hand, 18.7% acrylamide (contains 96% acrylamide and 4% N,N'-methylenebisacrylamide) water solution 1750m! , 0.23%
250 ml of N, N, N', N'-tetramethylethylenediamine aqueous solution and 500 ml of 0.28% ammonium persulfate aqueous solution were each prepared. Aqueous solution of these three types, bacterial cell suspension and phage solution 50
When the mixture was left to stand after mixing ml, the mixture solidified.

This solidified product was shaped into 2 to 3 [ff11] cubes using a mesh to obtain an immobilized product of M4S bacteria and phages.

The obtained fixed grains contained approximately 1 × 106 pieces/grain of M
It contained 4S bacteria and 1×105 phages/grain.

[Example] Example 1 To compare the control effects of the control method of the present invention and the conventional method of the invention using the same tobacco cultivation conditions such as variety, soil, season, weather, and fertilizer management. , conducted the following tests.

The test was conducted using Hakuenshu No. 1 as a tobacco plant, with 2 sides on each side.
8.5cm PVC resin pot (25 plants)
The seedlings grown in were tested. At the nursery, 6 weeks after sowing, seedlings that had grown to 8 to 9 leaves per plant were transplanted to their home country.

In Japan, the test was conducted in the Tobacco Blight-contaminated area within the Utsunomiya Testing Station of Japan Tobacco Inc., which was divided into the following five test areas. The number of transplanted seedlings per area is 1.
There were 80 pieces.

(1) M4Si and phage immobilized soil application area (
Method of the present invention) (2>M43i and phage seedling treatment area (Patent application 1986)
0-261354 method) (3) M4S seedling immobilized product soil application area (patent application No. 6
9 Method of No. 288013) (4) M2S concave seedling treatment area (International Publication WO 85/○3
Method of No. 519) (5) Untreated area (1) The area where the M 4 S 9 and phage immobilized product of the present invention were applied to soil was treated as follows. That is, the immobilized product prepared in Production Example 1 was mixed into compost when adding basal fertilizer two weeks before transplanting tobacco seedlings, and applied to a tobacco field at a concentration of 2.51 per 10 i. Transplantation of tobacco seedlings followed conventional practices.

(2> M4S bacteria and phage treatment area (FF
The method of Application No. 60-261354) was processed as follows. In other words, the live bacteria and concentration of M4S bacteria are I x 1
A suspension of M4S bacteria was prepared using sterilized water at a concentration of 0" cells/mj, and placed in a seedling treatment tank at a depth of 20 Tl. To this, tobacco seedlings 5 days before transplanting were placed in pots. The seedlings were left open for 1 hour and soaked.Furthermore, on the day before transplantation, phage solution (IXIO" pieces/m) was sprayed onto the seedlings at a distance of 100 m (100 m) per plant.

Transplantation of tobacco seedlings followed conventional practices.

(3) M 4300 immobilized product soil application area (patent application 1986)
-288013 method) was processed as follows.

That is, among the methods described in (1) above, λ 14S bacteria immobilized product (containing 4S bacteria: 1 x 10 6 cells/grain) prepared in the same manner without phage contamination ÷ % l (containing 4S bacteria: 1 x 106 cells/grain) 3 tobacco seedlings transplanted 2
Mixed with compost when adding basal fertilizer a week ago, 2 per 10 years
, 5j was applied to tobacco fields. Transplantation of tobacco seedlings followed conventional practices.

(4) Seedling treatment area for M4S bacteria (International Publication WO351035
Method No. 19) was processed as follows.

That is, I x 1010 live M4S bacteria/m
A suspension of M4S bacteria was prepared using sterilized water to a concentration of 1 ml, and this was placed in a seedling treatment tank to a depth of 2 g. Move on to this! Five-day-old tobacco seedlings were soaked in the pot for 1 hour. Transplantation of tobacco seedlings followed conventional practices.

(5) The untreated plot served as a control plot, and tobacco seedlings were transplanted according to conventional practices without any treatment.

After the seedlings were transplanted to the main field, the disease state of tobacco damping-off disease was investigated, and the average disease index was determined using the index shown below, and the control rate was calculated from this.

Index: 0: Healthy 1: One or two lower leaves are withered 3: Half of the leaves are withered 5: All leaves are yellowed and wilted 10: Blight control rate (%) The results of the survey 83 days and 103 days after transplanting tobacco seedlings It is shown in Table-1 and Table-2.

Table 1 Table 2 Example 2 Using the immobilized product prepared in Production Example 2, the occurrence of tomato bacterial wilt was investigated. In other words, 50% of the pathogenic Pseudomonas solanacearum was added to black soil so that the number of bacteria was 5x10' per 1g of dry soil.
Soil to which the immobilized material had been added at a ratio of 1:1 was placed in pots, and on the same day, 25 tomato seedlings (Fukui No. 100), 28 days after sowing, were transplanted into the pots, one seedling per pot. As a control, black soil containing only pathogenic Pseudomonas solanacearum seedlings was placed in pots, and 25 tomato seedlings were similarly transplanted, one per pot.

Sixty-five days after transplanting the seedlings, the incidence of tomato bacterial wilt disease was investigated, and the control rate was determined in the same manner as in Example 1.

The results are shown in Table-3. However, the test plots are (1) M4S bacteria and phage immobilized soil application plot (
method of the present invention) (2) Control group (no treatment).

Table 3 [Effects of the Invention] As is clear from the Examples, the method of the present invention makes it possible to further improve the effect of controlling soil diseases of Solanaceae plants.

Claims (1)

[Claims] Tobacco damping-off disease characterized by applying to the soil an immobilized product obtained by immobilizing a viable Pseudomonas solanacearum M4S strain and a bacteriophage that lyses both the bacteria and pathogenic Pseudomonas solanacearum. and methods for controlling bacterial wilt of solanaceous plants.