JPH0617291B2 - Soil disease control method for solanaceous plants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] Tobacco wilt and solanaceous wilt, which have caused great damage as soil-borne diseases of Solanaceae plants, are known as Pseudomonas solanarum.
cearum) is a disease that is difficult to control due to infection and parasitism of bacteria in plants.

The present invention relates to a method for controlling tobacco wilt and non-tobacco Solanaceae plants, for example, wilt disease of Solanaceae plants such as eggplant, tomato, potato and pepper.

[Prior Art] As a method for controlling tobacco wilt and bacterial wilt disease, soil sterilization using a soil disinfectant such as chloropicrin or methyl bromide has been widely practiced. However, the soil sterilization method using such a pesticide has a drawback that it sterilizes indiscriminately the beneficial microorganisms that live in the soil and turns the cultivated soil into "dead soil". Furthermore, the use of these pesticides may cause harmful effects on the natural environment, such as accumulation of harmful halides in soil.

Therefore, as a method for controlling soil-borne diseases of Solanaceae plants in place of the above-mentioned soil sterilizing and disinfecting agents, the present inventors have previously conducted non-pathogenic Pseudomonas solanasiarum M4S strain (Microtechnology Research Institute No. 700). ) Is inoculated into the root of a Solanaceae plant (International application published under the Patent Cooperation Treaty, International Publication No. WO85 / 03519) A non-pathogenic Pseudomonas solanacearum M4S strain live strain is a Solanaceae plant Of the bacteriophage that lyses the fungus and the pathogenic Pseudomonas solanacearum to the root of a solanaceous plant (Japanese Patent Application No. 60-261354). Regarding a method of immobilizing a live bacterium of the same strain using a polymer substance and applying the obtained immobilization product to soil (Japanese Patent Application No. 60-288013), etc. Performs invention is where the patent application.

[Problems to be Solved by the Invention] The method described in the above-mentioned prior art, that is, the method of inoculating a root of a solanaceous plant with a live bacterium of Pseudomonas solanaciarum M4S strain has a high effect of suppressing disease in the field. No, especially its sustainability. Then, two methods applied as improved methods thereof, namely, following the root inoculation of the live bacterium described above, a bacteriophage growth solution that lyses both the bacterium and pathogenic Pseudomonas solanacearum was applied to the root. The method for applying, and the above-mentioned method of immobilizing the viable bacterium using a polymer substance and applying the obtained immobilization product to the soil are all controlled in order to be put to practical use in a wide-area cultivation field. It is necessary to further enhance the effect.

[Means for Solving the Problems] The present invention is an improvement of the method of the invention described in the above-mentioned prior art, and the purpose thereof is to achieve the superior control effect of tobacco wilt disease and solanaceous plant blue. A method of controlling blight is provided. That is, the present invention was obtained by immobilizing a viable bacterium of a non-pathogenic bacterium, Pseudomonas solanaciarum M4S, and a bacteriophage that lyses both the bacterium and Pseudomonas solanaciarum, which is a pathogenic bacterium. A method for controlling tobacco wilt disease and solanaceous plant wilt disease, which comprises applying an immobilized product to soil.

The non-pathogenic bacterium used in the method of the present invention, Pseudomonas solanaciarum M4S strain (hereinafter abbreviated as M4S bacterium), is 1983 12
It has been deposited with the Institute of Microbial Technology, Institute of Industrial Technology, on March 14th. This was obtained by a sudden transition from a strain of Pseudomonas solanasiarum U-7 known as a bacterial wilt of tobacco, which grows well in competition with pathogenic allogeneic bacteria and inhibits the growth of pathogenic bacteria. In addition, it is a known strain having the characteristic that it has no pathogenicity to Solanaceae plants.

The method of culturing M4S bacteria is, for example, a known CPG medium (casamino acid 1 g, glucose 10 g, peptone 10 g, water 1000).
) And cultivate with shaking at 28-30 ° C for 48 hours. The culture broth thus obtained is harvested with a centrifuge to obtain wet cells.

In addition, the bacteriophage (hereinafter, abbreviated as "phage") used in the present invention is obtained by applying the method described in Microbiology Experimental Method (Microbial Research Method Round-table Conference, published by Kodansha Scientific in 1975). Can be separated. That is, for example, the stem of a tobacco infected with tobacco wilt is cut into narrow sides with a side of 1 cm or less, and 10 g of the stem is cut into 100
Suspend in m sterile water and shake for 1 hour. After that, the supernatant is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is passed through a Millipore filter (0.45 μm). Then liquid 0.
1 m is inoculated into 50 m of a known CPG medium containing pathogenic Pseudomonas solanaciarum and shake-cultured for 48 hours.
The obtained culture broth was centrifuged, the supernatant was passed through a Millipore filter, and then serially diluted, and 0.4 m of the diluted broth was used as a CPG containing pathogenic Pseudomonas solanacearum bacterium.
Suspend in 3 m of agar medium, pour this into a dish with a diameter of 9 cm, and culture at 30 ° C. for 24 hours. The phage is separated from the lytic plaque formed on the shearle. The isolated phage strain is suspended in 3 m of CPG agar medium containing M4S bacterium, poured into a dish having a diameter of 9 cm, cultivated at 30 ° C. for 24 hours, and the phage is separated again from the lytic plaque formed here. The isolated phage has the property of lysing both the M4S bacterium and the pathogenic Pseudomonas solanaciarum bacterium.

Mass culture of phage is similar to that of M4S bacteria. That is, shaking culture was performed at 28 to 30 ° C. for 36 to 72 hours in a liquid medium in which phages were inoculated simultaneously with M4S bacteria. The obtained culture solution is centrifuged at high speed (for example, 15,000 rpm),
The supernatant is used as a phage solution. In the immobilization described below, the number of phages per 10 m was 10 ⁶
Adjust to ~ 10 ⁹ and use.

Next, both the wet cells of M4S and the phage solution are immobilized using a polymer substance or its monomer. The method for immobilizing cells and phages may be a method known as a comprehensive method (“enzyme engineering”, edited by Saburo Fukui et al., P157-202).
That is, starch, which is a natural substance, and its derivatives,
Konnyaku flour and its purified products, aginic acid and its salts, gelatin, or polymeric substances such as algae-derived polysaccharide substances such as carrageenan are made into a sol, and the wet cells of M4S and phage liquid are added to form a gel. You can do it.
In addition, a monomer such as polyacrylamide or acrylamido acid copolymer may be added to the wet cells of M4S and the phage solution to cause gelation. In addition, polyvinyl alcohol,
A method in which a prepolymer such as a photocurable resin is added to wet cells of M4S bacteria and a phage solution and gelled to immobilize the prepolymer is also possible. Per one grain of the obtained immobilized product (average grain size: 2
The number of live M4S bacteria contained in 3 mm) is 1 × 10 ⁵
As described above, the number of living phages needs to be 1 × 10 ⁷ or more, preferably 1 × 10 ⁴ or more, preferably 1 × 10 ⁶ or more.

By applying the thus obtained immobilization product of M4S bacterium and phage to the soil, it exerts an excellent control effect against soil diseases of Solanaceae plants. Although the application method is not particularly limited, it is applied by thoroughly mixing with about 2 soils per 10 m ² from the day of applying basic fertilizer or the day of forming ridges to this field (about 2 to 4 weeks during this time). 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 plants, that is, bacterial wilt disease and wilt disease of Solanaceae plants other than tobacco, is considered as follows.

That is, the M4S bacterium contained in the immobilization product applied to the soil before seedling transplantation penetrates into the plant body, and the action of the M4S bacterium induces the defense reaction of the plant itself. Then, M
The phage immobilized with 4S bacteria migrates into the plant while lysing the M4S bacteria with the growth of the M4S bacteria (the phage has fungal parasitism and it is experimentally confirmed that the phage migrates into the plant). Has been). This phage inhibits the growth of pathogenic Pseudomonas solanasiarum that later invades and suppresses the onset of disease.

The method of the present invention is characterized in that the immobilized product of the M4S bacterium and the phage is mixed into the soil before transplanting seedlings. Live M4S bacteria in soil not only sequentially invade new roots after transplantation and exert their control effect, but also the phages immobilized together with M4S bacteria are accompanied by the growth of M4S bacteria.
It is possible to migrate and distribute in the plant one after another, and exert a sustainable control effect. Thus, according to the method of the present invention, the control effect of soil diseases of solanaceous plants can be sustained, and the control effect in this field can be further enhanced.

Next, a description will be given of production examples of immobilized products of M4S bacteria and phages used for carrying out the method of the present invention and examples of field tests.

[Production Example] Production Example 1 M4S bacteria was inoculated into a liquid medium containing casamino acid 0.1%, glucose 1%, and peptone 1%, and cultured at 30 ° C for 40 hours.
The culture broth was collected using a centrifuge to obtain wet bacterial cells. A liquid medium containing 0.1% casamino acid, 1% glucose and 1% peptone was inoculated with M4S bacteria and phage at the same time and cultured at 30 ° C for 60 hours. This culture solution was centrifuged, and the supernatant was passed through a Millipore filter (0.45 μm) to obtain a phage solution.

A bacterial cell suspension obtained by suspending 10 g of wet bacterial cells in 1 of demineralized water was mixed with 0.2 of phage solution and 3% of a 3% sodium alginate aqueous solution to prepare a sodium alginate suspension. This was added dropwise to a 1.5% aqueous solution of calcium chloride to obtain spherical M4S bacteria and phage immobilized with calcium alginate having a diameter of 2 to 3 mm.

The resulting immobilizate contained approximately 1 × 10 ⁶ M4S bacteria / grain and 1 × 10 ⁴ phage / grain.

Production Example 2 M4 was added to a liquid medium containing 1% yeast extract and 1.5% glucose.
S strain was inoculated and cultured at 30 ° C. for 48 hours. The culture broth was collected using a centrifuge to obtain wet bacterial cells. Further, a liquid medium containing 1% yeast extract and 1.5% glucose was simultaneously inoculated with M4S bacteria and phage, and cultured at 30 ° C. for 66 hours. The culture solution was centrifuged to obtain a phage solution as the supernatant.

A sterile cell suspension of 150 m was prepared by using sterilized water for 1 g of wet cells. On the other hand, 18.7% acrylamide (96% acrylamide and 4% N, N'-melene bisacrylamide) aqueous solution 750m, 0.23% N, N, N ', N'-tetramethylethylenediamine aqueous solution 250m and 0.28% excess 500 m of an ammonium sulfate aqueous solution was prepared. When these three types of aqueous solutions were mixed with the bacterial cell suspension and 50 m of the phage solution and then allowed to stand, the mixed solution solidified. This solidified material was made into a cube of 2 to 3 mm by using a mesh, and M4S
Immobilized products of fungi and phages were obtained.

The resulting immobilized product contains approximately 1 × 10 ⁶ M4S / grain.
It contained bacteria and 1 × 10 ⁵ phage / grain.

[Examples] Example 1 In order to compare superiority and inferiority of the control effect between the control method of the present invention and the conventional method described above, under the same conditions of tobacco cultivation such as variety, soil, time, and weather fertilization management. The following test was conducted.

The test uses Hakuenshu No. 1 as a tobacco plant, and one side is 2
Seedlings cultivated in a 8.5 cm vinyl chloride resin pot (25 plants) were tested. In the nursery, 6 weeks after sowing, seedlings that had grown to 8-9 leaves / row were transplanted to this field.

This field was set in a tobacco wilt disease-contaminated area in the Utsunomiya Experimental Station of Japan Tobacco Inc., and the test was carried out by dividing it into the following five test areas. The number of transplanted seedlings per ward is 18
It was set to 0.

(1) Soil application section of M4S bacterium and phage immobilized product (method of the present invention) (2) Seedling treatment section of M4S bacterium and phage (Japanese Patent Application No. 60)
-261354) (3) Soil application section of immobilized M4S bacteria (Japanese Patent Application No. 60-2
(Method of No. 88013) (4) Seedling treatment section of M4S bacterium (International Publication WO85 / 035
Method 19) (5) Untreated plot (1) The soil application plot of the M4S bacterium and the phage-immobilized product of the present invention was treated as follows. That is, the immobilization product prepared in the above Production Example 1 was mixed into compost at the time of basal fertilization 2 weeks before the transplantation of tobacco seedlings, and applied to a tobacco field so that the amount was 2.5 per 10 m ² . Transplantation of tobacco seedlings followed the practice.

(2) Seedling treatment section of M4S bacteria and phage (Japanese Patent Application No. 60)
No. 2631354) was processed as follows.
That is, a sterilized water was used to prepare a M4S bacterium suspension so that the concentration of the viable M4S bacterium was 1 × 10 ¹⁰ cells / m 2,
This was placed in a seedling treatment tank so that the depth was 2 cm. Tobacco seedlings 5 days before transplantation were dipped in the pot for 1 hour.
On the day before transplantation, the seedlings were subjected to phage solution (1 x 10 ⁸ cells /
m) was sprayed at 100 m per strain.
Transplantation of tobacco seedlings followed the practice.

(3) Soil application area of immobilized M4S bacteria (Japanese Patent Application No. 60-2
The method of No. 88013) was processed as follows. That is, among the methods described in (1) above, M4S bacterium-immobilized product (M4S) prepared in the same manner without mixing phages.
Bacteria: 1 × 10 ⁶ cells / grain) was mixed into the compost at the time of basic fertilization 2 weeks before the transplantation of tobacco seedlings, and applied to the tobacco field so that the amount was 2.5 per 10 m ² . Transplantation of tobacco seedlings followed the practice.

(4) Seedling treatment area of M4S bacteria (International Publication WO85 / 035
Method 19) was processed as follows. That is, M4
Prepare a suspension of M4S bacteria by using sterilized water so that the concentration of viable S bacteria is 1 × 10 ¹⁰ cells / m, and make a depth of 2 cm.
The seedling treatment tank was placed so that Tobacco seedlings 5 days before transplantation were dipped in the pot for 1 hour. Transplantation of tobacco seedlings followed the practice.

(5) The untreated plot was used as a control plot, and no treatment was performed, and tobacco seedlings were transplanted according to a customary practice.

After transplanting the seedlings into this field, the disease state of tobacco wilt was investigated. For the degree of disease, the average morbidity index was calculated using the index shown below, and the control rate was calculated from this.

Index 0: Healthy 1: 1-2 lower leaves withered 3: Half leaves withered 5: All leaves yellowed and withered 10: Withered Table 83 shows the results of the tobacco seedling 83 and 103 days after transplantation.
1 and Table-2.

Example 2 The occurrence of tomato wilt was examined using the immobilization product prepared in the above Production Example 2. That is, the soil to which the immobilization product was added at a ratio of 1/50 was added to the black soil containing the pathogenic Pseudomonas solanacearum bacterium so that the number of bacteria was 5 × 10 ³ per 1 g of dry soil. In a pot, 28 days after sowing, tomatoes (Fukuju 100
No.) seedlings were transplanted 25 per plant. As a control, black soil with only pathogenic Pseudomonas solanasiarum added was put in a pot, and similarly, 25 tomato seedlings were transplanted per pot.

65 days after the transplantation of the seedlings, the occurrence status of tomato wilt was investigated. The disease severity and the control rate were determined in the same manner as in Example 1.
The results are shown in Table-3. However, the test plots were (1) soil application plots of M4S bacteria and phage-immobilized product (method of the present invention) and (2) control plots (untreated).

[Effect of the Invention] As is clear from the examples, the method of the present invention makes it possible to further enhance the effect of controlling soil diseases of solanaceous plants.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12N 1/20 C12R 1:38) (56) Reference JP-A-53-79027 (JP, A) JP 59-62509 (JP, A) JP-A-60-180589 (JP, A)

Claims (2)

[Claims] 1. A Pseudomonas solanaciarum M isolated from a plant infected with Pseudomonas solanaciarum.
A method for controlling soil diseases of solanaceous plants, which comprises using a bacteriophage having the ability to lyse 4S bacterium (FERM BP-700) and Pseudomonas solanaciarum and an immobilized product of Pseudomonas solanaciarum M4S bacterium. 2. The method for controlling soil diseases of Solanaceae according to claim 1, wherein the soil diseases of Solanaceae are tobacco wilt or wilt of Solanaceae.