JP2003231606A - Method for controlling blight damage to plant of family brassicaceae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new control method which has high control effect on yellowing blight of Chinese cabbage being a blight damage to a plant of the family Brassicaceae and has no environmental pollution. <P>SOLUTION: A control agent comprising Pseudomonas vietnamiensis as active ingredient is used. Especially CGF4153 strain, CGF4164 strain and CGF5100 strain belonging to Pseudomonas vietnamiensis exhibit high control effect. The strains are immobilized by using a saccharide to give a formulation having especially high stability. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for controlling Verticillium disease of cruciferous plants using a bacterium belonging to the scientific name Pseudomonas vietnamiensis.

[0002]

2. Description of the Related Art Verticillium disease (pathogenic fungus; verticilli), which is a major soil disease of cruciferous plant diseases
um dahliae) is a soil fungal disease that occurs during the growth of cruciferous plants such as Chinese cabbage. Chinese cabbage yellowing disease
It occurs particularly frequently, hindering the stable production of Chinese cabbage. Symptoms include yellowing of leaves and poor growth during harvest, which may cause death. In addition, pathogenic bacteria
Individuals that have entered the middle ribs may lose leaves in the market and lose their commercial value significantly.

At present, several chemical agents are used as soil disinfectants for controlling these diseases. But,
These chemical agents include environmental issues, safety issues for users and neighboring residents, and reduction of pesticides in recent years.
There is a problem that it does not match the pesticide-free orientation.

Therefore, for the control of this disease, it has been desired to develop a control agent having a high control effect, no environmental pollution such as water pollution, and satisfying safety.

[0005]

An object of the present invention is to provide a method for controlling cruciferous plant diseases, which has a high control effect against Chinese cabbage yellowing disease, which is a cruciferous plant disease, and which has no environmental pollution. .

[0006]

Means for Solving the Problems The inventors of the present invention have made extensive studies to solve the above problems. As a result, microorganisms belonging to Pseudomonas vetonamiensis, especially CG
F4153 strain, CGF4164 strain, CGF5100
We have found that a formulation containing a strain as an active ingredient has a high control effect against verticillium diseases of Brassicaceae plants including yellow cabbage yellowing disease, and found that the above problems can be solved. Further, the present inventors have found the fact that a controlling agent having particularly high stability can be obtained by immobilizing these microorganisms on a carrier using a saccharide, and have completed the present invention.

Up to now, as a biological control using Pseudomonas vietnamiensis, it has been reported that control of the foot blight of legumes caused by Pishum has been carried out (Phytopathology 88, No. 9, S).
uppl., S46-S47, 1998), but there has been no control of verticillium disease in cruciferous plants.

[0008] The applicant of the present invention has found that Pseudomonas genus Vetonamiensis, in particular CGF4153 strain belonging to this strain, CG
The F4164 strain and the CGF5100 strain were found to have a high control effect against rice seed disease, which was disclosed as JP-A-2002-17343.

The present inventors have found that Pseudomona genus Pseudomona including these CGF4153 strain, CGF4164 strain, and CGF5100 strain.
s vietnamiensis) has a high antagonism against verticillium diseases of cruciferous plants such as Chinese cabbage yellowing disease and has an extremely strong control effect against these diseases. Is.

That is, the present invention is characterized by using a control agent containing Pseudomonas vietnamiensis as an active ingredient, and in particular, Pseudomonas vietnamiensis is CGF.
4153 strain, CGF4164 strain, CGF5100 strain, and these Pseudomonas vetonamiensis is a control agent immobilized on a carrier using saccharides, including Chinese cabbage The present invention provides a method for controlling diseases of cruciferous plants.

The present invention will be described in detail below. Microorganisms used in the present invention include Pseudomonas vetonamiensis, particularly CGF4153 strain, CGF4164 strain,
CGF5100 strain.

Pseudomonas vitonamiensis CGF
The 4153 strain, CGF4164 strain, and CGF5100 strain are selected from bacteria of about 7,000 strains isolated and collected from rice and vegetables, antibacterial activity against rice scab (Gibberella fujikuroi), rice seedling blast (Pyricularia oryzae), and selection by a seedling raising test. It is the strain obtained as a result. The selection method is as described in JP-A-2002-17343, and the outline thereof will be described below.

After washing the roots of the plants collected from the field with tap water, the roots were finely cut, put in sterilized water, and crushed with a mixer. The crushed liquid was appropriately diluted, applied to broth agar medium, and cultured. The colonies that appeared there were isolated and stored, and used as the test strain.

[0014] The antibacterial activity against rice scab seedling fungus and rice seedling blast fungus can be measured by Gibberella fuji
kuroi) or rice seedling blast fungus (Pyricularia oryzae) and the test strain were subjected to confrontation culture on PDA medium (potato dextrose medium). Culture is 25 ℃
I went there for a week.

Furthermore, a control test was carried out on rice sprouting seedlings for the strains having antibacterial activity obtained by the confrontation culture. The method was as follows: Dipping of 10 ⁸ cfu / ml diluted strain of the test strain with rice scab seedling disease-carrying rice was soaked for 24 hours at the time of germination, and after sowing, seedlings were raised for 3 weeks to investigate disease occurrence. as a result,
A strain that suppresses the development of rice banana seedling disease was found.

[0016] Therefore, the control effect against the seedling blast disease was examined for the strains which suppress the development of these rice scab seedling diseases. First, the filter paper wet chamber method (Blotter
test). After keeping the rice blast-bearing paddy treated with the bacterial solution at 25 ° C. in a petri dish for 4 days, it was observed by observing the presence or absence of conidial spores of the blast fungus under a stereoscopic microscope and measuring the sporulated paddy rate. . Then, a seedling-raising test for seedling blast disease was carried out on the strains which were found to be effective in the test by the filter paper wet chamber method (Blottertest).

[0017] As a result, Pseudomonas vetonamiensis CGF4153 strain, CGF4164, which has a strong disease suppressive effect on rice scab disease and rice blast fungus.
Three strains, CGF5100 strain, were selected.

Pseudomonas vetonamiensis CGF
The 4153 strain, the CGF4164 strain, and the CGF5100 strain were rod-shaped bacteria having a cell size of 1 to 3 μm as a result of morphological observation with an optical microscope and an electron microscope, and all had motility. Was. The Gram reaction was negative and did not form endospores.

Other bacteriological properties are shown below. (1) Cultural properties CGF4153 strain, CGF4164 strain, CGF51
The growth state of strain 00 on broth agar medium is shown below. The observation was performed after culturing at 30 ° C. for 3 days.

The colony morphology of CGF4153 and CGF4164 is cream-colored, circular, smooth on all edges, low-convex, and glossy, and the colony morphology of CGF5100 is cream-colored, round, slightly wavy on all edges, fried, and glossy. . It does not produce water-soluble pigments. (2) General properties

[0021]

[Table 1]

Due to the above bacteriological properties, CGF415
3, CGF4164, CGF5100 are all Gram-negative bacilli having motility and positive for catalase activity,
The bacterium was classified as a bacterium belonging to the genus Pseudomonas vetonamiensis because it was positive for oxidase activity, did not form endospores, and had nitrogenase activity.

These strains have been deposited at the Institute of Biotechnology, Institute of Industrial Technology, and have the following deposit numbers.

[0024] Pseudomonas vietnamiensis CGF4153: FERM-17842 Pseudomonas vietnamiensis CGF4164: FERM-17843 Pseudomonas vietnamiensis CGF5100: FERM-17844 Pseudomonas vietnamiensis CGF5100: FERM-17844 Pseudomonas vietnamiensis CGF4100: FERM-17844 Pseudomonas vietnamiensis CGF4100: FERM-17844 Bacteria belonging to the genus Petnamiensis can be used.

Cultivation of these bacteria belonging to the genus Pseudomonas vietamiensis and formulation as a control agent can be carried out by a conventional method, which will be described below by way of example.

The medium for culturing the strain used in the present invention is not particularly limited as long as the bacterium grows. Any natural medium or synthetic medium can be used as long as it is a medium that appropriately contains a carbon source, a nitrogen source, and an inorganic substance that can grow. Specific examples include 802 medium, broth medium, King B medium, PS medium and PDB medium. After culturing and proliferating in the medium as described above at 15 to 42 ° C., preferably 28 ° C. to 35 ° C. for 10 to 35 hours, the cells are concentrated by a centrifuge or a membrane concentrator to collect the cells, and the medium components are collected. remove. By this operation, the cell concentration is usually concentrated to about 1 to 50 × 10 ¹⁰ cfu / ml (cfu; colony forming unit).

As the control agent in the present invention, the live bacterium thus cultivated may be used as it is, but it is preferable to mix it with a carrier to prepare a solid preparation because it can be easily used as an agricultural chemical. The solid preparation may be called a granule, a powder or a wettable powder depending on its particle size and properties, and the preparation used in the present invention may be any of these.

Examples of the method for producing the preparation include a method in which a protective agent is added to viable bacteria, which is dried in a vacuum to be solidified and then mixed with a carrier to prepare a powder or granules.
In order to maintain the viability of the bacterium, it is particularly preferable to pre-freeze the microbial cell mixed with the protective agent before vacuum-drying and to freeze-dry the microbial cell while it is frozen.

Examples of the protective agent added during the production of the preparation include a mixture of a saccharide, sodium glutamate and a sodium phosphate buffer. By adding a protective agent to the bacterial cells, a preparation having high storage stability can be obtained, and in particular, by adding a saccharide, storage stability can be particularly enhanced. There is no particular limitation on the type of sugar, but saccharose, fructose, glucose and sorbitol are preferable, and sucrose is particularly preferable.
One kind of sugar may be used, or two or more kinds of sugar may be used at the same time. There is no particular limitation on the amount of sugar added. The protective agent may be mixed with the bacterial cells in the form of an aqueous solution, or may be mixed with the bacterial cells as a solid.

Examples of the carrier include mineral powders such as talc, kaolin, clay, calcium carbonate and diatomaceous earth, peat moss, high molecular compounds such as polyvinyl alcohol, natural high molecular compounds such as xanthan gum and alginic acid. To be The concentration of bacterial cells in the solid preparation is
It is set to 10 ⁵ cfu / g or more, preferably 10 ⁷ cfu / g or more.

By using the control agent of the present invention in the following manner, for example, it is possible to obtain an effective control effect against verticillium diseases such as Chinese cabbage yellowing disease without the problem of chemical damage. The method of use is not necessarily limited, but is exemplified below.

In the production of Chinese cabbage, generally, seedlings are sown in a seedling raising tray, seedlings are raised for 3 to 5 weeks, and then planted in a field. The control agent of the present invention is used by mixing it with seedling-raising soil at the time of sowing, soaking seedlings before planting in a dilute solution of the formulation in water, or by using a combination of both treatments.

When the control agent of the present invention is used by mixing it with the nursery soil, 1 g or more of the formulation is mixed with 1 liter of soil and the mixture is stirred so as to be uniform. The concentration of the bacteria in the soil is adjusted to preferably 10 ⁶ cfu / g or more, more preferably 10 ⁷ cfu / g or more.

When the raised seedlings are dipped in the present control agent diluting solution before planting, the fungus concentration of the diluting solution is 10 ⁶ cf.
It is prepared by mixing the above-mentioned preparation with water so that the concentration is u / ml or more, preferably 10 ⁷ cfu / ml or more. As the treatment method, there are a method of immersing the seedlings in a bacterial solution, a method of mixing the cell culture dilution solution with the seedling-raising soil at the time of seeding, and a method of pouring the seedlings being raised, or a combination thereof. There is. 10 minutes to 1 when soaking seedlings
During the day, it is preferable to perform immersion treatment in the bacterial solution. Mixing with the soil at the time of sowing is preferably such that the fungal concentration in the soil is 10 ⁵ cfu / g or more, more preferably 10 ⁷ cfu / g or more. In the case of irrigation, 10 ⁶ cfu
It is preferable to use a bacterial solution of 10 ⁷ c / ml or more.
Fu / ml or more is more preferable.

[0035]

EXAMPLES Examples will be shown below, but the present invention is not limited to the following examples.

The composition of the medium used in the examples is shown below. Broth Medium: Meat Extract 3g, Peptone 10g, NaCl 15
g, water 1 L, pH 7.0 PDA medium (potato dextrose medium): potato exudate 200 g, glucose 20 g, water 1 L, pH 5.6 [Example 1] Control test for Chinese cabbage yellowing disease Chinese cabbage yellowing fungus (84013) ) And Chinese cabbage yellowing fungus (MAF
F-103056) and PD Pseudomonas vetonamiensis
The measurement was carried out by carrying out a confrontational culture on medium A and observing the presence or absence of a growth inhibition zone of Chinese cabbage yellow rot caused by the antibacterial substance produced by the test bacteria. The culture was carried out at 25 ° C for 3 weeks.

The results are shown in Table 2. All three strains of Pseudomonas genus Vetonamiensis CGF4153, CGF4164, and CGF5100 strongly inhibited the growth of Chinese cabbage yellow leaf disease bacteria on PDA medium.

[0038]

[Table 2]

[Example 2] Control test against Chinese cabbage yellowing disease (CGF4153 strain) Pseudomonas genus Vetonamiensis CGF4153 strain was cultivated in a broth liquid medium for 24 hours, and the obtained bacterial cells were separated by centrifugation and tested. The suspension was prepared. In this suspension,
Chinese cabbage seedlings (variety: Toshikushi) were dipped and left for 24 hours while dipped in the suspension. Then, it was transplanted to a 10.5 cm polypot filled with contaminated soil mixed with Chinese cabbage yellowing fungus (strain: 84013). After about 45 days, the presence or absence of disease was investigated and the control effect was determined. The results are shown in Table 3. The concentration of the suspension of Pseudomonas genus Petnamiensis CGF4153 strain was 1 × 10 ⁸ cfu / ml. As a result, a high control effect was observed for Pseudomonas vitnamiensis CGF4153. Chinese cabbage yellowing test was evaluated by calculating the degree of disease from the degree of disease on the root. Root disease index 0; healthy; 1; browning of vascular bundles is observed in part of the main roots; 2; browning of vascular bundles is observed in most of the main roots and extends to the core; Here, disease severity = 100 × {Σ (value of index) × (number of individuals corresponding to each index)} ÷ {3 × (number of test strains)}. Control value = 100 × {(degree of disease in untreated section)-(degree of disease in treated section)} / (degree of disease in untreated section).

[0040]

[Table 3]

[Example 3] Control test against Chinese cabbage yellowing disease (CGF4164 and CGF5100 strains) Pseudomonas genus Vetonamiensis CGF4164 and CGF5100
The strain was cultured in a broth liquid medium for 24 hours, and the obtained bacterial cells were separated by centrifugation to prepare a suspension of the test bacteria. Chinese cabbage seedlings (variety: Toshikushi) were dipped in this suspension, and left soaked in the suspension for 24 hours. Then, 1 was filled with contaminated soil mixed with Chinese cabbage yellow fungus (strain: 84013)
Transplanted in a 0.5 cm poly pot. After about 45 days, the presence or absence of disease was investigated and the control effect was determined. The results are shown in Table 4. Pseudomonas vietnamiensis CGF4
The bacterial concentration of the suspension of strains 164 and 5100 was 10 ⁸ cfu / ml. As a result, Pseudomonas vietnamiensis CG
High control effect was recognized for F4164 and 5100.

[0042]

[Table 4]

[0043]

EFFECTS OF THE INVENTION By using the method for controlling Chinese cabbage yellowing disease of the present invention, it is possible to strongly suppress the onset of Chinese cabbage yellowing disease, and it is possible to obtain the same or higher effect than the chemical agents currently used. Play.

Further, the use of the control agent of the present invention does not cause environmental pollution by pesticides unlike existing chemical agents. Furthermore, the control agent of the present invention can be distributed in a stable state on the market.

Continued front page    (72) Inventor Masayuki Tsubouchi             2805 Imafuku Nakadai Centra, Kawagoe City, Saitama Prefecture             Le Glass Co., Ltd. (72) Inventor Masahiro Watanabe             2805 Imafuku Nakadai Centra, Kawagoe City, Saitama Prefecture             Le Glass Co., Ltd. F term (reference) 4H011 AA01 BB21 DA02 DA15 DD03

Claims (4)

[Claims] 1. A Pseudomonas genus Petnamiensis (Pse)
A method for controlling verticillium disease of cruciferous plants, which comprises using a control agent containing udomonas vietnamiensis) as an active ingredient. 2. Pseudomonas genus Petnamiensis (Pse)
udomonas vietnamiensis) is a CGF4153 strain, CG
The method for controlling verticillium disease of cruciferous plants according to claim 1, which is a F4164 strain or a CGF5100 strain. 3. The control agent is characterized in that Pseudomonas vietnamiensis (Pseudomonas vietnamiensis) is immobilized on a carrier using a saccharide.
Alternatively, the method for controlling Verticillium disease of the plant of the family Cruciferae according to claim 2. 4. The Brassicaceae plant is Chinese cabbage, according to any one of claims 1 to 3,
Method for controlling verticillium disease of Chinese cabbage.