JP4301920B2 - Control agent and control method for cruciferous plant diseases - Google Patents

Description

  The present invention relates to a control agent and a control method for cruciferous plant diseases using bacteria belonging to the genus Variovorax.

  Clubroot disease, which is a major soil disease of cruciferous plant diseases, is a soil filamentous fungal disease that occurs in more than 300 species of cruciferous plants such as Chinese cabbage, cabbage, cauliflower, broccoli. Clubroot disease hinders the stable production of these cruciferous crops. As a symptom, knots are generated on the roots around 20 days after planting, and when infected from the beginning, no cabbage is formed with Chinese cabbage or cabbage. It can be seen that it cannot be obtained.

  Currently, several chemical agents are used as soil disinfectants for the control of clubroot diseases of cruciferous plants. However, these chemical agents have environmental problems, safety problems for users and neighboring residents, and problems that do not meet the recent trend of reducing pesticides and pesticides by consumers. Furthermore, when the density of pathogenic bacteria is high, it is not uncommon for the effect to be inferior.

  Therefore, for the control of this disease, it is desired to develop a control agent that has a high control effect and satisfies environmental pollution such as water pollution and safety.

So far, there are filamentous fungi (Non-Patent Document 1) and Bacillus bacteria (Patent Document 1) as biological control methods for Brassicaceae plant clubroot. In addition, as a technique related to the present application, in Patent Document 2, a microorganism belonging to Pseudomonas vietnamiensis has a controlling effect on yellowing disease (Verticillium disease) of cruciferous plants such as Chinese cabbage. It is disclosed.
Nikkatsu Disease Bulletin Vol. 62, 1996, p. 281 JP 11-335217 A JP 2003-231606 A

  An object of the present invention is to provide a control agent and a control method that have a high control effect on Brassicaceae clubroot diseases such as Chinese cabbage clubroot disease and cabbage root clubroot disease that are cruciferous plant diseases, and have no environmental pollution. is there.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the Variophorax sp. CGF4526 strain (Variovorax sp. CGF4526) has a high control effect on clubroot of cruciferous plants such as Chinese cabbage. The present inventors have found that a control agent containing the present bacterium as an active ingredient can be brought into contact with a cruciferous plant to effectively control root-knot diseases.

  In addition, the present applicant has found a microorganism belonging to the genus Acidovorax delafieldii having a high control effect against clubroot of cruciferous plants, and has already filed a patent application (Japanese Patent Application 2002). -158565). In addition, a bacterium belonging to the genus Pseudomonas vietnamiensis having a high control effect against clubroot of a cruciferous plant has been found and a patent application has been filed (Japanese Patent Application No. 2003-171251).

  Varioborax sp. Strain CGF4526 (Variovorax sp. CGF4526) according to the present invention not only effectively controls root-knot disease of cruciferous plants, but also has excellent storage stability and promotes plant growth. In addition, it has high utility as an agrochemical.

  That is, the present invention provides a control agent for Brassicaceae plant root-knot disease, comprising the Variovorax genus (Variovorax) strain and its living bacteria as active ingredients, and control of Brassicaceae plant root-knot disease using the same. Is the method.

  The microorganism of the present invention is a Variovorax sp. CGF4526 strain having a high control effect against cruciferous root-knot disease.

  Varioborax sp. CGF4526 (Variovorax sp. CGF4526) was obtained from approximately 7000 bacterial strains isolated and collected from rice and vegetables, as well as antibacterial activity against filamentous fungi, and further selected by disease control tests using pot-cultivated Chinese cabbage It is a strain.

  Variovorax sp. CGF4526 (Variovorax sp. CGF4526) is a gonococcal 1 to 3 μm cell as a result of morphological observation with an optical microscope and an electron microscope. It had motility. Gram reaction was negative and no endospore was formed.

Other bacteriological properties are shown below.
1. Culture properties
The growth state of CGF4526 on nutrient agar is shown below. Observation was performed after culturing at 30 ° C. for 3 days.

The colony morphology of CGF4526 is cream, round, smooth all edges, low convex and glossy.
2. General properties

3.16s-rRNA Analysis The base sequence 1530 bp of 16s-rRNA was determined. As a result of the analysis of homology using the obtained 16s-rRNA base sequence, the highest homology was Variovorax paradoxus.

  Due to the above bacteriological properties, CGF4526 is a gram-negative gonococci with motility and is a bacterium belonging to the Pseudomonas group by being positive for catalase activity, positive for oxidase activity and not forming endospores. It was classified into bacteria belonging to Group II. Moreover, it was found from 16s-rRNA analysis that it belongs to the genus Varioborax. In the genus Varioborax, it is considered as Variovorax paradoxus, but since it has not yet been determined, it was named Variovorax sp.

The corresponding strain of the present invention has been deposited with National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, has obtained the following entrusted number.

Variovorax sp. CGF4526: FERM BP-10160

  If the control agent or the control method of the Brassicaceae plant clubroot disease in the present invention, the onset can be strongly suppressed against the Brassicaceae plant clubroot disease, which is equal to or better than the currently used chemical agents. There is an effect. In addition, the use of the control agent of the present invention does not cause environmental pollution by agricultural chemicals unlike existing chemical agents. Furthermore, the control agent of the present invention can be distributed in a stable state on the market.

Next, the culture of these bacteria and the preparation as a control agent can be carried out by a conventional method, and will be described below with examples. The medium used here is not particularly limited as long as the bacteria grow. Any natural or synthetic medium can be used as long as it is a medium appropriately containing a carbon source, a nitrogen source and an inorganic substance capable of growing. Examples of the medium include bouillon medium, King B medium, PS medium, and PDB medium. After culturing and growing in the above medium at 15 to 42 ° C., preferably 28 to 35 ° C. for 12 to 48 hours, the cells are concentrated by centrifugation or a membrane concentrator to remove the medium components. By this operation, the concentration of the bacterial cells is usually concentrated to about 1 to 50 × 10 ¹⁰ cfu / ml. As the control agent of the present invention, viable bacteria after culturing may be used as they are, but generally they are used as solid or liquid preparations usable as agricultural chemicals. Therefore, a protective agent comprising a saccharide, sodium glutamate, and a sodium phosphate buffer is added to the cultured wet cells and vacuum dried.

  In order to maintain the viability of the bacteria, it is preferable to pre-freeze the microbial cells mixed with the protective agent before vacuum drying and then vacuum-dry them while they are frozen. In addition, a protective agent may be mixed with a microbial cell in the state of aqueous solution, and may be mixed with solid.

    The microbial cells of the present invention are immobilized by using a saccharide consisting of one or more of saccharose, fructose, glucose and sorbitol as a protective agent, mixed with the microbial cells, and dried by a method such as vacuum drying or freezing vacuum. This can be done by drying.

The cultivated Varioborax sp. Strain CGF4526 can be mixed with an appropriate carrier to form a powder or granule. Carriers in this case include mineral powders such as talc, calcium carbonate, and diatomaceous earth, peat moss, polymer compounds such as polyvinyl alcohol, and natural polymer compounds such as xanthan gum and alginic acid. The concentration of the bacterial cells is 10 ⁵ cfu / g or more, preferably 10 ⁷ cfu / g or more. In the case of a wettable powder, it is 10 ⁹ cfu / g or more, preferably 10 ¹⁰ cfu / g or more.

  In general, the production of cruciferous plants such as Chinese cabbage, cabbage, cauliflower, broccoli is sown in a seedling tray, and after 3 to 5 weeks of seedling, they are planted in a field. The control agent of the present invention can be used without any problem of chemical damage. The control agent of the present invention can be mixed with seedling culture soil at the time of sowing or can be poured into seeds before and after sowing. Furthermore, it is possible to immerse the seedling roots before planting in the diluted solution, or pour them into seedlings during or just before planting. It is preferable to combine the treatments at the time of sowing and planting because the establishment of the fungus at the root becomes more efficient.

When using the control agent of this invention by the said method, in the case of the mixing process to raising seedling culture soil, 1g or more is mixed per 1L of soil, and it stirs so that it may become uniform. The bacterial concentration in the culture medium is adjusted to be 10 ⁶ cfu / g or more, preferably 10 ⁷ cfu / g or more.

In addition, when soaking seedlings before and after sowing or before planting, adjust the bacterial concentration in the diluted solution to 10 ⁷ cfu / ml or more, preferably 10 ⁸ cfu / ml or more. To do.

  Next, selection of the Varioborax genus CGF4526 will be described in detail. CGF4526 is an antibacterial activity against fungi, Fusarium oxysporum and Verticillium daliae, among fungi isolated and collected from rice, vegetables and weeds, and yellowing using Chinese cabbage seedlings As a result of selection by the pot test of rot and clubroot, it was found that it has a controlling effect against Chinese cabbage yellowing disease and cruciferous plant clubroot. Specifically, after the plant roots collected from the field were washed with tap water, the roots were finely cut, put into sterilized water, and crushed with a mixer. The crushed liquid was appropriately diluted, applied to a bouillon agar medium, and cultured. The colonies that appeared there were isolated and stored, and used as test strains.

  Antibacterial activity against tomato wilt fungus (Fusarium oxysporum) and Chinese cabbage yellow rot fungus (Verticillium daliae) was measured using PDA medium (potato dextrose medium) with Fusarium oxysporum and tomato wilt fungus (Verticillium daliae). ) Measured by performing counterculture on The culture was performed at 25 ° C. for 1 week or 3 weeks.

Furthermore, a control test was conducted on Chinese cabbage yellowing disease and Chinese cabbage root-knot disease with respect to the strain having antibacterial activity obtained by anti-cultivation. In the method, the roots of Chinese cabbage seedlings were immersed in a diluted solution of 10 ⁸ cfu / ml of the test strain for 24 hours and planted in contaminated soil. As a result, it was found that the clubroot was strongly suppressed.

EXAMPLES Next, examples will be shown, but the present invention is not limited to the following examples. The composition of the medium used in the examples is shown below.
Bouillon medium: meat extract 3g, peptone 10g, NaCl 15g, water 1L, pH7.0
PDA medium (potato dextrose medium): potato exudate 200 g, glucose 20 g, water 1 L, pH 5.6).

[Example 1] Control test against Chinese cabbage root-knot disease Varioborax sp. CGF4526 strain was cultured in bouillon liquid medium for 24 hours, and the resulting cells were separated by centrifugation to prepare a suspension of the test bacteria. . In this suspension, Chinese cabbage seedling (variety: New ideal Megumi) was immersed and left in the suspension for 24 hours. Then planted in contaminated soil. As the contaminated soil, field soil was used in which Chinese cabbage roots (with root knots) affected by Chinese cabbage root koji were homogenized and mixed. After 33 days, the presence or absence of the disease was investigated and the control effect was determined. The results are shown in Table 2.

The cell concentration of the suspension of the Varioborax sp. Strain CGF4526 was 1 × 10 ⁸ cfu / ml. As a result, a high control effect was observed in the varioborax genus CGF4526. In the test of Chinese cabbage root-knot disease, the disease severity was calculated from the degree of root-knot formation at the root and evaluated.
Root disease index 0; healthy, 1; 10% or less of the roots show attachment of the humps 2; 10% to 50% of the roots show attachment of the humps; 3; 50% or more of the roots Adhesion is recognized.
Incidence of disease = 100 × {Σ (the value of the exponent) × (number body number corresponding to each index)} ÷ {3 × (test試株number)}.

[Example 2] Control test against broccoli root-knot disease Varioborax sp. CGF4526 strain was cultured in bouillon liquid medium for 24 hours, and the resulting cells were separated by centrifugation to prepare a suspension of the test bacteria. . Broccoli seedlings (variety: Green Emperor) were immersed in this suspension and left for 24 hours while being immersed in the suspension. Then, it transplanted to the 10.5cm polypot filled with the contaminated soil which mixed the crushing liquid of the root of a cabbage root-knot disease. About 30 days later, the presence or absence of disease was investigated, and the control effect was determined. The results are shown in Table 3. The cell concentration of the suspension of the Varioborax sp. Strain CGF4526 was 10 ⁸ cfu / ml. As a result, a high control effect was observed in the varioborax genus CGF4526.

[Example 3] Control test against Chinese cabbage root-knot disease (field test)
Varioborax sp. Strain CGF4526 was cultured in a broth liquid medium for 24 hours, and the resulting cells were separated by centrifugation, and mixed with the seed soil at a bacterial concentration of 10 ⁸ cfu / g. Chinese cabbage seeds (variety: New ideal Megumi) were sown and grown for 3 weeks. In the same manner, a suspension (10 ⁸ cfu / ml) of the cultivated varioborax bacterium CGF4526 was prepared, and the Chinese cabbage seedling was immersed and left in the suspension for 24 hours. After that, the plants were planted in a field mixed with the crushed solution of root-knot disease. After about 65 days, the presence or absence of disease was investigated and the control effect was determined. The results are shown in Table 4. As a result, a high control effect was observed in the varioborax genus CGF4526.

Claims (4)

Varioborax bacterium CGF4526 strain (FERM BP-10160) having an antagonistic activity against the clubroot disease of cruciferous plants. A control agent comprising, as an active ingredient, a varioborax genus CGF4526 strain (FERM BP-10160) having an antagonistic activity against the clubroot disease of cruciferous plants. A method for controlling a clubroot disease of a cruciferous plant, which comprises contacting a Clioflorax genus CGF4526 strain (FERM BP-10160) with a cruciferous plant. The control method according to claim 3, wherein the cruciferous plant is Chinese cabbage, cabbage, cauliflower or broccoli.