JP2760775B2 - How to control soft rot - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the scientific name Elvinia
Carotobora (Erwinia carotovor)
The present invention relates to a method for immobilizing bacteria belonging to a) and spraying the immobilized product to control soft rot.

[0002] Plants targeted for disease control in the present invention include a large number of plants such as Chinese cabbage, cabbage, celery, lettuce, carrot, radish, wasabi, potato, tobacco, tomato, and cyclamen. Soft rot
disease) is the target disease.

[0003]

As a method for controlling so-called soft rot caused by bacteria of Erwinia carotobora, spraying of an antibiotic preparation such as streptomycin or a copper agent such as bordeaux solution is generally performed.

[0004] However, when these agents are used, their controlling effect is not satisfactory, and
Killing even beneficial bacteria other than pathogens,
There is a problem of environmental pollution and also a problem of phytotoxicity. Also,
With respect to antibiotics, there is the emergence of bacteria having resistance thereto, and these are problems.

[0005] Erwinia carotobola bacteria have the ability to produce pectin-degrading enzymes that cause soft decay of storage tissues of many plants and degrade pectin substances that function as intercellular junctions of plant tissues. Has been reported to exist.

[0006] The occurrence of this soft rot is sometimes observed even in a field in which a crop serving as a host of the fungus has not been produced for more than 5 years. The general ecology of this bacterial body is that, for example, in the case of frequently occurring Chinese cabbage, this bacterium grows around the root from about 40 days after sowing, and its presence in all places such as rhizosphere soil and leaves. Will be recognized.

[0007] If the Chinese cabbage is damaged by typhoons, insects, or daily work, bacteria enter from there.
As long as the weather conditions are met, the concentration of pathogenic bacteria rises overnight, and lesions become visible. In order to prevent these diseases, if it becomes possible to grow Erwinia carotobola bacteria, which are nonpathogenic to pathogenic bacteria, in the same manner as pathogenic strains in rhizosphere soil and leaves, It is possible to control the growth of certain bacteria and control these soft rots.

[0008]

Means for Solving the Problems Based on the above considerations, the present inventors have conducted intensive studies, and as a result, among the mutant-treated strains of Erwinia carotobola, they competed with the pathogenic strain of the same strain. Strains that grow well and have no pathogenicity are selected, and viable bacteria of Erwinia carotobola bacterium lacking these pathogenicities are applied to the roots or leaves of the target plants for soft rot. The inventors have found that the disease can be controlled, and have reached the present invention.

That is, the present invention relates to a method of mixing a soft-rot fungus whose pathogenicity has been deleted by mutation or mutation treatment with an immobilized protective agent comprising saccharides or beef extract, sodium glutamate and sodium phosphate buffer, and drying under vacuum or This is a method for controlling soft rot, which comprises freeze-drying and immobilizing the product, dissolving the immobilized product with water, and applying the product to crops.

[0010] Such bacteria mutate the pathogenesis of soft rot to produce a pathogenic deletion strain. As a mutation treatment method, a method using a commonly used mutation reagent, for example, ethyl methanesulfonyl, nitrosoguanidine, ultraviolet light, etc. [Microorganism Experiment Method, pages 288 to 306, published by Kodansha (1)
982)] is known, and processing may be performed according to these.

The screening for the pathogenic deletion strain is carried out by
A strain having a reduced pectinase secretion ability was picked up and subjected to a pathogenicity test using Chinese cabbage slices. In the test, a leaf section of Chinese cabbage was scratched, a test solution of high concentration was applied, and the mixture was allowed to stand in a water bath at 28 ° C for 24 hours in the presence of water, and then the presence or absence of the lesion was measured. The presence or absence was determined.

[0012] Among these pathogenic deletion strains, a strain having a high ability to prevent lesions has been deposited with the Japan Institute of Microorganisms and given the following deposit number. Elvinia carotobola subsp. Carotobola CGE6M14 Microtechnical Laboratory Bacteria No. 10998 (FERM P-10998) Erwinia carotobola subsp. No. 11000 (FERM P-11000) Erwinia carotobola subspiro carotobola CGE11M5 Microtechnical Laboratories No. 11001 (FERM P-11001) Erwinia carotobola subspiro carotobola CGE234M403 Microtechnical Laboratories No. 11792 (FERM P-1192) The present invention also provides a method for immobilizing the soft-rot fungus lacking these pathogenicity and using the same to control soft-rot. It is.

The immobilized protective agent for the cells of the present invention includes a saccharide or beef extract comprising one or more of saccharose, glucose, fructose and sorbitol, and an immobilized protective agent comprising sodium glutamate and sodium phosphate buffer. Is performed by mixing with bacterial cells and vacuum drying or freeze vacuum drying.

Hereinafter, the present invention will be described in detail. First, the pathogenic deletion strain of the soft rot fungus is cultured in an appropriate medium. As used herein, for example, the liquid medium is not particularly limited as long as the bacteria can grow, and a commonly used medium such as the following 802 medium or broth medium is used.
After culturing at -35 ° C for 10-35 hours to proliferate, the cells are collected by centrifugation to remove the medium components. By such an operation, the cell concentration is usually concentrated to about 2 to 3 × 10 ¹¹ cfu / g. Then, a protective agent consisting of a saccharide or beef extract, sodium glutamate and sodium phosphate buffer is added to the wet cells, followed by vacuum drying. It is preferable to pre-freeze the cells mixed with the protective agent before vacuum drying, and then vacuum-dry the cells in a frozen state in order to maintain the survival rate of the cells.

The protective agent may be mixed with the cells in the form of an aqueous solution, or may be mixed as a solid.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. The composition of the medium used in the examples is shown below. 802 medium: 10 g of polypeptone, 2 g of yeast extract, M
gSO ₄ · 7H ₂ O1g, water 1L, (in the case of plates, agar 15 g) pH 7.0 broth: meat extract 3g, peptone 10 g: NaC
15 g, 1 L of water, pH 7.0

[0017]

EXAMPLES Example 1 Erwinia carotobola CGE10M2 in 802 medium
[Microtechnical Research Bacteria No. 11000 (FERM P-1100
0). ] At 30 ° C for 15 minutes.
Cultured for hours. The culture was collected using a centrifuge to obtain a cell concentrate (3.0 × 10 ¹¹ cfu / mL of bacterial cells). Protective agent [40% (w
/ W) saccharose, 2% (w / w) sodium glutamate, 0.1 M sodium phosphate buffer pH 7.0]
After mixing well with 25 μL, the mixture was put in an ampoule tube, stuffed with absorbent cotton, dried at 100 mtorr for 2.5 hours, sealed, stored at room temperature, and examined with time.

As a result, the viable cell counts after 10, 30, and 90 days were 4%, 4%, and 4%, respectively, as compared to those before drying. Prior to drying, the samples were pre-frozen using dry ice-ethanol cryogen. As a result, 1
Viable cell counts after 0, 30, and 90 days were 39%, 35%, and 35%, respectively, as compared to those before drying.

Table 1 shows the results when the saccharose concentration was changed and when no saccharose was added.

[0020]

[Table 1]

Example 2 In the same manner as in Example 1, the survival rate of the bacterium was measured when glucose, fructose, sorbitol and beef extract were used at various concentrations instead of the saccharose saccharide. Table 2 shows the results.

[0022]

[Table 2]

Example 3 A box having a width of 32 cm, a length of 60 cm and a height of 18 cm was filled with cultivated soil in which red clay and mulch were mixed at a ratio of 2: 1. Twelve seedlings of bok choy were transplanted into this box. 1 after transplant
After 0 days, the cell-immobilized product shown in No. 4 in Table 2 of Example 2 was stored at room temperature for 10 days, and diluted with water to 60 mL.
And sprayed this. The bacterial concentration at this time was 8.8 × 1
Was 0 ⁷ cfu / mL. Twenty-one days after spraying, the outer leaves of bok choy were taken and the bacterial concentration was measured. As a result, bacteria having an average of 1.2 × 10 ⁴ cfu / cm ² were established.

Example 4 Three Chinese cabbage seedlings were transplanted in the same box as in Example 3, and CGE234M4 was obtained by the method of No. 6 shown in Table 1 of Example 1.
03 [Microbial Deposit No. 11792 (FERMP-11
792). And then fixed at room temperature for 10 days.
L and sprayed. The bacterial concentration at this time was 8.8 × 10 ⁷
cfu / mL. Eighteen days after spraying, the outer leaves of Chinese cabbage were taken and the bacterial concentration was measured. As a result, 5.2 × 10 ³ c
Fu / cm ² bacteria were established. Growing these, 7
Until harvest after 0 days, no occurrence of soft rot was observed at all.

On the other hand, in the case where the immobilized material was not sprayed at the time of cultivation in the adjacent box, the occurrence of soft rot with a disease degree of 33.3 was observed.

[0026]

[Formula 1] Disease severity = [Σ (number of diseased strains by index x index) / total number of surveyed stocks x
3] × 100 Example 5 The cells CGE234M403 were cultured in a 10 L jar fermenter for 36 hours using 4 L of a broth medium supplemented with glucose and bolipeptone. Then, this part was centrifuged to obtain 35 g of wet cells. 40% of this
40g of sucrose, pre-freeze, 150mt
Drying was performed at orr for 40 hours. As a result, the cells 25
g was obtained. At this time, the viable cell concentration of the cell body was 2.3 × 10
It was ¹¹ cfu / g. It was stored at room temperature and examined for changes over time. As a result, the respective bacterial counts after 10, 30, and 90 days were 27%, 25%, and 25%, respectively, as compared with those before drying.

[0027]

According to the method of the present invention, a pathogenic deletion strain can be stably immobilized. By using this as a means for controlling soft rot, it is possible to efficiently perform safe control without chemical injury. Can be.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) A01N 63/00 C12N 1/20 C12N 11/02 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A soft rot fungus whose pathogenicity has been deleted by mutation or mutation treatment is mixed with an immobilized protective agent comprising saccharides or beef extract, sodium glutamate and sodium phosphate buffer, and dried under vacuum or freeze-vacuum. A method for controlling soft rot, comprising dissolving the immobilized product in water and applying the solution to crops. 2. The method for controlling soft rot according to claim 1, wherein the saccharide is saccharose, glucose, fructose and / or sorbitol.