JPH02149506A - Composition for control of plant disease - Google Patents

Abstract

PURPOSE:To obtain the subject composition exhibiting existence of component fungus body by a shade of color of red-pink generated from resazurin, by containing resazurin into control agent of plant disease containing bacterium belonging to genus Pseudomonas. CONSTITUTION:Resazurin (especially water-soluble salt of said compound is preferable) is contained in a bacterium belonging to genus Pseudomonas and having anti-fungus activity against pathogenic fungi of plant (e.g. Pseudomonas cepacia ATCC10856) to obtain a composition for control of plant disease. Said composition is capable of exhibiting existence of microorganism in itself and plant disease by soil such as black rot or fusarium wilt is correctly and efficiently controlled. For instance, the aimed composition is inoculated into plant having affinity with said component fungus body, e.g. genus Allium fistulosum L., and said inoculated plant is interplanted with the target plant with advantageously using small amount of the aimed composition.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a composition for controlling plant diseases containing bacterial cells as an active ingredient, and more specifically, the present invention relates to a composition for controlling plant diseases containing bacterial cells as an active ingredient. The present invention relates to a composition for controlling plant diseases that indicates their presence and survival.

[Prior Art 2] Problems to be Solved by the Invention Continuous cultivation of highly profitable crops such as vegetables is generally considered difficult due to deterioration of the physical and chemical properties of the soil and the occurrence of soil pests and diseases.

In recent years, various methods have been studied for controlling soil pathogenic bacteria using microorganisms that have antibacterial activity against the pathogens.

For example, to control tomato canker,
Bacteria that exhibit antibacterial activity against the pathogen Corynebacterium michiganens, such as Pseudomonas cepacia, Pseudomonas gladiolii, Pseudomonas avenae, and Pseudomonas gourmae, are effective (Shinki et al. 2 Japanese Society of Plant Pathology Report, Vol. 52, No. 5, pp. 835-842, December 1985). In addition, the isolated strain M-2 was identified as Pseudomonas gladiolii.
196 is a fungus of the genus Fusarium, a fungus of the genus Verticillium,
Bacteria of the genus Alternaria, Bacteria of the genus Corticium, and Rhizoctonia (Ri)
20Ctonia), Piricularia (P
yricularia), SukL/c7tium (S
Bacteria of the genus Clerotiun, Pythium
m) Genus bacteria.

Macrohomina genus bacteria, Cot letotrichium
M-2196 exhibits antibacterial activity against bacteria of the genus Botrytis and Corynebacterium.
is also known to be effective in controlling plant diseases caused by these pathogens, such as strawberry chlorosis, cucumber vine blight, watermelon vine blight, chive blight, tomato canker blight, and konjac blight. (chemistry and biology,
Vol. 26, No. 5, pp. 284-286, 1988).

On the other hand, the present inventors have discovered that strains belonging to Pseudomonas cepacia, Pseudomonas gladiolii, Pseudomonas gourmae, Pseudomonas planchleyi, Pseudomonas cariophieri, Pseudomonas solanacearum, Pseudomonas avenae, and Pseudomonas cadrea are Fusarium Oxysporum shows antibacterial activity,
They discovered that soil diseases caused by Fusarium oxysporum, such as rot, yellow wilt, vine splitting, leaf wilt, damping-off, and dry rot, can be controlled, and based on this knowledge, they developed an invention. It was completed and a patent application was filed.

Bacteria used as active ingredients of plant disease control agents (hereinafter referred to as component bacteria) must be alive at the time of use. In order to preserve these component bacteria alive, serum, various sugars such as glucose and fructose, protective agents such as serine and dimethyl sulfoxide are added, and low temperature preservation, frozen preservation or vacuum freeze-drying is possible. It is done.

However, in these conventional methods, the color of the product is only the color of the component bacteria and the protective agent, which is diluted with water and becomes almost colorless when used. If this is not known, it is not possible to obtain an appropriate amount of the component microorganisms, and it is difficult to efficiently use a plant disease control agent containing the component microorganisms.

Therefore, there has been a desire for a preparation that shows the presence and survival of this component bacterial cell.

[Means and effects for solving the problem] The present inventors
Regarding formulations of plant disease control agents containing bacterial cells belonging to the genus Pseudomonas, we investigated various methods that could easily indicate the presence and survival of the component bacteria. The present inventors have discovered that by containing resazurin, it is possible to indicate the presence of the component bacteria based on the shade of red or pale pink color developed by resazurin, and have completed the present invention.

That is, the present invention is a composition for controlling plant diseases, which contains at least a bacterium belonging to the genus Pseudomonas and having antibacterial activity against plant pathogens, and resazurin.

The bacteria used in the present invention belong to the genus Pseudomonas,
Any bacteria may be used as long as it has antibacterial activity against plant pathogens, and typical examples include Pseudomonas cepacia, Pseudomonas gladiolii, and Pseudomonas gourmae.

There are strains belonging to Pseudomonas bluntii, Pseudomonas cariophieri, Pseudomonas solanacearum, Pseudomonas avenae, Pseudomonas cadrea, and the like. Furthermore, mutant strains derived from these strains belonging to the genus Pseudomonas may also be used.

Representative examples of these strains include Pseudomonas cepacia ATCC10856, Pseudomonas cepacia ATCC13945, and Pseudomonas cepacia ATCC10856, Pseudomonas cepacia ATCC13945,
C17460, ATCC17616, ATCC17
759 (=jCM2799), same ATCC1?762.
ATCC17765, ATCC25416 (=JC
M5964=IF014074), same ATCC256
08゜ ATCC25609, ATCC25610,
Same ATCC27515゜ Same ATCC29351, Same AT
CC29352, ATCC29424゜ATCC3
5130, ATCC35254, ATCC3927
7゜ ATCC39356, JCM 2800. Same J
CM 2801 and IFO 3739, Pseudomonas gladioli ATCC 19302, ATCC 1
0247, same ATCC10248 (=IF013700
), ATCC10854 and ATCC254
17. Pseudomonas gourmae NIAES 1169
(ATCC33617), Pseudomonas bluntii JCM5492, Pseudomonas cariophieri-AT
CC54218 (DSM 50341), ATCC1
1441, IFO12950゜IFO12951,
IFO12952, IFO12953, IFO1
3591 and IF013694. Pseudomonas solanacearum ATCC11696, Pseudomonas solanacearum ATCC1069
2, ATCC 11697, ATCC 25237, DSM 1993 and DSM 50905. Pseudomonas avenae ATCC 19860 and Pseudomonas scadrea ATCC 33619 and ATCC 10
200 etc.

The mycological properties of these bacteria are described in Bergey's Manual of Systematic Bacteriology.
Manual of 5yste+natic Ba
cteriology) Volume 1 Editor: Kri
eg) and Holt: Willta+ns & Wilkin
(1984), and International Journal of Systematic Bacteriology (In
International Journal of S
systematic Bacteriology),
37, pp. 144-152 (1987).

Of these strains, NIAES is the National Institute of Agriculture and Environmental Technology of the Ministry of Agriculture, Forestry and Fisheries.
te of Agro-Environmental 5
science, 3-1-1 Kannondai, Tsukuba City, Ibaraki Prefecture).
JCM is the RIKEN Microbial System Collection Facility (JCM).
An Co11ection of Microor
-ganisms, 2-1 Hirosawa, Wako City, Saitama Prefecture), ATC
C is for American Type Culture Collection (Ame
rican Type Culture Co11ec
tion, 12031ParklawnKoive R
ockville, Maryland 20852°U, S, A);
elungvon Mikroganismen, G
risebachstrasse 8. Gott-n
gen, Federal Republic of
Germany).

The composition for controlling plant diseases of the present invention (hereinafter sometimes simply referred to as composition) may contain at least component bacterial cells.

The component microbial cells used as a component of the composition of the present invention include wet microbial cells separated from a culture solution by ordinary solid-liquid separation means such as centrifugation and filtration, and the wet microbial cells stored at low temperatures. Treated bacterial cells that have undergone treatments such as , freezing and lyophilization are suitable. The cryopreserved, frozen or lyophilized bacterial cells may be, for example, a mixture with commonly used preservatives such as serum, sugars and glycerin.

In a preferred composition of the present invention, for example, resazurin is added to a culture solution, wet component bacterial cells separated from the culture solution, and a solution obtained by reconstitution of the above-mentioned treated bacterial cells in water. There are suspensions obtained by resuspending the above-mentioned wet component bacterial cells and treated cylinders in an aqueous resazurin solution, and frozen products and freeze-dried products of such suspensions.

There is no particular limit to the concentration of the component hedrons in this suspension, but in practice it is usually about 10'' to 10' pieces/mI.

As resazurin, resazurin itself and a water-soluble salt of resazurin such as resazurin sodium can be used, but for practical purposes, a water-soluble salt of resazurin is preferred (both resazurin itself and a water-soluble salt of resazurin are used). (Sometimes referred to as resazurins).

Resazurins can be used as solids and as aqueous solutions.

There is no particular restriction on the amount of resazurin used as long as it does not adversely affect the component bacterial cells.

When the composition of the present invention is a suspension, the concentration of resazurin is preferably 1wt% or less as resazurin, particularly preferably 0.5% or less, and 0.001 to 0.001% by weight.
More preferably, it is about 0.1 wt%.

In addition, when the composition of the present invention is a wet bacterial strain or a treated bacterial cell, the concentration of resazurin is usually used in practice at a concentration of 10 wt % or less as resazurin.

The pH of this suspension is such that the component bacteria do not die in a short period of time or the antibacterial activity does not decrease significantly in a short period of time.
It varies depending on the type of component hedron and culture conditions, and although it cannot be generally specified, in practice, it is usually about 5 to 8,
Preferably it is about 6 to 7.5.

In order to keep the component bacterial cells in the above suspension alive for a long period of time, this suspension should be stored at a low temperature until it is used, or this suspension should be frozen or freeze-dried. It is preferable to do so.

For convenience of use, it is preferable to encapsulate the component bacterial cells and resazurin.

During these cryopreservations, freezing and lyophilization, conventional preservation protectants such as serum, sugars such as glucose and fructose, glycerin and dimethyl sulfoxide may be present in the M suspension.

The conditions for cryopreservation, freezing and lyophilization are no different from those for ordinary microorganisms.

Among these treatments, in order not to damage the component bacteria,
Freeze-drying is preferred, particularly vacuum freeze-drying.

Vacuum freeze-drying is carried out in a conventional manner. For example, a certain amount of a plant disease control composition, which is a suspension containing component bacterial cells, resazurin, and a protective agent (e.g., 5% sucrose), is placed in a sterilized ampoule, frozen at a low temperature such as -80°C, and then , freeze-dry, 1. seal the ampoule under vacuum to create an ampoule containing the 111 product for plant disease control.

In any case, the composition of the present invention exhibits a red to pale pink color in the presence of water, indicating the presence of bacteria as a color.

During storage of the composition for controlling plant diseases, the component bacterial cells and resazurin may separate in the ampoule. Use by diluting with water, or
After thoroughly mixing the contents of the ampoule, take a predetermined amount from it and dilute with water if necessary. In order to use accurate nails of the component bacteria and from the point of view of workability, it is particularly preferred to use the entire ampoule diluted with water.

In addition, the component bacterial cell concentration of the bacterial solution at the time of application is usually 10'-
It is generally used at a concentration of about 10'''/ml.

Plant diseases that can be controlled by the present composition for controlling plant diseases include various soil diseases such as sagebrush, tomato, etc.
Strawberries, chives, rakkyo, mitsuba, burdock, asparagus, onions, cabbage.

Kyu 1 hachisha, loofah, eggplant, melon, lotus.

Dry rot that occurs in vegetables such as watermelon, radish, spinach, celery, chorogi, taro, yam, ginger, udon, etc. Single plant blight, leaf wilt, damping off, yellow yellow disease, vine splitting disease, root rot disease, There are half blight and @septic diseases.

In order to prevent and eliminate these plant diseases, the present composition for controlling plant diseases can be diluted with a necessary amount of water and applied by scattering, spraying, etc. on soil and plants.

However, in this method, the component bacterial cells gradually die in nature, so a large amount of the plant disease control composition is required.

Therefore, in order to obtain a large plant disease control effect with a relatively small amount, plants that have an affinity for the component microorganisms, such as plants of the genus Allium, are inoculated with the present plant disease control composition, and the inoculated plants are injected with pathogenic bacteria. By co-planting with plants to be controlled (hereinafter referred to as target plants), the component bacteria are grown in the soil, and the antibacterial action of the component hedrons is used to prevent and eliminate the target pathogenic bacteria. .

Alternatively, the target plant and a plant having an affinity for the body component microorganisms may be co-planted in advance, and then the composition for controlling plant diseases of the present invention may be inoculated.

If pathogenic bacteria occur in target plants such as tomatoes by co-planting plants inoculated with bacteria, which are the component bacteria of the composition of the present invention, they may inhabit the roots of the co-planted plants such as green onions. The bacteria of the present invention exhibiting an antibacterial effect against pathogenic bacteria of target plants, making it possible to control pathogenic bacteria more efficiently.

[Examples] Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example】 1 text In an Erlenmeyer flask, broth culture medium (manufactured by Difco).

200ml of Difco) was added and autoclaved at 120°C. Nine pieces of this medium were prepared. Each strain of Pseudomonas was inoculated into each of these, and cultured with rotational shaking at 30°C for 1 day.

Put the culture solution from each flask into a sterilized centrifuge tube,
Centrifugation was performed at 00XG for 20 minutes, the supernatant liquid was discarded, and each of the resulting bacterial cells was combined with 5 ml of filter-sterilized horse serum containing 5% sucrose and 1 wt% resazurin sodium (resazurin), which had been filter-sterilized. (approximately 0.9 wt%) aqueous solution-1 tO, 5 ml was mixed aseptically to obtain a suspension. 0.2+nl of this suspension was placed in a sterilized ampoule, frozen at -80°C, and then freeze-dried in vacuum, and the ampoule was sealed in a vacuum to obtain an ampoule containing the plant disease control composition.

I opened this ampoule that had been stored at 5°C for 6 months.
The bacterial solution was diluted with one liter of sterile water, and the number of viable bacteria and color of the bacterial solution were examined. The results are shown in Table 1. In addition, on a potato dextrose agar medium (manufactured by Hinaga Pharmaceutical Co., Ltd.), each of the above VI solutions containing bacteria of the genus Pseudomonas and Fusarium oxysporum fnisbi lycovir sisii (
Fusaricunoxysporum f, sp,
Fusarium oxysporum was inoculated with J-3 (pathogen) and cultured at 25°C for 7 days.
The growth status of bacteria in J-3 was observed. The results are shown in Table 1.

In Table 1, strains that inhibited both colony formation and hypha growth of Fusarium oxysporum J-3 were classified as having antibacterial activity (+10).

On the other hand, those that inhibited the formation of Fusarium oxysporum J-3 pigment-producing colonies but did not inhibit the growth of hyphae were rated as active (+).

(Margin below) Table 1 Example 2 Pseudomonas gourmae NIAES1169 prepared in the same manner as Example 1 and stored at 5°C for 6 months.
The ampoule containing the plant disease control composition was opened and diluted with 1 liter of sterilized water (the number of viable bacteria was 1.8 x 10').
However, it showed a pale pink color.

Chive scales were dipped and inoculated in the solution.

Then, when planting tomatoes, this inoculated chive is used for about 2
Mixed planting at around 0 cm. Table 2 shows the results of the control test for tomato root rot wilt.

Table 2 In addition, The color of the night is all pale pink Met.

Example 3 When prepared in the same manner as in Example 1 and diluted with sterilized water at 5° C. for 6 months (viable cell count 1.0×10′]/ml), it showed a pale pink color. Chive scales were immersed and inoculated in the solution.

Then, when planting strawberries, this inoculated green onion is applied to about 2
Mixed planting at around 0 cm. Table 3 shows the results of the strawberry chlorosis control test.

Table 3 [Effects of the Invention] The composition for controlling plant diseases of the present invention can indicate the presence of microorganisms therein. , vine split disease, leaf wilt, damping-off, and dry rot.

It becomes easier to more accurately and efficiently control soil diseases of plants such as canker disease and white silk disease, and it becomes possible to significantly improve the productivity of cultivation of vegetables and the like.

Claims (1)

[Claims] 1. A composition for controlling plant diseases, comprising at least a bacterium belonging to the genus Pseudomonas and having antibacterial activity against plant pathogens, and resazurin.