JPH0477405A - Controller of microorganismic blight of plant and control of the same - Google Patents

Abstract

PURPOSE:To obtain the title controller effective for plant blight caused by plant fungi belonging to the genus Botrytis and/or Penicillium, also for fruits and vegetables during storage, comprising cells of new strain, Pseudomonas cepacia D-212, or a cultured material thereof as an active ingredient. CONSTITUTION:A controller for preventing plant blights caused by plant fungi belonging to the genus Botrytis and/or Penicillium, comprising cells of new strain, Pseudomonas cepacia D-202 (FERM P-11, 559), or a cultured material thereof as an active ingredient. By using the new cells having antagonism on plant blights, occurrence of rot disease in storage of vegetables and fruits caused by the genus Botrytis and/or Penicillium can be effectively controlled. When the controller is used for beets, reduction in amount of sugar, which is resulted from the formation of putrid substances can be prevented and decrease in workability of sugar manufacturing process can also be suppressed.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a control agent and method for controlling plant diseases, and more specifically, to control plant diseases caused by Botrytis and/or Penicillium from Shutomonas cepacia ( Ps
The present invention relates to a pesticidal agent and a pesticidal method using a new strain of bacteria belonging to Eudomonas cepacia.

(Prior Art) Plant diseases caused by plant pathogenic bacteria belonging to the genus Botrytis and Penicillium are well known, and this plant disease attacks many types of plants from vegetables to fruits. The damage has been severe. These pests are generally controlled by adding chemicals such as bactericides, but adding chemicals to foods is not preferable.

"Botrytis""graymold" and "storage rot"
There are a wide variety of fruits, including mandarin oranges, cucurbits, tomatoes, green peppers, strawberries, green onions, lettuce, and sugar beets. In addition, "storage rot disease" caused by Penicillium spp.
It occurs due to increases in humidity, etc., and often occurs in sugar beets and mandarin oranges. In particular, the rear part of sugar beet, which is the raw material for beet sugar, is transported directly to the sugar factory after being harvested, but most of it is stored in a dumping ground, and the storage period is over three months in some cases. It may happen. During storage,
It is mainly affected by "storage rot disease" caused by the genus Botrytis and Penicillium. For this reason, the sugar content contained in sugar beets is significantly reduced during storage, and various operations at sugar refineries are made difficult.

(Problems to be Solved by the Invention) The present invention uses chemical agents such as fungicides as a method of controlling "storage rot disease" that occurs during storage of vegetables and fruits after harvest as described above. Since this is not permitted in Japan, the aim is to prevent the outbreak of pathogenic bacteria by using microorganisms that exhibit antagonistic activity.

(Means for Solving the Problems) The inventors conducted extensive research on the actual situation of vegetables and fruits affected by the above-mentioned "storage rot disease" and found that the storage rot disease of sugar beet is mainly caused by Botrytis cinerea. (7ci
nerea) and Penicillium.

It was discovered that it is Penicilliu+s Hiromi 匣憇 (Hirokatsu Uchino et al., Sugar beet Research Bulletin, 1984). Similarly, tangerines, cucurbits, tomatoes,
Gray mold on peppers, strawberries, lettuce, etc. is caused by the genus Botrytis, and storage rot on tangerines is caused by the genus Penicillium.As a result of further research on how to deal with this disease, we found that the use of antagonistic microorganisms is effective. I learned that it can be controlled.

In addition, by carefully observing sugar beets after harvesting at the heap, they discovered that the storage performance of sugar beets differed depending on the harvesting area. It was found that this difference was largely due to the difference in microbiota living in the soil attached to the sugar beets.

That is, the present inventors searched widely in nature for microorganisms with strong antagonistic activity against the genus Botrytis and Penicillium, and as a result, isolated and purified a bacterial strain with extremely strong antagonistic activity from the soil of a sugar beet cultivation field in the Tokachi region of Hokkaido. The bacterial strain was obtained. In addition.

As a separation medium, diluted meat broth (DNB) plate (composition:
Meat extract 0.01%, peptone 0.01%, salt o, o
os%.

A low-nutrient medium with an extremely low concentration of nutrients called agar (2.0%, pH 7.0) was used. In addition, in the process of obtaining the strain, the inventors investigated the antagonistic activity at a relatively low temperature of 8°C, which is close to the actual storage conditions of sugar beets, and confirmed that the antagonistic activity was sufficient even at low temperatures. There is.

Next, various properties of this strain will be described.

(a) Morphological properties Cell shape and size Presence or absence of polymorphism of cells Motile presence Presence or absence of spores Durham staining rods, 0.7-1. I
Glossy Good growth Uniformly suspended, forming a film on top Liquefied alkaline, no coagulation/reducing properties of lidmus Meat juice agar slant culture Meat juice liquid culture Meat juice gelatin puncture culture Lidomus milk (C) Physiological properties Reduction of nitrate Denitrification Reaction MR test vP test Production of indole Production of hydrogen sulfide Hydrolysis of starch Utilization of citric acid Utilization of inorganic nitrogen sources Positive Negative Negative Negative Negative Negative Positive Positive Production of pigment Urease Oxidase Catalase Growth temperature range Growth PH range Attitude towards oxygen OF Test Production of acids and gases from sugars L - Arabinose D - Xylose D - Glucose D - Mannose Slow Fructose D - Galactose Maltose Sucrose Lactose Negative Positive Positive Positive 4-42°C 5-8 Aerobic oxidative acid production Positive Positive Positive Positive Positive Negative Positive Negative Gas production Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Utilization of decomposed malonic acid Decomposition of arginine Decarboxylation of lysine positive positive negative positive levulinic acid assimilation positive ON Ace acylamidase β-galactosidase negative negative positive (e) Pathogenicity to vegetables and fruits Sugar beet None Summer mandarin orange None Sweet potato None Tomato none Cucumber Note: Pathogenicity to vegetables and fruits is based on the inoculation method for vegetable soft rot.

The above properties are described in Bergey's Manual of
Systematic Bacteriology V
ol, 1 (1984), it is a Durham-negative aerobic bacillus with one or more polar flagella,
Since the oxidase activity and catalase activity are positive and the OF test is oxidative, the present bacteria is P.
It belongs to the genus Seudomonas, has the ability to accumulate poly-β-hydroxybutyrate (P)IB), has no ability to hydrolyze arginine, has no denitrification ability, and has the ability to assimilate levulinic acid and 2,3-butylene gellicol. Unlike its closely related species, Pseudomonas M.
It was identified as Pseudomonas spp.

The detailed properties of the present invention bacterium are described in Bergey's Manua.
l of Systematic Bacteriolo
gy Vol. 1 (1984), it is different in that it grows at 4°C, and it also differs in that it has plant disease antagonistic activity.
It has been named nas cepacia D-202 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 11559.

As is clear from the test examples below, the new bacterial strain according to the present invention is a strain of the Botrytis genus that causes "storage rot" and "gray mold" that occur during storage after harvesting vegetables and fruits. It shows extremely strong antagonistic activity not only against Penicillium filamentous fungi alone, but also against both of these fungi.Moreover, this strong antagonistic activity is effective not only at room temperature but also at low temperatures below 10°C.
It is extremely unique in that it is well maintained even at low temperatures such as 4°C and 8°C, where microorganisms are normally unable to actively function. In addition, although the effect of drugs lasts only for a short period of time, the bacteria of the present invention have the advantageous feature that they can continue to survive for a long period of time and exhibit antagonistic effects even in small amounts of bacterial cells.

Therefore, the present invention is applicable to vast low-temperature layers such as outdoor storage areas, refrigerated warehouses, and bulk coolers for harvested vegetables and fruits, as well as ships and aircraft for transporting these materials maintained at low temperatures, as well as homes and shops. It is possible to prevent fruits and vegetables stored in refrigerators, coolers, low-temperature showcases, etc. in restaurants from rotting.

To carry out the present invention, the cells, culture and/or processed products thereof (concentrates, suspensions, dried products, diluted products, etc.) of one bacterial strain are sprayed or sprayed onto target vegetables and fruits. You can shake it, apply it, or soak vegetables and fruits in it. If necessary, the cells of the present bacterial strain may be sprayed or sprayed into the storage atmosphere, and any system in which one bacterial strain comes into contact with vegetables or fruits may be used as appropriate.

Vegetables and fruits treated with this strain can be heated to kill the strain, and can be washed away with water, and this strain is originally a bacterium that grows widely in general fields and is stored. It has the characteristic of not showing any pathogenicity to the vegetables and fruits inside, and is a safe antagonistic microorganism against storage spoilage.

Next, the present invention will be further explained using test examples.

Test Example 1 The present invention strain Pseudoxronas cepacia
D-202 (FERM P-11559) shows extremely strong antagonistic activity against Botrytis and Penicillium, which are the causes of "storage rot" that occurs during storage after harvesting vegetables and fruits. This is Botrytis isolated from rotting sugar beet, whose pathogenicity has been confirmed.
cinerea and Penicillium exp.
Ansum growth inhibition effect test at 25℃ and 8℃
It was done under the environment. The results are shown in Table 1.

In the test, one strain of the present invention was placed in PY medium (composition: peptone 0.5%).
, yeast extract 0.3%, meat extract 0.3%, glucose 1.0%, pH 7.0-7.2) and cultured with shaking at 25°C for 2 days in a reciprocating shaking incubator. Culture solution 40
μQ was placed on a paper disk (diameter 8 m5) on a PGA medium (composition: 20% potato infusion, 2% glucose, 2% agar), and after the liquid permeated the agar plate, pathogen spores were sprayed on each individual plate. 3 in a constant temperature room at 25℃ and 8℃
After culturing for days, the size of the inhibition zone was measured.

As shown in Table 1 above, one strain of the present invention exhibits good antagonistic activity against both Botrytis and Penicillium.

Test Example 2 Based on the above results, a storage test was further conducted in which the culture solution of the present invention was applied to individual sugar beets. The test was carried out using the bacteria of the present invention in diluted meat broth (DNB) medium (composition: meat extract 0.01%, peptone 0.01%, 1 salt o, oos%, pH 7.0).
A seed culture was prepared by culturing with shaking at 25°C for 24 hours. Spread 1Q of this seed culture into 100ml of King's B medium (
Composition: 2% peptone, 1% glycerin, K, HPO,
0.15%, Mg5O, -78200, 15%, pH 7
.

100 mU of the culture solution of the present bacteria was centrifuged at 10,000 rpm (rotation for 7 minutes) for 10 minutes to collect the bacterial cells, and diluted with sterile water to 100 tQ to prepare a solution containing only the "bacterial cells". At the same time, the supernatant portion was used as a "filter" for testing. In addition, the culture solution before separating the bacterial cells and the filter was used as a "suspension" for the test.

Prepare 80 sugar beets, divide them into 4 parts of 20 each, apply the above-mentioned antagonistic antibacterial suspension, bacterial cells, and filter to about 2% of the weight each, and seal them in a plastic bag. Ta. The remaining 20 pieces were placed in a plastic bag and sealed without treatment as a control. The sugar beets prepared in this manner were stored in a constant temperature room at 8° C., and the occurrence of mold and rot in the sugar beets was investigated after 2 months and 4 months of storage. As a result, as shown in Table 2, a strong effect was observed in the "suspension" and "bacterial cells", and the effect was also confirmed in r-filtering j.

Table 2 2 months 4 months 2 months 4 months no treatment 0.51 0.77 0,17
The sum of the products of 0.50 divided by the total number of individuals.

As shown in Table 2 above, the treatment with the culture solution of the present invention bacteria significantly reduces the occurrence of "mold" and "rottenness" during the storage period compared to the untreated case, and the degree of occurrence is one-fifth. It has the following properties, and its antagonistic activity against pathogenic bacteria that causes rotting disease during plant storage is extremely effective.

The microorganism of the present invention may be cultured in a conventional manner according to techniques commonly used for culturing microorganisms, particularly bacteria. Although a solid medium can be used as the medium, it is preferable to use a liquid medium to which necessary components in addition to a carbon source and a nitrogen source are added for aerated agitation culture or shaking culture.

As a liquid medium, for example, a meat extract medium (composition: meat extract 1%, peptone 1%, salt 0.5%, pH 7.0-7.
2) Or King's B medium (composition: Beptone 2%, glycerin 1%, K,) lPo, 0.15%, MgSO4
・7H200, 15%, pH 7,2) can be used, and if necessary, yeast extract (0,5%) can be added to the meat extract medium, and King's B medium can be prepared by adding glycerin to the same amount of manganese. You can also replace it with knit.

The microorganism of the present invention may be used as it is without dilution after being cultured using a liquid culture medium as described above. Alternatively, the culture solution can be centrifuged and the bacterial cells or filtration can be used separately.

When using bacterial cells, they can be suspended in sterile water or the like and used as a suspension, or they can be lyophilized and used as a powder. Alternatively, the centrifuged bacterial cells can be frozen and stored in a freezer, then thawed and diluted with sterile water before use.

(Effect) The fungus of the present invention exhibits an antagonistic effect on the genus Botrytis and Penicillium by culturing it in a liquid culture medium and using the culture solution, filtered, bacterial cells, etc. on plants to be stored.

(Example) Examples of the present invention will be shown below.

Example 1 Pseudomonas cepacia (Pseudomonas cepacia) as an antagonistic microorganism
dosonas Insho Bangaki) D-202 (FERM P
-11559) in diluted meat broth (DNB) medium (composition: meat extract 0.01%, peptone 0.01%, salt o, oos
%, pH 7.0) and cultured with shaking at 25° C. for 24 hours to prepare seed bacteria. 100mQ of this inoculum was added to 10Ω King's B medium (composition: peptone 2%, glycerin 1%, K
, HPo, 0.15%, Mg5O, 7H200,1
5%, pH 7.2) in a 20 m capacity fermenter, and cultured with aeration at 25° C. for 2 days to prepare a culture solution.

Two piles of sugar beets with a total weight of 10 tons were made on a sheet, and 10Q of the above-mentioned suspension of Pseudomonas cepacia D-202 was sprinkled over the entire surface of the pile on one side, and on the other side. The pile was covered with a sheet and the temperature inside the pile was maintained at 8 to 12°C for storage.

After about 2 months of storage, there will be mold, and after the 4th month, there will be mold.
and investigated the occurrence of "corruption." In addition, the investigation is based on Pyle's 5
100 sugar beets were randomly collected from different locations. As shown in Table 3, the occurrence of "mold" and "rot" was extremely low compared to the untreated control.

Table 3 Degree of mold occurrence Degree of rot 2 months 4 months 4 months without treatment 0.61 1.39 0.82 Example 2 Prepare 60 each of fresh and healthy summer mandarin oranges, Japanese radish, tomatoes, and cucumbers. gave three cuts and bruises with a knife, tomatoes and cucumbers gave only bruises, and strawberries did not cause any damage.

Next, the antagonistic microorganism culture solution in Example 1 was applied to each of the 30 individuals in an amount of about 2% of the individual's weight over the entire surface of the individual, and then placed in a plastic bag and sealed. On the other hand, as a control, 30 individuals not treated with antagonistic microorganisms were also placed in a plastic bag and sealed. The samples were stored at 8 to 15°C for 1 to 2 weeks and examined for the presence or absence of mold growth. As a result, as shown in Table 5,
An extremely strong effect was observed on summer mandarin oranges, and the effect was also observed on strawberries, tomatoes, and cucumbers. From these results, the invented fungus not only affects sugar beet storage rot disease, but also Rutaceae and Rutaceae.
Storage rot disease of vegetables and fruits of Rosaceae, Solanaceae, and Cucurbitaceae”
It was also found to be effective against.

Table 4 Number of mold-infested individuals/Total number of individuals Untreated 30/30 26/30 16/30 24/3
0 (Effects of the Invention) The present invention has been achieved by using a culture solution, a culture filter, or an isolated bacterial cell using a new bacterial strain that has an antagonistic activity against plant pathogenic bacteria of the genus Botrytis and Penicillium belonging to Pseudomonas cepacia. It is highly effective in effectively controlling the occurrence of "storage rot diseases" caused by Botrytis and Penicillium in vegetables, fruits, etc.

In particular, sugar beets are susceptible to such "storage rot diseases."
By being able to prevent this, it is possible to prevent a decrease in sugar content due to putrefaction, and also to prevent a decrease in workability in the sugar manufacturing process due to putrefactive substances.

In addition, it is now possible to prevent other vegetables and fruits from rotting not only at high temperatures or room temperature, but also at low temperatures, making it possible to prevent rotting during low-temperature storage or transportation. Therefore, the present invention is a great blessing not only to the fruit horticulture industry but also to the distribution industry.

According to the present invention, harvested vegetables and fruits do not rot, so there is no loss of fruit, and therefore agricultural income increases, which in turn greatly contributes to solving the world's food problems.

Moreover, it is a novel plant disease control system that applies extremely safe biotechnology.

Agent Patent Attorney 1) Parent Male

Claims (4)

[Claims] (1) New strain Pseudomonas cepacia (￥Pseudomonas￥￥cepacia￥) D
A pest control agent for controlling plant diseases caused by plant pathogenic bacteria of the genus Botrytis and/or Penicillium, which contains cells or a culture of No. -202 as an active ingredient. (2) The culture is Pseudomonas cepacia D-202
The pesticidal agent according to claim 1, which is a bacterial cell, a culture solution, a culture filter, or a mixture of one or more of these. (3) A method for controlling plant diseases, which comprises controlling plant diseases caused by plant pathogenic bacteria of the genus Botrytis and/or Penicillium using Pseudomonas cepacia D-202 strain. (4) After culturing strain D-202, which belongs to Pseudomonas cepacia and exhibits antagonistic activity against plant pathogens of the genus Botrytis and/or Penicillium, using a culture solution, culture filter, and/or cultured bacterial cells, A method for controlling plant diseases, which comprises controlling plant diseases caused by plant pathogenic bacteria of the genus Botrytis and/or Penicillium.