JP2007332069A - Method for sterilizing xanthomonas campestris pv. campestris in seed of plant of family brassicaceae using ozone gas - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for sterilizing Xanthomonas campestris pv. campestris in seeds of plant of the family Brassicaceae, with which the seeds are sterilized for a short time without requiring drying the seeds after the sterilization. <P>SOLUTION: The method for sterilizing Xanthomonas campestris pv. campestris comprises treating seeds of plant of the family Brassicaceae with ozone gas. The seeds are preferably rolled during the sterilization. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for sterilizing black rot fungi in cruciferous plant seeds.

  There are many diseases in vegetable fields, and their control is extremely difficult. In particular, diseases caused by plant bacteria are mainly controlled by copper agents, but their effects are limited. Recently, the use of pesticides has attracted attention to safety and environmental pollution issues for humans. It is desired to reduce the number of uses and the amount used. On the other hand, as a source of plant diseases, seeds are contaminated and there are many seed-borne diseases. Therefore, seed disinfection has attracted attention from the viewpoint that healthy seed production is extremely important.

  The cruciferous plant black rot fungus (Xanthomonas campestris pv. Campestris) invade cruciferous vegetables such as Chinese cabbage, chingensai, turnip, komatsuna, cabbage, cauliflower and broccoli, causing great damage. This disease is known to transmit seeds and seed disinfection is considered extremely important. In seed disinfection, depending on the type of vegetables, the type of pathogen, etc., pesticide dressing, soaking in pesticides, dry heat treatment, hot water treatment, etc. are performed, but registered pesticides for bacterial diseases such as black rot are Absent. Furthermore, even if there are highly effective pesticides, it takes a lot of labor, place and time to dry after disinfection, and it may affect germination rate by chemical treatment, so even if the disinfection effect is high, it is practical Is currently not available. In addition, in the case of hot water treatment, drying is necessary, and attention must be paid to the influence on germination.

  At present, there are currently no seed disinfectants that target bacterial bacterial diseases such as black rot of cruciferous seeds. In order to dry the wet seeds, it is usually necessary to carry out a dry heat treatment for about 4 days, so a method in which the seeds get wet in the sterilization treatment step is not preferable.

  On the other hand, seed sterilization methods using the sterilizing power of ozone are disclosed in Japanese Patent Application Laid-Open Nos. 5-2181808 (Patent Document 1), Japanese Patent Application Laid-Open No. 11-158015 (Patent Document 2), and the like. The technique described in Patent Document 1 is characterized by performing tests using seeds of “de-de” using ozone water and sterilizing and promoting germination of seeds. Has the problem that it needs to be dried. The technique described in Patent Document 2 is intended to sterilize microorganisms such as Escherichia coli and Salmonella in seeds of crops cultivated in greenhouse horticulture such as sprouts and radish. The technique described in Patent Document 2 is characterized in that microorganisms attached to seeds are sterilized by bringing the ozone gas phase into contact with the seeds in the presence of a small amount of water (6 to 20%). This method also has a problem that it is necessary to dry the seeds after the treatment. The reason why the sterilization treatment of seeds described in Patent Documents 1 and 2 is performed in the presence of ozone and water is because it is generally known that sterilization by ozone is likely to proceed in the presence of water. it is conceivable that. Patent Documents 1 and 2 do not disclose a technique for sterilizing cruciferous plant seeds infected with black rot fungi.

Japanese Patent Application Laid-Open No. 5-21808 Japanese Patent Laid-Open No. 11-158015

  An object of the present invention is to provide a method for sterilizing black rot fungi in cruciferous plant seeds, which does not require drying of the seeds after sterilization treatment and can be sterilized in a short time.

  The present inventors have surprisingly found that cruciferous plant seeds contaminated with black rot fungi can be sterilized by treatment with ozone gas (gas), thereby completing the present invention.

That is, the present invention includes the following inventions.
(1) A method for sterilizing black rot fungi in a seed of a cruciferous plant, wherein the seed of the cruciferous plant is treated with ozone gas.
(2) The method according to (1), wherein the seed is rolled during the treatment.

  The seeds of cruciferous plants sterilized by the method of the present invention do not require drying. The seed can be sterilized in a relatively short time. In addition, since ozone gas is converted into oxygen and discharged in the case of disposal, it can be said that the method of the present invention has little adverse effect on the environment.

  Examples of the cruciferous plant seeds that can kill black rot fungi by the method of the present invention include seeds such as Chinese cabbage, chingensai, turnip, komatsuna, cabbage, cauliflower, broccoli, caylan, saicin, tarsai, radish, etc. Is not limited. The seed used in the present invention is a seed whose surface is not wet (ie, the surface is dry).

In the present invention, black rot fungus refers to Xanthomonas campestris pv. Campestris.
According to the present invention, a method for sterilizing black rot fungi in seeds of cruciferous plants is characterized by treating the seeds of cruciferous plants with ozone gas.

  The ozone concentration of the ozone gas used in the present invention is not particularly limited as long as it is a concentration effective for sterilizing black rot fungi, but a higher concentration is more preferable. This is because the higher the ozone concentration, the higher the sterilizing effect and the shorter the time. The ozone gas can be generated using an ozone generator. In order to generate high-concentration ozone gas, it is more preferable to use an ozone generator capable of generating ozone gas having a concentration of 500 ppm or more. Commercially available ozone generators include EO-1 manufactured by Techjam Co., Ltd. (ozone generation 50 to 100 mg / h, ozone concentration 80 to 150 ppm, flow rate 1 liter / min), EO-4 (ozone generation 1,000) mg / h, ozone concentration at the gas outlet 500 to 900 ppm, flow rate 5 liters / minute), ED-OG-A7TM from Ecodesign Co., Ltd. (ozone generation amount 1,500 mg / h, ozone concentration 500 ppm, raw material gas flow rate 30 liters / minute) ) And the like. A suitable ozone gas concentration range is that when ozone gas is injected into a container having a capacity of about 85 L for 45 minutes or more, preferably 1 hour or more, more preferably 2 hours or more using these commercially available ozone generators. It can also be defined as the range of ozone concentration in the container.

  The ozone gas used in the present invention is substantially free of water. That is, seed treatment with ozone gas is performed under dry conditions. “Substantially free of water” means that water is not added artificially or exogenously.

  The treatment with ozone gas is performed by bringing the seeds of cruciferous plants into contact with ozone gas in an atmosphere of ozone gas. In order to increase the chance of contact, it is preferred that the seeds being treated are rolled continuously or at regular time intervals. When rolling is not performed, it is preferable to spread the seeds so that four or more layers do not overlap and stand still. The ozone gas may be appropriately flowed.

  The treatment of the cruciferous plant seeds with ozone gas is preferably performed at a temperature at which the seeds are normally stored, for example, at room temperature (around 20 ° C.). The treatment time is preferably 0.5 to 5 hours.

Outline of Experimental Procedure In the following experiment, an ozone generator, EO-1 manufactured by Techjam Corporation was used as an ozone generator. The capacity of EO-1 is an ozone generation amount of 50 to 100 mg / h, an ozone concentration at the generation port of 80 to 150 ppm, and a flow rate of 1 liter / min. The ozone treatment experiment was conducted in an acrylic desiccator (length 74 x width 34 x height 34 cm). A small hole was made in this desiccator, a tube connected to the ozone generation port of the ozone generator was inserted, and ozone gas was injected into the inside (see FIG. 1).

  After injecting ozone gas into the desiccator for 45 minutes in advance, seeds were inserted and ozone gas was injected for a predetermined time. The black rot fungus on the medium was treated for 1 minute, 10 minutes, 30 minutes, 1 hour and 2 hours. When treating black rot-contaminated seeds, a small amount of seeds were placed in a single, double or triple manner, or treated with ozone gas for 1 hour using a large amount of seeds.

  When the seeds were put into a net container and subjected to ozone gas treatment, the seeds were placed in the container shown in FIG. 2, and the seeds were moved by moving the container at intervals of 10 to 15 minutes and exposed to ozone gas.

The treatment with ozone gas was performed at room temperature (20 to 25 ° C.).
The black rot fungus on the petri dish treated with ozone gas in Example 1 was placed in a constant temperature room at 25 ° C. and cultured for 5 days, and then the number of colonies of the black rot fungus was investigated.

  When investigating the fungus in the seeds, the treated and untreated seeds (10 ml) are ground in a mortar, added with physiological saline, centrifuged for 20 minutes (2,000 rpm), and the supernatant (if necessary) Was diluted in a black rot fungus selection medium (NV medium) and cultured in a thermostatic chamber at 25 ° C. for 5 days, and then the black rot fungus was examined from the shape of the colonies.

Example 1 Bactericidal Effect of Ozone Gas against Black Rot Fungus on Medium Medium Brassica black rot fungus (Xanthomonas campestris pv. Campestris) was spread on an NV medium, dried, and then placed in a desiccator filled with ozone gas. After culturing in a constant temperature room at 25 ° C. for 5 days, the number of colonies of black rot fungi was examined. The results are shown in Table 1, FIG. 3 and FIG.

  The black rot fungus was partially sterilized by treatment for 1 minute (FIG. 4), but was sterilized by treatment for 10 minutes or more and could not grow on the medium (Table 1 and FIG. 3). It was revealed that the pathogenic bacteria of black rot were sterilized by ozone gas treatment in 10 minutes.

Example 2 Disinfection effect of artificially contaminated seed of black rot fungus of Chingensai with ozone gas (1)
To prepare artificial seeds of black rot fungus, cultivate black rot fungus on potato, dextrose, agar medium (PDA medium), dilute the fungus concentration to 4 × 10 ¹⁰ cfu / ml, and let the seeds of chingensai for 15 minutes Soaked. The seeds filtered with gauze were spread on paper and dried naturally to obtain artificially contaminated seeds.

  We investigated the disinfection effect against black rot fungus by ozone gas treatment using artificially contaminated seeds of Chingensai. First, in the desiccator filled with ozone gas for 45 minutes, Chingensai seeds, which are artificially contaminated with black rot fungi, were placed and treated for 1 hour. Contaminated seeds are divided into three sections, treated with ozone gas using three types: (1) seeds with one layer, (2) seeds with two or three layers, and (3) seeds with contaminated seeds embedded in a large number of seeds. did.

  The treated seed (10 ml) is ground in a mortar, added with physiological saline and centrifuged for 20 minutes (2,000 rpm), and the supernatant is diluted 100 times or 10 times, and the supernatant or diluent Was placed on a black rot fungus selection medium (NV medium) and cultured in a constant temperature room at 25 ° C. for 5 days, and then the black rot fungus was examined from the shape of the colony. The results are shown in Table 2, FIG. 5 and FIG.

  As a result of the investigation, in a test conducted using artificially contaminated seeds of Chingensai, ozone gas reaches the seeds if the seed is a thin layer of 2 to 3 layers, and the number of black rot fungi is greatly reduced by ozone gas treatment Was recognized. Since artificially contaminated seeds contaminated with high concentrations were used, they were not sterilized until no bacteria were detected from the seeds. However, most of the black rot fungi on the contaminated seeds were sterilized and were found to have a high disinfection effect. (Table 2, FIGS. 5 and 6).

Example 3 Disinfection effect of artificially contaminated seeds of Chingensai black rot fungus by ozone gas (2)
We investigated the disinfection effect against black rot fungus by ozone gas treatment using artificial polluted seeds of Chingensai. The artificial contamination seed of Chingensai was placed in a desiccator filled with ozone gas using (1) a seed with one layer and (2) a seed placed in a net container and treated for 1 hour. During the treatment, the seed was moved by moving the container at intervals of 10 to 15 minutes so that the entire surface of the seed could come into contact with ozone gas. The seeds after the treatment were examined for black rot fungus by the same method as described above. The results are shown in Table 3 and FIG.

  As a result of the investigation, in the seeds treated with ozone gas, black rot fungi were detected in both (1) seeds in one layer and (2) seeds in the net container, but the number of colonies was extremely small, and ozone gas It shows that the disinfection effect by is high.

Example 4 Disinfection effect of black rot fungus naturally contaminated seed of Chinese cabbage Using the black rot fungus naturally contaminated seed of Chinese cabbage, the disinfection effect against black rot fungus by ozone gas treatment was investigated. Naturally contaminated Chinese cabbage seeds were placed in a net container. During the treatment, the seeds were moved by moving the container at intervals of 10 to 15 minutes so that the entire surface of the seeds could come into contact with ozone gas, and treated with ozone gas for 1 hour. The seeds after the treatment were examined for black rot fungus by the same method as described above. The results are shown in Table 4 and FIG.

  Ten black rot fungi per petri dish were detected from untreated Chinese cabbage seeds, but the detection of the fungi was significantly reduced by moving the seeds and treating with ozone gas. Although it was not possible to sterilize until no fungus was detected, it was shown that natural polluted seeds also have a high disinfection effect by ozone gas treatment. As a result, it was shown that the ozone gas treatment of seeds has a high disinfection effect against black rot fungi.

Experimental Example 5 Influence of ozone gas treatment on germination rate of Chingensai seeds After treatment of Chingensai seeds with ozone gas for 1 hour, the germination rate was investigated. Seeds treated with ozone gas and untreated are placed on a petri dish with a diameter of 9 cm, seeds are placed on the floor, 4 ml of water is added, cultured in a constant temperature room at 25 ° C, 8 hours light, 16 hours dark for 3 days to germinate The rate was investigated. As a result, normal germination was observed in the germination rate of chingensai seeds treated with ozone, which was the same as that of untreated seeds. The results are shown in Table 5 and FIG.

Experimental Example 6 Influence of ozone gas treatment on germination rate of turnip seeds After treatment of turnip seeds with ozone gas for 1 hour and 3 hours, the germination rate was investigated. The ozone gas treatment is the same as that of Chingensai. As a result, the germination rate of turnip seeds treated with ozone for 1 hour and 3 hours was not different from that of untreated, and normal germination was observed.

The desiccator connected to the ozone generator used for seed disinfection is shown. Fig. 2 shows a mesh container for stirring seeds. A black rot fungus is transplanted to a selective medium, treated with ozone gas for 10 minutes to 2 hours, and a colony of the fungus after culturing in a constant temperature box is shown. The black rot fungus is transplanted to the selective medium, treated with ozone gas for 1 to 10 minutes, and the colony of the fungus after culturing in a constant temperature box is shown. Chingensai artificially contaminated seeds are treated with ozone gas for 1 hour as a single layer, two layers, or a lump, and the seeds are ground, centrifuged, and then 10 times diluted supernatant is tested for black rot using selective medium It is a figure which shows the result of having performed. Chingensai artificially contaminated seeds are treated with ozone gas for one hour as a single layer, two layers, or a lump, and the seeds are ground, centrifuged, and then 100 times diluted supernatant is tested for black rot using selective medium It is a figure which shows the result of having performed. Place artificial seeds of Chingensai artificially in a net container, move the container from time to time, move the seeds for 1 hour so that they are exposed to ozone gas, grind the seeds, centrifuge, and then dilute the supernatant 100 times It is a figure which shows the result of having examined the black rot fungus using the selective culture medium. It is a figure which shows the result of having test | inspected black-rot fungus using the selective culture medium for the Chinese cabbage natural polluted seed for 1 hour, grind | pulverizing the seed and centrifuging and centrifuging the supernatant. It is a figure which shows the result of investigating the influence which germin seeds treat with germinating for 1 hour and germinate.

Claims (2)

  A method for sterilizing black rot fungi in cruciferous plant seeds, characterized by treating the cruciferous plant seeds with ozone gas.   The method according to claim 1, wherein the seeds are rolled during the treatment.

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