JP4606131B2 - Rice seedling disease control agent and control method - Google Patents

Description

  The present invention relates to a rice seedling disease control agent and control method using a bacterium belonging to the scientific name Bacillus simplex.

  Bacterial wilt of rice seedlings and bacterial wilt of rice wilt, which are bacterial wilt diseases of rice seedlings, are seed-borne diseases that occur during rice seedling raising.

  Particularly in warming seedlings, these bacterial wilt diseases cause seedlings to decay, die or germinate poorly, and seedlings with these diseases cannot be transplanted to Honda.

  Currently, several chemical agents such as oxolinic acid, inorganic copper agent, and kasugamycin are used as seed disinfectants for controlling these diseases. However, these chemical agents have problems such as phytotoxicity, waste liquid treatment after disinfection, the emergence of resistant bacteria to seed disinfectants, and the fact that they do not meet the recent trend toward reducing pesticides and pesticide-free consumers. There is.

  Therefore, for the control of these diseases, it has been desired to develop a control agent having a high control effect and free from environmental pollution such as water pollution.

On the other hand, regarding the biological control method, Pseudomonas sp. CAB-02 and Pseudomonas sp. Patent Document 2 discloses Tricoderma atroviride and the like.
JP-A-9-124427 Japanese Patent Laid-Open No. 11-225745

  The microorganisms disclosed in Patent Document 1 and Patent Document 2 have a drawback that storage stability is somewhat difficult when a preparation is made, and the period is limited to circulation at room temperature.

  An object of the present invention is to control rice seedling diseases that are highly effective in storage and storage stability and are free from environmental pollution. It is to provide an agent and a control method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have a high control effect on the rice seedling bacterial blight disease and the rice blight bacterial disease that are the bacterial bacterial disease of rice seedlings. The present inventors have found a microorganism belonging to the Bacillus simplex having the present invention, and have reached the present invention.

  The Bacillus genus plexus is a strain selected from the results of seedling test for rice seedling bacterial disease and rice wilt bacterial disease from about 7000 bacterial strains isolated and collected from rice and vegetables. In addition to strongly suppressing the onset of bacterial wilt of rice, it has excellent storage stability and is highly practical as an agricultural chemical. There have been no examples of biocontrol using Bacillus simplex so far.

That is, the present invention comprises a Bacillus simplex CGF2856 strain and a live bacterium as an active ingredient, which is a bacterial wilt disease of rice seedlings, And a method for controlling rice seedling diseases using the same.

  The microorganism of the present invention is a Bacillus simplex CGF2856 strain having a high control effect against rice seedling blight and rice blight bacteria.

  The bacteriological properties of CGF2856 strain are shown below.

  As a result of morphological observation with an optical microscope and an electron microscope, the size of the cells was 1 to 3 μm bacilli, there was no cell polymorphism, and none of them had motility. Gram reaction was positive and formed endospores.

Other bacteriological properties are shown below.
1. Culture properties
The growth state of CGF2856 on nutrient agar is shown below. Observation was performed after culturing at 30 ° C. for 3 days.

The colony morphology of CGF2856 is cream, round, raised half-lens, fringe, smooth surface, opaque, viscous butter-like.
2. General properties

３．16ＳｒＤＮＡ分析
16ＳｒＤＮＡ（16ＳｒＲＮＡ遺伝子）の塩基配列1503ｂｐ（塩基対）を決定した。解析は得られた16ＳｒＤＮＡの塩基配列を用い、対象としてInt.J.Syst.Bacteriol.,1989,39,93-94に記載の、バチルス属シンプレクスの16ＳｒＤＮＡの塩基配列と相同性検索を行った。その結果、相同率99.93％でバチルス属シンプレクスの16ＳｒＤＮＡに高い相同率を示し、両者の16ＳｒＤＮＡ間の相違点は複合塩基で１塩基のみであった。

3. 16S rDNA analysis
The base sequence 1503 bp (base pair) of 16S rDNA (16S rRNA gene) was determined. For the analysis, the obtained 16S rDNA base sequence was used to search for homology with the base sequence of Bacillus simplex 16S rDNA described in Int. J. Syst. Bacteriol., 1989, 39, 93-94. As a result, the homology rate of 99.93% was high, and the 16S rDNA of the Bacillus simplex showed a high homology rate.

  Due to the above bacteriological properties, CGF2856 is a Gram-positive bacilli that has no motility and belongs to the genus Bacillus by viability under aerobic conditions, positive catalase activity, negative oxidase activity, and forming spores. Classified as bacteria. Moreover, it was identified as Bacillus simplex from 16S rDNA analysis.

  The relevant strain of the present invention has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and has the following deposit number.

  Bacillus simplex CGF2856 (FERM P-20267)

  With the use of the rice seedling disease control agent or method according to the present invention, it is possible to strongly suppress the onset of rice seedling bacterial rot and rice wilt bacterial disease, which is equivalent to or better than currently used chemical agents. The effect of.

  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, PS (Potato sucrose) medium, PDB (Potato dextrose broth) medium, and the like.

The culture can also be performed using a solid medium obtained by adding agar to these mediums. Cultivate in the medium as described above at 15 to 42 ° C., preferably 28 to 35 ° C. for 24 to 36 hours, proliferate and then concentrate by centrifuge or membrane concentrator to collect and remove medium components . By this operation, the concentration of the bacterial cells is usually concentrated to about 1 to 50 × 10 ¹⁰ cfu / ml.

  Then, a protective agent consisting of a saccharide, sodium glutamate, and a sodium phosphate buffer is added to the wet cells and vacuum dried.

  In order to maintain the survival rate of 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.

  For immobilization of the Bacillus simplex strain of the present invention, it is preferable to use a saccharide consisting of one or more of saccharose, fructose, glucose and sorbitol as a protective agent. This can be done by drying in a method.

  This bacterium, which is a Gram-positive spore-forming bacterium, Bacillus simplex, is converted from nutrients to spores and then centrifuged or concentrated, or the concentrated cells by centrifugation or membrane concentration are converted from nutrients to spores. After the conversion, the cells can be dried by a dryer or a spray dryer.

  As a general method for converting Bacillus bacterium nutrients to spore form, in the literature “Science of Microorganisms” Vol. 3, page 25, the nutrients such as glucose on the medium are starved by dilution or washing. It is disclosed to do.

  The Bacillus simplex converted to a spore shape can then be stored and distributed as a stable preparation by adding mineral powder, a surfactant or a stabilizer.

  In general, rice seedlings are soaked in cold water for about one week (sowing) before sowing (sowing), and the seed soak is sufficiently absorbed with water, and then soaked in water at around 30 ° C. (germination) for seeding for one day.

  The control agent of the present invention is a rice seedling blight and rice blight bacterial disease control agent that can be used widely without any problem of phytotoxicity and no limitation on the time of use. The control agent of the present invention, soaking seeds at the time of sowing or sprouting, wet seeding before sowing, before germination or before sowing, mixing with soil or covering soil, or sowing Later, the diluted solution of the control agent is poured into the soil and used.

When the control agent of the present invention is used in the above method, in the case of immersion treatment or soil irrigation treatment, the concentration of bacteria in the immersion liquid is 10 ⁶ cfu / ml or more, preferably 10 ⁷ cfu / ml or more. adjust. In the wet dressing treatment method, the control agent of the present invention having a weight of 0.1% or more, preferably 1% or more with respect to the dry rice cake is uniformly applied to the candy. When the soil is mixed with the soil or cover soil, 1 g or more of the present control agent is mixed with 1 L of the culture soil and stirred to be uniform.

  In addition, the temperature of the diluting solution when the seed pod is dipped in the present control agent diluting solution at the time of soaking or germination is 10 ° C. to 35 ° C., preferably 15 ° C. to 30 ° C., and instantaneously to 48 hours, preferably 1 Process for 24 hours.

  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.

The Bacillus simplex CGF2856 strain cultured here can be mixed with a carrier (a bulking agent) to form a powder or a granule. The types of carriers in this case include mineral powders such as talc, kaolin, clay, calcium carbonate and diatomaceous earth, peat moss, and polymer compounds such as polyvinyl alcohol, and natural polymer compounds such as xanthan gum and alginic acid. And can be used with or without saccharides. In the case of a liquid preparation, the concentration of bacterial cells is 10 ⁶ cfu / ml or more, preferably 10 ⁷ cfu / ml or more. In the case of a solid (wettable powder, powder), it is 10 ⁶ cfu / g or more, preferably 10 ⁷ cfu / g or more.

  Next, the selection of Bacillus simplex CGF2856 strain will be described in detail. The Bacillus simplex CGF2856 strain was selected from about 7000 strains of bacteria isolated and collected from rice and vegetables as a result of seedling growth tests for rice seedling bacterial blight and rice blight bacterial disease. 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.

About the obtained preservation | save strain, the prevention | control test with respect to rice seedling bacterial disease and rice bacterial bacterial disease was done. In the method, a 10 ⁸ cfu / ml diluted solution of the test strain was immersed in a rice seedling-borne bacterial disease-carrying bacterium for 24 hours before sowing, and after sowing, seedlings were grown for 2 weeks to investigate the disease. As a result, several strains that suppress the pathogenesis of rice seedling blight were confirmed.

  Then, the control effect with respect to a rice wilt bacterial disease was examined about the strain which suppresses the onset of these rice seedling wilt.

  As a result, a Bacillus simplex CGF2856 strain having a strong disease-suppressing effect against rice seedling blight and rice blight bacteria was selected.

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

Inhibitory effect of CGF2856 strain on bacterial wilt of rice seedlings
After the CGF2856 strain was grown in a broth culture cell solution, the supernatant was removed by centrifugation and the concentration of CGF2856 in the cell solution was adjusted to 10 ⁸ cfu / ml. Rice seedling bacterial disease contaminated culm was put into this bacterial solution, and pre-soaking treatment was performed at 15 ° C. for 24 hours. Then, in accordance with a conventional method, seeding was carried out after soaking and germination, and seedlings were raised in a glass greenhouse for 2 weeks.

  As a result, as shown in Table 2, in the untreated group, the disease was severely ill and most seedlings died, but in the CGF2856-treated group, almost no disease was observed, and a high disease-inhibiting effect was observed. The rice seedling bacterial disease test was evaluated by calculating the diseased seedling rate, disease severity, and control value from the degree of seedling breeding.

Disease index 5: Withering, 3: Disease and plant height is less than 1/2 of healthy, 1: Disease and plant height is 1/2 or more of healthy, 0: Healthy diseased seedling rate (%) = 100 x {(number of diseased seedlings ) ÷ (Total number of surveyed seedlings)}
Incidence of disease = 100 × {Σ (the value of the exponent) × (number body number corresponding to each index)} ÷ {5 × (survey number seedlings)}
Control value = 100 × {(morbidity in untreated area) − (morbidity in treated area)} ÷ (morbidity in untreated area)

Inhibitory effect of CGF2856 strain on bacterial wilt of rice seedlings

Inhibitory effect of CGF2856 strain on bacterial wilt of rice
After the CGF2856 strain was grown in a broth culture cell solution, the supernatant was removed by centrifugation and the concentration of CGF2856 in the cell solution was adjusted to 10 ⁸ cfu / ml. Rice seedling bacterial disease contaminated culm was put into this bacterial solution, and pre-soaking treatment was performed at 15 ° C. for 24 hours. Then, in accordance with a conventional method, seeding was carried out after soaking and germination, and seedlings were raised in a glass greenhouse for 2 weeks.

  As a result, as shown in Table 3, in the untreated group, the disease was severely ill and most seedlings died, but in the CGF2856-treated group, almost no disease was observed, and a high disease-inhibiting effect was observed. The rice bacterial wilt disease test was evaluated by calculating the diseased seedling rate, disease severity, and control value from the degree of seedling breeding. In addition, about the diseased seedling rate, disease incidence, and the calculation method of the control value, it carried out similarly to Example 1.

Inhibitory effect of CGF2856 strain on bacterial wilt of rice

Inhibitory effect of CGF2856 strain on bacterial wilt of rice seedlings
Using the preparation of CGF2856 strain, we investigated the disease-suppressing effect against rice seedling bacterial disease. Preparation of the preparation was performed by appropriately diluting the cells cultured and dried by the above-mentioned method with a carrier (a bulking agent) and adjusting the concentration of the bacteria to 2 × 10 ⁹ cfu / g. The CGF2856 preparation was treated with a 200-fold diluted solution at 15 ° C. for 24 hours before soaking.

  The results are shown in Table 4. The CGF2856 preparation also showed a high disease suppression effect. The CGF2856 preparation was tested for bacterial wilt of rice seedlings by calculating the diseased seedling rate, disease severity, and control value from the seedling growth level. In addition, about the diseased seedling rate, disease incidence, and the calculation method of the control value, it carried out similarly to Example 1.

Inhibitory effect of CGF2856 strain on bacterial wilt of rice seedlings

Claims (5)

A control agent comprising Bacillus simplex CGF2856 strain as an active ingredient, which has the ability to suppress the disease of rice seedlings. The control agent according to claim 1, which is immobilized using a saccharide. The method for controlling rice seedlings diseases consists of using the controlling agent according to claim 1 or 2. Bacillus simplex CGF2856 strain having the ability to suppress the disease against rice seedling diseases. Claim 4 rice seedlings disease rice bacterial seedling blight according to a rice paddy bacterial grain rot, bacterial strains according to claim 4.