JPH04295407A - Microorganism capable of controlling disease injury of gramineous crop and method for controlling disease injury - Google Patents

Abstract

PURPOSE:To provide a microorganism capable of controlling disease injury of gramineous crops and a method for controlling the disease injury by using the aforementioned microorganism. CONSTITUTION:New Pseudomonas glumae N7503 (FERM P-12105) effective in controlling disease injury of gramineous crops. A method for controlling the disease injury of the gramineous crops, especially controlling rice plant seedling rot by inoculating the aforementioned strain into rice plant seeds or adding and mixing the strain in soil is provided.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides a novel microorganism effective for controlling diseases of grass crops, particularly rice seedling rot and rice blast blight, and a method for controlling diseases of grass crops using this microorganism. Regarding.

0002

[Prior art] Pseudomonas blight of rice
Bacterial blight of rice, a disease caused by Nas glumae), is widely distributed in rice-growing regions in Southeast Asia, including Japan, Korea, and China, and causes serious damage to rice yields. Initially, this pathogenic bacterium was thought to cause disease only in rice ears, but subsequent research has revealed that this bacterium is also pathogenic to young rice seedlings. Damage to young seedlings has become a serious problem in Japan due to the spread of seedling boxes based on the special characteristics of wet rice cultivation. Therefore, a wide range of research has been carried out on the rice bacterium blight disease fungus, including elucidation of its developmental ecology, relationship between cultivation methods and disease onset, and development of control methods. However, the route of transmission and the mechanism of invasive infection are still unclear, and no accurate control method has yet been established. Currently, control measures include removal of diseased firs by salt water selection, seed disinfection with various chemicals, and kasugamycin and
Examples include application of captan hydrating agents, polycarbamate hydrating agents, etc. in seedling boxes.

0003

[Problems to be Solved by the Invention] However, these drugs have unstable effects, differ in effectiveness depending on the bacterial strain, and can cause growth-suppressing drug damage, so further study is required before putting them into practical use. The current situation is that it must be done. The use of resistant varieties is also being considered, but it is not clear whether or not resistant genes for this bacterial disease exist in rice. The current situation is that the pest control methods used to date are not sufficiently effective.
Development of new control methods is desired.

[0004] In recent years, as a means of controlling soil-borne plant diseases, the development of biological control methods more based on nature has become important. Among these, research on the use of rhizosphere microorganisms capable of producing various antibacterial substances is being carried out around the world, and several successful cases have been reported. There are also many studies using non-pathogenic mutant strains that have lost or eliminated pathogenicity from pathogenic bacteria. Some of these non-pathogenic strains proliferate within the host in the same way as pathogenic strains, and exhibit almost the same ecological behavior as pathogenic strains. By pre-treating a host plant with such a non-pathogenic strain, it is possible to prevent the invasion of subsequently inoculated pathogenic bacteria or to suppress their proliferation, thereby suppressing the onset of the disease. Based on this idea, the present inventors have repeatedly investigated a method for biologically controlling rice rice blast blight by using a non-pathogenic rice rice blast blight bacterium, and have now accomplished the present invention. Ta.

[0005]

[Means for Solving the Problems] That is, the present invention provides a novel non-pathogenic Pseudomonas glume that is effective for disease control of grass crops.
related to mae). Furthermore, the present invention relates to a method for controlling diseases of gramineous crops by inoculating rice seeds with this novel Pseudomonas gourmand or spraying it on the soil. The diseases of gramineous crops in the present invention are:
It refers to rice seedling rot and/or rice rice blight. The novel Pseudomonas gourmet in the present invention has been deposited with the FIKEN Co., Ltd. as FAIKEN Deposit No. 12105. Furthermore, in the present invention, strains obtained by mutating this strain by radiation, chemicals, etc. are also included in the strain of the present invention, as long as they can control diseases of grass crops and are not pathogenic.

[0006] When inoculating rice seeds with this new Pseudomonas gourmet, the strain concentration is 108 to 1012.
This is preferably carried out by immersing rice seeds in a cfu/ml suspension. In addition, new Pseudomonas
When controlling by spraying Gourmet on the soil, 107 to 1
It is preferable to spray at a bacterial cell concentration of 0.010 cfu/g-soil.

[0007] The present inventors selected rice plants infected with rice blight from rice grown in rice fields in the Fukuoka region, immersed them in a 70% ethanol aqueous solution for several seconds, and then reduced the effective chlorine concentration to 3. % sodium hypochlorite aqueous solution for 5 minutes, rinsed with sterile distilled water, and ground it.
Expand on A plate culture medium and add 47 rice blight bacteria from now on.
The bacterial strain was isolated.

These rice blight bacteria have the following mycological properties. It is rod-shaped and has polar hairs. It is aerobic, and the suitable growth temperature is 30-35℃, and can grow even at 40℃. Colonies appear grayish white. Can be grown on King's medium. Gelatin liquefaction, nitrate reduction, litmus milk reduction, ammonia production,
Hydrogen sulfide production, catalase, and lecithinase were all positive. Indole production, MR reaction, oxidase are
Both are negative.

Next, among these, the only difference in mycological properties is that it is not pathogenic not only to rice but also to other plants, and in other respects it shows the same properties as rice blight. A non-pathogenic rice blight bacterium was selected.

This selection will be explained in more detail. 1. Selection of a non-pathogenic rice blight bacterium Bacterial blight bacterium is a pathogenic bacterium that attacks rice seedlings and ears, so even if it is effective in suppressing the onset of the disease, it cannot be used as is for biological control. It is a danger. However, this bacterium tends to lose its pathogenicity in vitro, and may lose its pathogenicity during storage or subculture. On the other hand, the present inventors have discovered that this bacterium is not only pathogenic to rice but also has the property of rotting tissue sections of potatoes, carrots, etc. Therefore, we investigated the pathogenicity of this fungus to various crops in order to select strains that are non-pathogenic to all crops.

As plant materials, potatoes (May Queen), carrots (variety unknown), tomato seedlings (Toko), and rice seeds (Asominori) were used in the experiment. The decomposition ability of various vegetable tissues was tested in the following manner. Mix various vegetable tissues with 3% sodium hypochlorite (antiformin).
Sterilize the surface by soaking for 5 minutes, then rinse thoroughly with sterile water, cut into pieces approximately 5 ml thick with a sterile scalpel, place in a 9 cm diameter sterile petri dish lined with filter paper, and add 5 ml of sterile water. 48 at 30°C on PSA slants.
Place 1 hour of cultured rice bacterium bacterium on the center of the cut surface.
After inoculating a platinum loopful amount, the plants were allowed to stand at 30°C for 48 hours, and the presence or absence of putrefaction ability was examined. To examine the pathogenicity of tomato seedlings,
Each strain of rice bacterium blight was cultured on a PSA slant medium at 30°C for 48 hours and was scraped off from the slant with a sterilized platinum loop, and then applied to the stems of tomato seedlings (3 weeks after sowing) whose surface had been previously sterilized with 70% ethanol. This was done by multi-needle inoculation. The pathogenicity to rice was examined as follows. That is, seeds of the test variety Asominori were disinfected with 3% sodium hypochlorite (antiformin) for 30 minutes, thoroughly washed with water, and soaked at 25° C. for 2 days. Take 60 of these seeds in a sterile petri dish with a diameter of 9 cm, and add a concentration of about 109
15 ml of cfu/ml bacterial suspension was injected and inoculated by immersion at 30° C. for 24 hours. After that, the seeds were sown in a 60 x 60 x 45 mm plastic container filled with 80 g of sterilized compost (manufactured by Mitsui Toatsu) in an autoclave and 30 ml of sterile distilled water. In an inoculation box at 32℃ (humidity 100%)
Seedlings were grown for 2 days. Furthermore, the presence and severity of disease onset was investigated on the 15th day after inoculation after greening in an air-conditioned greenhouse at 25°C.

0012

[Table 1]

The results are shown in Table 1. There were 15 strains that were not pathogenic to rice. No pathogenicity to tomatoes was observed in any of the tested strains. Tomatoes that do not exhibit decomposition ability for various vegetable tissues,
8 bacterial strains that are not pathogenic to rice, namely N.
7504, N7503, YN7810, YN7805,
YN7825, 750, 752, and 805 were selected. Furthermore, a high correlation was observed between the spoilage ability for potatoes and the pathogenicity for rice among the 47 strains tested. It is thought that these 15 bacterial species lost their pathogenicity to rice during subculture or storage. These non-pathogenic bacteria do not exhibit pathogenicity to other plants such as potatoes.

Next, it was investigated whether these non-pathogenic bacteria could suppress the onset of rice seedling rot caused by Bacillus blight of rice. 2. Effect of suppressing the onset of rice seedling rot caused by non-pathogenic rice bacterium blight
u82-34-2) and 5 non-pathogenic strains (N750
3, N750, YN7810, YN7825, 805)
was used in an experiment. Asominori rice variety was used. The effect of suppressing the onset of rice seedling rot caused by non-pathogenic rice bacterium blight was tested using the following method. That is, water-preserved bacteria were transplanted onto a YPDA slant medium, cultured at 30°C for 48 hours, and then suspended in 10 ml of sterile distilled water (concentration: approximately 109 cf
u/ml), add this to 200ml of YPD liquid medium,
Shaking culture was performed at 30°C for 48 hours. After that, 3,600
Centrifugation was performed at xg for 20 minutes, and the resulting bacterial cells were suspended in sterile distilled water to a concentration of about 1010 cfu/ml, and methylcellulose was added to the suspension to a concentration of 1.5%. Rice seeds were surface sterilized with 3% sodium hypochlorite (antiformin) for 30 minutes, thoroughly washed with sterile water, and immersed in a bacterial suspension containing methylcellulose at 30°C for 24 hours. As a control, seeds soaked in a 1.5% methylcellulose solution containing no bacteria were used. The pathogenic strain was cultured in the same manner as the non-pathogenic strain, and the concentration was adjusted to approximately 108 cfu/ml with sterile water, and this was used as the inoculation source. Bacterial concentrations of non-pathogenic and pathogenic strains were determined at YP for each experiment.
It was determined using a DA plate medium and according to the dilution plate method. Rice seeds immersed in a non-pathogenic bacterial strain solution were placed in a 60 x 60 x 45 m container containing about 80 g of sterilized mixed soil (manufactured by Mitsui Toatsu).
Sow 60 seeds each in a plastic container of
0 g), then 10 ml of a bacterial suspension of a pathogenic strain as an inoculum was inoculated by irrigation. The treated containers were placed in an inoculation box (humidity: 100%) at 32°C for 48 hours to germinate, and then placed in an air-conditioned greenhouse at 25-30°C or a plant growth chamber at 28°C for greening. Irrigation was performed once a day. The disease severity was determined on the 15th day after inoculation. All experiments were repeated two or more times. As shown in Figure 1, the severity of the disease is tested in six stages from 0 to 5, depending on the severity of the disease symptoms.
Incidence level 0 means healthy seedlings, 1 means the same height as healthy seedlings but chlorosis appears on the leaf blade, 2 means seedlings that are shorter than healthy seedlings and have necrosis at the base of the leaf sheath, 3 means chlorosis on the leaf blade. In addition to necrosis at the base of the leaf sheath, the whole body exhibits an abnormal morphology; in 4, only the first true leaf has developed and the entire leaf has defoliated; and in 5, it has rotted and died). The severity of the disease was determined using the average value of 60 grains in each group.

The results of the experiment are shown in Table 2. As is clear from this table, it was revealed that soaking rice seeds with a non-pathogenic bacterial strain suppressed the onset of rice seedling rot. However, the degree of the effect of suppressing the onset of the disease differs markedly depending on the combination of non-pathogenic and pathogenic strains, ranging from no effect at all to a high level of effect of suppressing the onset of the disease. That is, for pathogenic strain So-1, non-pathogenic strains N7503 and N75
0, YN7825, and 805 suppressed the onset of the disease. On the other hand, no inhibitory effect was obtained even when treated with YN7810. Against pathogenic strain 2, N7503 and 805 were found to be effective in suppressing the onset of the disease, but N750 and YN7810
, YN7825 did not inhibit it at all. For pathogenic strain Kyu82-34-2, non-pathogenic strain N75
The disease onset suppressive effect was observed only when treated with 03, and non-pathogenic strains N750, YN7810, YN7825, and 8
Treatment with 05 did not suppress the onset of the disease. Furthermore, for pathogenic strain Ku8111, N7503, YN78
10 and 805 were found to have a disease onset suppressing effect, N750 had a weak effect, and YN7825 had no effect. As a result, it was confirmed that the onset of rice seedling rot can be suppressed by treating rice seeds with the non-pathogenic strain N7503.

[0016]

[Table 2]

3. Relationship between bacterial concentration for immersion treatment and disease onset suppressing effect We therefore investigated the relationship between the bacterial concentration of this non-pathogenic strain N7503 and disease onset suppressing effect. N7503 was used as a non-pathogenic strain, and So-1 was used as a pathogenic strain. The disease onset suppressive effect was tested according to the method described above. That is, each strain stored in water was spread on a YPDA plate medium to isolate a single colony, and this was inoculated into a Sakaguchi flask containing 200 ml of YPD liquid medium, and incubated at 30°C.
Culture with shaking for 48 hours, then centrifuge (8,000 x g,
For about 10 minutes, the bacterial cells obtained by
Suspend to a concentration of 10 cfu/ml and serially dilute it 10 times to obtain approximately 1010, 108, 106
A cfu/ml bacterial suspension was made. Add 1 methyl cellulose to each suspension of non-pathogenic strain N7503 at each concentration.
．． Rice seeds were surface sterilized with 3% sodium hypochlorite (antiformin) and then soaked in this solution, and left to stand at 30°C for 24 hours. After sowing 60 seeds each in a container and covering with 20 g of soil, each concentration of pathogenic strain So-1 was added.
10 ml of bacterial suspension was inoculated by irrigation, placed in an inoculation box at 30°C for 48 hours, placed in a plant growth chamber at 28°C with 12 hours of light per day, and assayed for disease severity on the 15th day. went.

The results are shown in Table 3. As is clear from Table 3, the concentration of the non-pathogenic strain was 1010 cfu/ml.
When rice seeds were treated with this bacterial suspension, a high disease-inhibiting effect was shown even when the concentration of pathogenic bacterial strains in the soil was as high as 1010 cfu/g. However, the bacterial concentration of non-pathogenic strains was below 108 cfu/ml, and
If the bacterial concentration of the pathogenic strain is as low as 106 cfu/g, the disease onset suppression effect cannot be obtained, and in order to obtain the effect, a high concentration (
It became clear that treatment with a suspension of a non-pathogenic strain (1010 cfu/ml) was necessary.

[0019]

[Table 3]

Furthermore, in the present invention, the following test was conducted in order to prove that Pseudomonas gourmet N7503 of the present invention can be used for disease control of grass crops.

[Test 1] Effect of non-pathogenic N7503 strain on disease onset against various pathogenic bacterial strains The disease-suppressing effect of non-pathogenic strain N7503 shows a similar high disease onset suppressive effect on many other pathogenic strains. We considered whether or not this is the case.

That is, among the strains of rice bacterium blight disease preserved in the Department of Plant Pathology, Kyushu University, Ku, which exhibits strong pathogenicity,
8106, Ku8121, III, 8001, 801
A total of 5 strains of 7, I, and Ku8105 were tested, and N7503
The effectiveness of the bacterial strain in suppressing disease onset was investigated. The disease onset suppression test was conducted by the method described above. As shown in Table 4, the results revealed that the non-pathogenic strain N7503 was a strain that had a strong effect on suppressing the onset of disease against all the pathogenic strains tested.

[0022]

[Table 4]

[0023]

[Test Example 2] Comparison of the effect of suppressing disease onset with kasugamycin/captan hydrating powder The effect of the N7503 strain of the present invention on controlling rice seedling rot and the conventional rice seedling box application agent that is effective in controlling rice seedling rot We compared the control effects of kasugamycin and captan hydrating agents.

That is, an experiment was conducted using Asominori as a rice variety with a combination of pathogenic strain So-1 and non-pathogenic strain N7503. The disease onset suppression test was conducted according to the method described above. Kasugamycin captan hydrating agent is 1
The treatment was carried out by pouring 10 ml of the 200-fold diluted solution (1.2 g of kasugamycin + 1.2 mg of captan in terms of bulk) into the sterilized soil after sowing and before covering the soil.

The results are shown in Table 5. The disease-inhibiting effect obtained by pre-treating rice seeds with N7503 is as follows:
Add kasugamycin captan hydrating agent to 0.00% per soil.
The effect of suppressing disease onset was almost the same as that obtained when applied at 4 mg. However, in the plots treated with kasugamycin/captan hydrating agent, some growth inhibition was observed, such as lower seedling height, compared to plots treated with N7503 pretreatment or 1.5% methylcellulose alone. was there. The pre-treatment with a non-pathogenic bacterial strain was almost as effective as the control agent, and there was almost no difference in growth between the untreated and untreated plots.

[0026]

[Table 5]

[0027]

[Test 3] Differences among cultivars in the effect of suppressing disease onset It was revealed that the non-pathogenic injected strain of the present invention, rice quality Asominori, significantly suppresses the onset of rice seedling rot. We investigated whether it is also recognized in rice varieties.

[0028] Namely, as rice varieties, Asominori,
Tachi style, Yellow ball, Chugoku No. 45, Kuju, Aichi Asahi, Norin 2
A total of nine varieties were tested: No. 9, IR64, and Inabawase. Bacteria include N7503, YN7810 and 805 as non-pathogenic strains, and So-1 and Kyu82 as pathogenic strains.
-34-2 and 2 were used. Seed treatment with a non-pathogenic strain, inoculation with a pathogenic strain, and efficacy testing were carried out in the same manner as described above for the disease suppression test.

The results are shown in Tables 6 and 7. The experimental results revealed that there are differences among cultivars in the effect of non-pathogenic strains on disease onset. In the combination of non-pathogenic strain 805 and pathogenic strain Kyu82-34-2, a disease suppression effect was observed in Tachifu, Ogyoku, Chugoku No. 45, and Kuju, but other varieties (Asominori, Aichi Asahi,
Norin No. 29, IR64, Inabawase) did not suppress the disease onset at all. In the combination of non-pathogenic strain 805 and pathogenic strain 2, no disease suppression effect was observed in Aichi Asahi and Chugoku No. 45, slight disease suppression was observed in Norin No. 29, and in other six varieties (Asominori, Tachi style, yellow ball,
It showed a high effect of suppressing the onset of disease in the following plants (Kuju, IR64, Inabawase). In addition, the combination of non-pathogenic strain YN7810 and pathogenic strain So-1 did not have any effect on suppressing the onset of disease in Tachikaze, Kuju, and Inabawase, and did not suppress the disease in other varieties (Asominori, Ogyoku, Chugoku No. 45, Aichi). Asahi,
Norin No. 29, IR64) had a high disease-inhibiting effect. It became clear that the combination of non-pathogenic strain N7503 and pathogenic strain So-1 suppressed disease onset in all varieties. From the above, it was revealed that the expression of the disease suppressing effect involves a specific relationship between the non-pathogenic bacterial strain, the pathogenic bacterial strain, and the rice variety.

[0030]

[Table 6]

[Table 7]

[0031]

[Test 4] Examination of the mechanism of suppressing the onset of rice seedling rot disease 1.
Furthermore, in the present invention, in order to understand the mechanism of suppressing the onset of rice seedling rot caused by the non-pathogenic Bacterium blight of rice, we first investigated the antibacterial activity of the non-pathogenic bacterial strain against the pathogenic strain. i.e., non-pathogenic strains N7503, N750, 805
, YN7810, YN7825 and pathogenic strain Ku81
No. 11,2, So-1, and Kyu82-34-2 were tested. The productivity and activity of antibacterial substances were assayed using a YPDA plate medium using two methods: plate chloroform method and UV irradiation. UV irradiation was performed for 3 hours from a height of 30 cm using a 15 W germicidal lamp (Toshiba GL-15) after removing the top lid of the petri dish in which colony formation was observed after culturing. Thereafter, the colonies were allowed to stand at 30°C for 24 hours, and a pathogenic strain as an indicator bacterium was overlaid. After culturing at 30°C, antibacterial substance productivity was examined based on the presence or absence of an inhibition zone formed around the colony.

As a result, the non-pathogenic strain N750 tested
Antibacterial activity was observed in 3 and N750. The width of the inhibition zone formed by these strains was as small as several mm. However, no direct relationship was observed between the antibacterial activity of non-pathogenic strains against pathogenic strains on the culture medium and the effect of suppressing disease onset.

2. Next, we investigated the effects of known plant pathogenic bacteria and saprophytic bacteria other than rice seedling rot in suppressing the onset of rice seedling rot. Agrobacterium, a plant pathogenic bacterium preserved in the Department of Plant Pathology, Kyushu University
tumefaciens Ku7411, Erwi
nia carotovora subsp. ca
rotovora N7129, Clavivact
er michiganense pv. mich
iganense N6601, Bacillus s
ubtilis ATCC 6633, Pseud
omonas syringae pv. syri
ngae I, and Pseudomonas fl.
Uoresense p-15 was tested. Each of these bacteria was cultured on a PSA slant medium at 30°C for 48 hours, then transferred to a Sakaguchi flask containing 200 ml of YPD liquid medium, cultured at 30°C for 48 hours, and centrifuged (8,0°C).
00 × g, 20 minutes), and then
The suspension was suspended in sterilized water to a concentration of 10 cfu/ml, used for pretreatment, and subjected to the above-described disease suppression test.

As a result, the effect of suppressing the onset of rice seedling rot was demonstrated only by the non-pathogenic rice blight bacterium. It became clear that all other bacteria tested had no effect on suppressing the onset of disease.

3. We also investigated the effect of suppressing disease onset when rice seeds were treated with the culture filtrate of a non-pathogenic strain. That is, non-pathogenic strain N7503 was cultured in YPD liquid medium (
200 ml) and cultured with shaking at 30°C for 4 days,
Centrifuge (8,000 x g, 20 minutes) and remove the supernatant with a pore size of 0.2.
A non-pathogenic strain culture filtrate was obtained by completely removing bacteria through a μm membrane filter. Surface-sterilized rice seeds were immersed in this culture filtrate for 24 hours, and then sown on contaminated soil inoculated with pathogenic strain So-1 at a concentration of 2.6 x 107 cfu per gram of soil. We investigated the degree.

As a result, the degree of disease onset was almost the same whether the culture filtrate was used as a stock solution or diluted to 1/100, and no effect of the culture filtrate on suppressing disease onset was observed.

4. Furthermore, we investigated whether the effect of suppressing the onset of rice seedling rot caused by non-pathogenic rice bacterium blight was also observed when the dead bacteria were used. A non-pathogenic strain N7503 and a pathogenic strain So-1 were used as test strains. The non-pathogenic strain N7503, which had been cultured on a PSA slant medium at 30°C for 48 hours, was suspended in sterile distilled water to a concentration of approximately 1010 cfu/ml, and then killed bacteria were prepared using the following three methods. . ■Heat treatment at 100℃ for 10 minutes, ■Pour 2 to 3 ml of the bacterial suspension into a sterile Petri dish, and use a 15W germicidal lamp (Toshiba GL-15) to incubate 30 cm.
Irradiate for 4 hours from a height of
ml beaker and add about 200ml of chloroform.
The mixture was placed in a 2,000 ml beaker containing 100 ml of water, sealed with aluminum foil, and steamed for 4 hours while stirring with a stirrer. The disease onset suppression test was conducted by the method described above.

[0038] In all cases, no effect on suppressing disease onset was observed at all. From this, it became clear that the disease-inhibiting effect of non-pathogenic strains was exhibited only by viable bacteria.

5. Furthermore, we investigated the effects of differences in seed treatment and inoculation methods on disease suppression effects. In other words, the non-pathogenic strain N75 is recognized to have a high disease-suppressing effect.
The experiment was carried out using a combination of 03 and pathogenic strain So-1. A mixture of non-pathogenic and pathogenic bacterial strains (concentration N7503)
:6.3×109 cfu/ml, So-1:2.3×
Methylcellulose was added to 1.5% to individual solutions of 107 cfu/ml), non-pathogenic bacterial strains, and pathogenic bacterial strains, and surface-sterilized rice seeds were immersed in these solutions for 30 min.
It was kept at ℃ for 24 hours. The treated seeds were sown 60 seeds each in a container containing mixed soil at 100°C, kept in an inoculation box at 32°C and 100% humidity for 48 hours, and then placed in a plant growth chamber at 28°C and irradiated for 12 hours a day. 1 after sowing.
On the 5th day, the severity of the disease was tested. Also, the concentration is 6.3×1
09 cfu/ml non-pathogenic strain suspension and concentration 2.
Mix 10 ml of a 7 x 107 cfu/ml suspension of pathogenic bacterial strains per container (100 g of mixed soil),
After sowing surface-sterilized seeds in this soil, the disease severity was investigated on the 15th day of sowing in the same manner as above. Furthermore, the effect of suppressing disease onset when treated with a non-pathogenic strain after inoculation with a pathogenic strain was tested using the following method. Surface-sterilized rice seeds were treated with a bacterial suspension of pathogenic strain So-1 (concentration: 2.
3 x 108 cfu/ml) for various times (10 min, 2
4, 48, 72, 96, 102 hours) After immersion inoculation, 10
Sow 60 seeds each in a container containing 0g of compost,
Bacterial suspension of non-pathogenic strain N7503 (concentration: approx. 109
-10 cfu/ml) in 10 ml portions and placed in an inoculation box kept at 32°C and 100% humidity for 48 hours, then 2
The seedlings were placed in a plant growth chamber at 8° C. and illuminated for 12 hours a day. Post-onset assays were performed on the 15th day after seeding.

The results of the experiment are shown in Tables 8 and 9. As shown in Table 8, in the plot where rice seeds were coated with a pathogenic strain alone, all the solids rotted and died completely, whereas when rice seeds were coated with a mixture of a non-pathogenic and pathogenic strain. When coated, the disease onset degree was 0.7, and a high disease onset suppressive effect was observed. Also, the concentration is 2.3×106 c
The disease severity was 3.6 when the soil was sown with the pathogenic strain at a concentration of 6.3 x 108 cfu/g. When present, the disease severity was 0.6, showing a remarkable suppressive effect. Furthermore, as is clear from Table 9, the pathogenic strain So-
1 was inoculated into rice seeds by immersion, and the disease onset was also significantly suppressed when the non-pathogenic strain N7503 was sown in soil containing about 109 cfu per gram of soil. Furthermore, it has been revealed that even when rice seeds are soaked and inoculated in a bacterial suspension of a pathogenic strain for 120 hours, the onset of disease can be suppressed if the rice seeds are sown in soil supplemented with a non-pathogenic strain. From this result, in order to prevent the onset of the disease, the effect can be fully achieved by coating rice seeds with the non-pathogenic strain of the present invention or adding the non-pathogenic strain of the present invention to the soil in which rice seeds are sown. Can be done.

[0041]

[Table 8]

[Table 9]

6. Furthermore, in order to clarify the mechanism of the effect of non-pathogenic bacterial strains on suppressing the onset of rice seedling rot, we investigated non-pathogenic bacterial strains and pathogenic bacterial strains in rice germination liquid and in sterile water with extremely limited nutrients. We examined the competitive relationship with N7503 as a non-pathogenic strain, and streptomycin-resistant So-1-S as a pathogenic strain.
R was used. The rice germination solution was prepared as follows. That is, the seeds were surface sterilized with 3% sodium hypochlorite (antiformin) for 90 minutes, thoroughly washed with sterile water, and then placed in a 100 ml triangular container containing 50 ml of sterile water.
Place each grain in a plant growth chamber with 12 hours of light per day at 30°C for 7 days, then remove the rice plants with sterile tweezers and filter with a membrane filter (pore size 0.2 μm).
The solution that was completely sterilized by passing through the rice was used as the rice germination solution. Pour 28 ml of this germination solution into 100 ml triangular containers, and add the test bacteria to the predetermined concentration (N7503: 1×
10cfu/ml, So-1-SR: 3 x 10cfu/
After inoculating the cells to a volume of 1.5 ml, the cells were incubated statically at 30°C.
YPDA and streptomycin 500 ppm over time
Bacterial quantification was performed by dilution plate method using YPDA-containing medium. As a control, sterile distilled water was used instead of rice germination solution.

[0043] When the pathogenic strain So-1-SR was inoculated into rice germination liquid alone at 3 x 10 cfu/ml and cultured, the number of bacteria was 1 x 107 cfu on the second day of culture.
/ml, and a slight decrease was observed after that, but culture 1
The bacteria survived at a nearly constant amount of about 106 cfu/ml until the 5th day. When mixed culture with non-pathogenic strain N7503, the growth of pathogenic strain So-1-SR was significantly suppressed,
On the 2nd day of culture, the number of bacteria was about 1/100 of that of the single culture, on the 5th day of culture, it was about 1/10, and then remained at about 1/10 until the 15th day. The growth of the non-pathogenic strain N7503 was not reduced by mixed culture with the pathogenic strain So-1-SR, and showed almost the same growth pattern as in the case of monoculture. In addition, a tendency similar to the growth pattern in rice germination liquid was observed in sterile distilled water. That is,
In the mixed culture, the growth of the pathogenic strain So-1-SR was suppressed, and the amount of bacteria remained at about 1/10 of that in the case of single culture from the 2nd day to the 15th day of culture.

7. Furthermore, by treating rice seeds with a non-pathogenic strain, infection and onset of rice seedling rot caused by a pathogenic strain can be suppressed. In this case, we investigated how pathogenic and non-pathogenic bacterial strains fade in rice. As for the bacterial strains, non-pathogenic strain N7503 and pathogenic strain 2-SR were used as a combination that was found to be effective in suppressing disease onset, and non-pathogenic strain YN7810 and pathogenic strain 2-SR were used as a combination that was not found to be effective. The outer and inner glumes were removed from the seeds of Asominori rice variety, surface sterilized with 3% sodium hypochlorite (antiformin) for 90 minutes, and thoroughly washed with sterile distilled water. Add these seeds to 100ml containing 20ml of 0.5% agar.
Sow 10 seeds each in a triangular container of
2 ml of a mixed solution (approximately 108 cfu/ml) and 2 ml of a 2-fold dilution of each bacterial solution were inoculated. Thereafter, seedlings were grown at 25° C. under 34,000 to 40,000 lux illumination, and each bacteria was quantified at 2-day intervals using a YPDA plate medium and a YPDA plate medium containing 100 ppm of streptomycin.

[0045] As a result, in the plots inoculated with pathogenic strain 2-SR alone, the bacteria proliferated over time, reaching about 107 cfu per seed on the 10th day after inoculation. However, the non-pathogenic strain N7, which shows the effect of suppressing disease onset,
In mixed culture with 503, the pathogenic strain 2-SR produced 1 x 105 c per seed even on the 10th day after inoculation.
The value remained at fu/ml, which was almost the same value as the amount of bacteria at the time of inoculation. In addition, in mixed culture with the non-pathogenic strain YN7810, which does not show any disease-suppressing effect, the pathogenic strain 2-SR proliferates in the same way as in the case of monoculture, and on the 10th day after inoculation, 1× A bacterial load of 107 cfu was reached. Both non-pathogenic strains remained constant at approximately 107 cfu per seed up to day 10 in both single and mixed cultures. As described above, in combinations of bacterial strains that have been found to be effective in suppressing the onset of disease, there was a tendency for the growth of pathogenic strains to be suppressed by mixed culture with non-pathogenic strains.

8. Furthermore, it has been reported that the rice blight bacterium produces toxins in the culture medium. However, it has become clear that toxic substances are produced in the culture medium regardless of whether they are pathogenic or not. On the other hand, since non-pathogenic strains do not inhibit rice growth in soil, non-pathogenic strains neutralize or detoxify toxins produced by pathogenic strains under conditions involving rice and soil. It is highly possible that the onset of the disease may be suppressed. The following experiment was conducted to clarify this point.

Detoxification of toxins by non-pathogenic bacterial strains was investigated using the following method. In other words, add 1,000 ml of distilled water to 500 g of mixed soil, and add 200 ml of distilled water while stirring occasionally.
After standing for 4 hours, an aqueous extract of the mixed culture soil was obtained by suction filtration. This was dispensed in 18 ml portions into 100 ml triangular containers and sterilized under pressure. Add 2 sterile rice seeds that have been surface sterilized with 3% sodium hypochlorite (antiformin).
Add 0 tablets at a time, and add pathogenic bacterial strain 2 (bacterial concentration: approximately 109 cf
2 ml of each microorganism suspension was inoculated and the seedlings were grown at 25° C. under 34,000 to 40,000 lux illumination for 10 days. After that, remove the rice seeds and give 10,000×g
After centrifugation for 20 minutes, the resulting supernatant was treated with a 0.2 μm membrane filter to obtain a completely sterilized culture filtrate of pathogenic strain 2. Put 2 ml of this culture filtrate into a sterile test tube, and add YPDA slant medium to it at 30°C for 4 hours.
The non-pathogenic strain N7503 cultured for 8 hours was
The cells were inoculated at fu/ml and cultured at 25°C. On the 10th day of culture, bacterial cells were removed from the culture filtrate by centrifugation at 10,000 x g for 20 minutes, completely sterilized by passing it through a 0.2 μm membrane filter, and 2 ml of each filtrate was sterilized. Dispense into test tubes, add 2 surface sterilized rice seeds to each, and heat at 25℃, 34,000~40℃.
The seedlings were grown under conditions of ,000 lux, and on the 10th day, the length and slenderness of the seedlings were measured to examine the presence or absence of detoxification.

As a result, strong growth inhibition was observed in the culture filtrate of pathogenic strain 2, with a seedling length of 6.8 mm and a root length of 1.0 mm, whereas it was inoculated with non-pathogenic strain N7503 for 10 days. In the cultured filtrate, the seedling length was 91.6 mm and the root length was 83.0 mm, showing almost the same growth as the control plot, and it became clear that the activity of the toxin was significantly reduced.

Next, examples of the present invention will be shown.

Example 1 Pseudomonas gourmet strain N7503 was suspended in sterile distilled water to a cell concentration of approximately 1010 cfu/ml, and methylcellulose was added to the suspension to a concentration of 1.5%. Sixty grains of Asominori rice seeds were immersed in 100 ml of this suspension at 30° C. for 24 hours. These seeds were sown in a container containing approximately 80 g of mixed soil (manufactured by Mitsui Toatsu Co., Ltd.) that had been sterilized at 120°C for 30 minutes, and after covering with 20 g of soil, a suspension of pathogenic bacterial strains (approximately 108 cfu/ml) was added.
10 ml of was injected. Although 15 days had passed, no disease onset was observed. On the other hand, when a similar test was conducted on seeds that were not inoculated with the N7503 strain, all of them developed disease.

[0050]

[Example 2] Pseudomonas gourmet N7503 strain was added to soil sterilized at 120°C for 30 minutes at 6.3
cfu/g-soil and pathogenic Pseudomonas gourmet were sprayed and mixed at a concentration of 2.3 x 106 cfu/g-soil. Asominori seeds were sown in this soil. The seeds then germinated and grew, but no disease was observed. On the other hand, in the plot where N7503 was not mixed and only pathogenic bacteria were mixed, all of the plants became sick.

[0051]

[Example 3] Rice variety Asominori was sown, and on the 26th day, 5 plants were transplanted into Wagner pots and allowed to grow. Pseudomonas gourmet N.
Suspension of 7503 strain (bacterial cell concentration 2.5 x 1010cf
u/ml) and a mixture (1:1
), a 2-fold diluted suspension of the N7503 strain, or a 2-fold diluted suspension of the pathogenic strain, each in an amount of 50 ml per pot. After inoculation, the plants were covered with vinyl for 3 days, and on the 20th day, the panicles were examined for the presence or absence of disease, and the percentage of affected plants and the average degree of disease per panicle were calculated. The results are shown in Table 10.

[0052]

[Table 10] Inoculated bacterial strains
Disease rate (%) Average disease severity per panicle
2-fold dilution of N7503
0
0 N7503:So-1 mixed liquid 40.0
5 N7503: Ku8
111 mixed liquid 35.0
15 So-1 2-fold dilution 90.0
20 Ku8111 2-fold dilution 90.0
38 Blank
0
0

[Brief explanation of the drawing]

[Figure 1] An indicator of rice seed disease caused by Pseudomonas gourmet. 0-5N indicates necrosis.
indicates chlorosis.

Claims (4)

[Claims] Claim 1: A novel non-pathogenic strain of Pseudomonas gourmet that is effective for disease control of grass crops.
onas glumae) N7503 (Feikoken Bacterial Serial No. 12105). [Claim 2] New non-pathogenic Pseudomonas
Gourmet (Pseudomonas glumae) N
A method for controlling diseases of gramineous crops, the method comprising inoculating rice seeds with 7503 (Feikoken Bokuyori No. 12105). [Claim 3] New non-pathogenic Pseudomonas
Gourmet (Pseudomonas glumae) N
A method for controlling diseases of gramineous crops, which comprises spraying 7503 (KAIKEN BIKYORI NO. 12105) on the soil. Claim 4: Claims (2)-(
The method described in any of 3)