JPH0749364B2 - Disease control microorganisms for grass crops and disease control method - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel microorganism effective for controlling diseases of grass crops, particularly rice seedling rot and rice bacterial blight, and a method for controlling diseases of grass crops using this microorganism. Regarding

[0002]

2. Description of the Related Art Pseudomo
rice bacterial blight, which is a disease caused by nas glumae ), is
It is widely distributed in the rice growing regions of Southeast Asia and seriously damages the yield of rice. Initially, this pathogenic bacterium was thought to be a bacterium that causes disease only in the rice ears,
Subsequent studies have revealed that this bacterium is also pathogenic to rice seedlings. The damage to the seedlings is
The spread of nursery boxes based on the peculiarities of paddy rice cultivation has become a serious problem in Japan. Therefore, a wide range of research has been conducted so far on the rice blight fungus, such as elucidation of the ecology of development, relationship between cultivation method and disease, and development of control method. However, there are still unclear points about the transmission route and the mechanism of invasion and infection, and an appropriate control method has not been established yet. Currently, as control measures, removal of diseased rice by salt water selection, seed disinfection with various chemicals, and application of seedling boxes such as kasugamycin / captan wettable powder and polycarbamate wettable powder are mentioned.

[0003]

However, these drugs are unstable in their effects, have different effects depending on the disease-causing strain, and show phytotoxicity for growth inhibition.
At present, it is necessary to further study before putting it into practical use. It is also considered to use resistant varieties, but it is not clear whether or not the resistance gene for this bacterial disease is present in rice. As described above, the control methods that have been used up to now are not sufficiently effective, and the development of new control methods is desired.

In recent years, as one of the means for controlling soil-borne plant diseases, the development of a more naturally standing biological control method has been emphasized. In particular, research on utilization of various antibacterial substance-producing rhizosphere microorganisms is being advanced in various countries around the world, and some successful cases have been reported. There are also many studies using non-pathogenic mutant strains that have lost or eliminated pathogenicity from pathogenic bacteria. Among these non-pathogenic strains, there are some that grow in the host similarly to the pathogenic strain and behave ecologically almost the same as the pathogenic strain. By pre-treating the host plant with such a non-pathogenic strain in advance, it is possible that the pathogenesis may be suppressed by preventing the invasion of the pathogenic bacteria inoculated thereafter or by suppressing the growth. Based on such an idea, the present inventors have repeatedly studied a method of biologically controlling rice blast disease by utilizing a non-pathogenic rice blast disease bacterium, leading to the present invention. It was

[0005]

[Means for Solving the Problems] That is, the present invention provides a novel Pseudomonas gluma ( Pseudomonas gluma) that is effective for controlling diseases of gramineous crops and has no pathogenicity.
e ). Furthermore, the present invention also relates to a method for controlling the disease of grasses by inoculating rice seeds with this novel Pseudomonas gourmet or spraying it on soil. The disease of the grass family crops in the present invention refers to rice seedling rot and / or rice blight bacterial disease. The novel Pseudomonas gourmet in the present invention is
It has been deposited as Microorganism Research Institute No. 12105. Further, in the present invention, strains obtained by mutating this strain with radiation, chemicals, etc. are also included in the strains of the present invention as long as they can control diseases of grass crops and have no pathogenicity.

When inoculating rice seeds with this novel Pseudomonas gourmet, the strain concentration is 10 ⁸ to 10 ¹² cfu /
It is preferable to immerse rice seeds in ml of the suspension. In addition, when spraying new Pseudomonas gourmet on soil to control it, 10 ⁷ to 10 ¹⁰ cfu /
It is preferable to spray so that the cell concentration of g-soil may be adjusted.

The present inventors selected rice cultivated by rice bacterial blight, from rice cultivated in rice fields in the Fukuoka region, dipped it in a 70% ethanol aqueous solution for several seconds, and then the effective chlorine concentration of 3 % Sodium hypochlorite aqueous solution for 5 minutes, rinse with sterile distilled water, and then mash.
It is spread on A plate medium and 47 rice germs are killed from now on.
The strain was isolated.

These rice blast bacteria have the following mycological properties. It is rod-shaped and has polar hairs. It is aerobic and can grow at temperatures of 30-35 ℃ and 40 ℃. The colony is grayish white. Can grow on King's medium. Gelatin liquefaction, nitrate reduction, litmus milk reduction, ammonia production, hydrogen sulfide production, catalase, lecithinase are all positive. Indole production, MR reaction, and oxidase were all negative.

Next, among these, the mycological properties differ only in that it has no pathogenicity not only to rice but also to other plants, and in other respects, it shows the same properties as the rice bacterial blight. Non-pathogenic rice blast bacterium was selected.

This selection will be described in more detail. 1. Selection of non-pathogenic rice blast fungus Since rice blast fungus is a pathogenic bacterium that infects rice seedlings and ears, it is not possible to use it for biological control as it is even if a disease suppressing effect is observed. It is a danger. However, this bacterium has a property of easily losing pathogenicity in vitro, and may lose pathogenicity during storage or during subculture. On the other hand, the present inventors have found that this bacterium has pathogenicity to rice and also has a property of spoiling tissue sections such as potato and carrot.
Therefore, the pathogenicity of this fungus to various crops was examined in order to select non-pathogenic strains for all crops.

As the plant material, potato (make-in), carrot (unknown variety), tomato seedling (Toko), and rice seed (asominori) were tested. The assay of putrefaction ability for various vegetable tissues was performed by the following method. 1% of various vegetable tissues in 3% sodium hypochlorite (antiformin)
Perform surface sterilization by immersing for 5 minutes, then wash thoroughly with sterilized water, cut to a thickness of about 5 ml with a sterilizing knife, put this on a sterile petri dish with a diameter of 9 cm covered with filter paper, and put 5 ml of sterilized water. , PSA on slant medium at 30 ℃
After inoculating the center portion of the cut surface with 1 platinum loop amount of the rice blast fungus cultivated for 8 hours, the mixture was allowed to stand at 30 ° C. for 48 hours and examined for spoilage ability. To investigate the pathogenicity of tomato seedlings, the tomato seedlings were sterilized from the slope by sterilizing each strain of the rice bacterial blight fungus cultivated in PSA slope medium at 30 ° C for 48 hours and sterilized with 70% ethanol in advance. It was carried out by inoculating multiple needles on the stem (3 weeks after sowing). The examination of the pathogenicity to rice was performed as follows.
That is, seeds of the test variety Asominori were sterilized with 3% sodium hypochlorite (antiformin) for 30 minutes, washed thoroughly with water, and soaked at 25 ° C. for 2 days. 60 seeds were placed in a sterile petri dish with a diameter of 9 cm and the concentration was about 10 ⁹ c.
Inject 15 ml of fu / ml bacterial suspension and incubate at 30 ° C for 2
It was inoculated by immersion for 4 hours. Then, put 80g of Kamiai culture soil (manufactured by Mitsui Toatsu) sterilized in an autoclave, sow it in a plastic container of 60 x 60 x 45 mm, which was irrigated with 30 ml of sterile distilled water, and cover it with the same culture medium so that the seeds would cover. Then, the seedlings were raised in a 32 ° C. inoculation box (humidity 100%) for 2 days. Furthermore, the presence and extent of disease was investigated 15 days 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 did not show pathogenicity to rice. No pathogenicity to tomato was observed in any of the tested strains. Tomatoes that do not show putrefaction against various vegetable tissues,
Eight strains that are not pathogenic to rice, namely N
7504, N7503, YN7810, YN7805
YN7825, 750, 752, 805 could be selected.
In addition, a high correlation was observed between the putrefaction ability against potato and the pathogenicity against rice in the 47 strains tested. These 15 bacterial species are considered to have lost the pathogenicity to rice during subculture or storage. And these non-pathogenic bacteria do not show pathogenicity to other plants such as potato.

Next, it was examined whether or not these non-pathogenic bacteria could suppress the onset of rice seedling rot caused by the rice blast fungus. 2. Suppressive effect of rice seedling rot caused by non-pathogenic rice bacterial pathogens 4 pathogenic strains (Ku8111, 2, So-1, Ky)
u82-34-2) and 5 non-pathogenic strains (N750
3, N750, YN7810, YN7825, 805)
Was used in the experiment. The rice variety used was Asominori. The following method was used to test the inhibitory effect of non-pathogenic rice blast fungus on rice seedling rot. That is, a water-preserved bacterium was transplanted to a YPDA slant medium, cultured at 30 ° C. for 48 hours, and then suspended in 10 ml of sterilized distilled water (concentration: about 10 ⁹ cfu).
/ Ml), add this to 200 ml of YPD liquid medium, and add 3
It was shake-cultured at 0 ° C. for 48 hours. Then 3,600x
After centrifugation at g for 20 minutes, the obtained bacterial cells were suspended in sterile distilled water so as to have a concentration of about 10 ¹⁰ cfu / ml, and methylcellulose was added thereto at a concentration of 1.5%. Rice seeds 3% with 3% sodium hypochlorite (antiformin)
After surface sterilization for 0 minutes, the surface was thoroughly washed with sterilized water and immersed in a bacterial suspension containing methylcellulose at 30 ° C. for 24 hours. As a control, seeds soaked in 1.5% methylcellulose solution containing no bacteria were used. After culturing the pathogenic strain in the same manner as the non-pathogenic strain, it was adjusted with sterile water to a concentration of about 10 ⁸ cfu / ml, and this was used as the inoculum. The concentration of non-pathogenic strains and pathogenic strains is YPDA for each experiment.
It was determined according to the dilution plate method using a plate medium. Rice seeds soaked in the non-pathogenic strain liquid are 60 x 60 x 45 mm containing about 80 g of sterilized Kumiai Baidou (manufactured by Mitsui Toatsu).
60 seeds are sown in each plastic container and covered with soil (20
g) and then adding 1 to the bacterial suspension of the pathogenic strain as the inoculum.
Inoculation was performed by irrigation in 0 ml increments. Treated containers at 32 ° C for 4
After being placed in an inoculation box (humidity: 100%) for 8 hours for germination, it was placed in an air-conditioned greenhouse or a plant growing chamber at 28 ° C at 25 to 30 ° C for greening. Irrigation was performed once a day. The degree of disease was determined on the 15th day after inoculation. All experiments were repeated two more times. As shown in Fig. 1, the test of the degree of disease is divided into 6 stages of 0 to 5 depending on the severity of the symptom (0 is a healthy seedling, 1 is a healthy seedling and the height does not change, but chlorosis is present in the leaf blades). What appeared, 2 was shorter than healthy seedlings, necrosis at the base of leaf sheath was seen, 3 was chlorosis of leaf blade, necrosis at the base of leaf sheath, and whole body had abnormal morphology, 4 was this Leaves that have developed only the 1st leaf and are vegetative as a whole, and 5 have rotted and died)
The disease level was shown as 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 dipping treatment of rice seeds with a non-pathogenic strain suppresses the onset of rice seedling rot. However, the degree of disease-controlling effect remarkably differs depending on the combination of the non-pathogenic strain-pathogenic strain, and there existed those having no disease-controlling effect at all and those showing high disease-controlling effect. That is, for the pathogenic strain So-1, non-pathogenic strains N7503, N75
0, YN7825, 805 suppressed the onset.
On the other hand, the treatment with YN7810 did not produce any inhibitory effect. As for the pathogenic strain 2, N7503 and 805 were found to have disease suppressive effects, but N750 and YN781
0, YN7825 was not suppressed at all. For pathogenic strain Kyu82-34-2, non-pathogenic strain N7
The disease suppressive effect is observed only when treated with 503,
Non-pathogenic strains N750, YN7810, YN7825,
Treatment with 805 did not suppress the disease. Furthermore, for pathogenic strain Ku8111, N7503, YN7
A disease suppressive effect was observed in 810 and 805, weak in N750, and no effect in YN7825. As a result, it was confirmed that treatment of rice seeds with the non-pathogenic strain N7503 can suppress the onset of rice seedling rot.

[0016]

[Table 2]

3. Relationship between bacterial concentration for soaking treatment and disease suppressive effect Therefore, the relationship between bacterial concentration of this non-pathogenic strain N7503 and disease suppressive effect was examined. N7503 was tested as a non-pathogenic strain, and So-1 was tested as a pathogenic strain. The test for the disease suppressive effect was performed according to the method described above. That is, each strain stored in water was spread on a YPDA plate medium to separate single colonies, and 200 ml of this was isolated.
Inoculate into a Sakaguchi flask containing YPD liquid medium of
Shake culture for 48 hours at ℃ and centrifuge (8,000 x
g, 20 minutes), the cells obtained were suspended in sterilized distilled water to a concentration of about 10 ¹⁰ cfu / ml, and the cells were serially diluted 10-fold to obtain about 10 ¹⁰ , 10 ⁸ , 10 ⁶ cf.
A u / ml bacterial suspension was made. 1.5% of methylcellulose was added to the suspension of the non-pathogenic strain N7503 at each concentration.
% Rice seeds, surface-sterilized with 3% sodium hypochlorite (antiformin), soaked in the seeds and allowed to stand at 30 ° C for 24 hours, then sterilized
60 seeds were sown in a container containing 0 g and covered with 20 g of a soil culture medium, and then 10 ml of a bacterial suspension of the pathogenic strain So-1 at each concentration was irrigated and inoculated at 48 ° C at 30 ° C.
It was placed in a time inoculation box, placed in a plant growing chamber adjusted to illumination at 28 ° C. for 12 hours a day, and the disease degree was tested on the 15th day.

The results are shown in Table 3. As is clear from Table 3, when rice seeds were treated with a bacterial suspension having a non-pathogenic strain concentration of 10 ¹⁰ cfu / ml, the bacterial concentration of the pathogenic strain in the soil was as high as 10 ¹⁰ cfu / g. Even at a high concentration, a high disease suppressive effect was shown. However, when the bacterial concentration of the non-pathogenic strain is 10 ⁸ cfu / ml or less and the bacterial concentration of the pathogenic strain is as low as 10 ⁶ cfu / g, the disease suppressing effect cannot be obtained, and in order to obtain the effect, High concentration (10 ¹⁰ c
It has become clear that it is necessary to treat with a suspension of non-pathogenic strains (fu / ml).

[0019]

[Table 3]

Further, in the present invention, the following tests were carried out in order to prove that Pseudomonas gourmet N7503 of the present invention can be used for disease control of gramineous crops.

[Test 1] Non-pathogenic strain N7503 strain suppressive effect against various pathogenic strains The non-pathogenic strain N7503 has the same disease suppressive effect against many other pathogenic strains as well. Whether or not it was examined.

That is, among the strains of the rice bacterial blight fungus stored in the Department of Plant Pathology, Kyushu University, Ku showing a strong pathogenicity
8106, Ku8121, III, 8001, 8017,
A total of 5 strains of I and Ku8105 were tested, and the disease suppressing effect of N7503 strain was examined. The disease suppression test was carried out by the method described above. As a result, as shown in Table 4, it was revealed that the non-pathogenic strain N7503 is a strain having a strong disease suppressive effect against all the pathogenic strains tested.

[0022]

[Table 4]

[0023]

[Test Example 2] Comparison of disease suppressive effect with Kasugamycin-Captan wettable powder Reported as a rice seedling box application agent effective for controlling rice seedling rot of the N7503 strain of the present invention and conventional rice seedling rot Compared with the control effect of the existing Kasugamycin-Captan wettable powder.

That is, an experiment was conducted using a combination of the pathogenic strain So-1 and the non-pathogenic strain N7503 and using Asominori as a rice variety. The disease suppression test was based on the above method. Kasugamycin captan wettable powder is 1
10 ml of 200-fold diluted solution (1.2 g of kasugamycin in terms of drug substance + 1.2 mg of captan) per container was treated by irrigating sterilized soil after seeding and before covering with soil.

The results are shown in Table 5. The disease suppressive effect obtained by pretreating rice seeds with N7503 is
Kasugamycin captan wettable powder was added in an amount of 0.
The effect was almost the same as that obtained when 4 mg was applied. However, in the group treated with Kasugamycin / Captan wettable powder, compared with the group pretreated with N7503 or treated with only 1.5% methylcellulose, the growth of rice seedlings is low, and some growth inhibition is observed. was there. Those pretreated with non-pathogenic strains,
The effect was almost the same as that of the control agent, and its growth was almost the same as that of the untreated section.

[0026]

[Table 5]

[0027]

[Test 3] nonpathogenic Note strains of varietal differences invention onset suppressing effect, since rice products species Asominori revealed that significantly suppress the onset of rice seedling rot disease, the effect of other It was examined whether or not it was also recognized in the rice varieties.

That is, as rice varieties, Asominori,
Sword style, yellow jade, China No. 45, kyuju, Asahi Aichi, Norin 2
A total of 9 varieties of No. 9, IR64 and Inaba Wase were tested. Bacteria were N7503, YN7810 and 805 as non-pathogenic strains, and So-1, Kyu82 as pathogenic strains.
-34-2 and 2 were used. Seed treatment with a non-pathogenic strain, inoculation of a pathogenic strain, and assay of the effect were carried out by the method shown in the above disease suppression test.

The results are shown in Tables 6 and 7. From the experimental results, it was clarified that there are differences among the varieties in the disease suppressive effect of non-pathogenic strains. In the combination of the non-pathogenic strain 805 and the pathogenic strain Kyu82-34-2, the disease suppressive effect was observed in sword wind, yellow jade, Chinese No. 45 and kyuju, but other varieties (Asominori, Aichi Asahi,
In Norin 29, IR64, Inabawase), the disease onset was not suppressed at all. In the combination of the non-pathogenic strain 805 and the pathogenic strain 2, Aichi Asahi and China No. 45 showed no disease control effect, and Norin 29 showed some disease control, and other 6 varieties (Asominori, Sword style, yellow ball,
(Kuju, IR64, Inabawase) showed a high disease suppressive effect. Further, in the combination of the non-pathogenic strain YN7810 and the pathogenic strain So-1, no disease suppressive effect was obtained in sword wind, kuju, and inabawase, and other varieties (Asominori, Yellow jade, China No. 45, Aichi) Asahi, Norin 29, IR64) showed a high disease suppressive effect. Non-pathogenic strain N7503 and pathogenic strain So-1
It was clarified that the combination of 2) suppressed the disease in all varieties. From the above, it was revealed that a specific relationship among the nonpathogenic strain, the pathogenic strain and the rice variety is involved in the expression of the disease suppressing effect.

[0030]

[Table 6]

[Table 7]

[0031]

[Test 4] Examination of the mechanism for suppressing the development of rice seedling rot 1. Further, in the present invention, in order to know the mechanism of suppressing rice seedling spoilage rot onset by nonpathogenic rice bacterial pathogens, first, the antibacterial activity of nonpathogenic strains against pathogenic strains was examined. That is, non-pathogenic strains N7503, N750,
805, YN7810, YN7825 and pathogenic strain K
u8111, 2, So-1, and Kyu82-34-2 were tested. YPDA for assaying antibacterial productivity and activity
Using a plate medium, the plate chloroform method and UV irradiation were carried out in two ways. The UV irradiation was performed for 3 hours from a height of 30 cm with a 15 W sterilizing lamp (Toshiba GL-15) after removing the upper lid of the petri dish in which colony formation was observed after culture. Then, the mixture was allowed to stand at 30 ° C. for 24 hours, overlaid with a pathogenic strain that is an indicator bacterium, and after culturing at 30 ° C., antibacterial substance productivity was examined by the presence or absence of an inhibition zone formed around colonies.

As a result, the tested non-pathogenic strain N750 was tested.
3 and N750 showed antibacterial activity. 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 the non-pathogenic strain on the medium against the pathogenic strain and the disease suppressive effect.

2. Next, the inhibitory effects of rice plant seedling rot on the known plant pathogenic bacteria and saprophytic fungi other than the rice bacterial pathogen were examined. Agrobacterium, a phytopathogenic bacterium preserved in the Department of Plant Pathology, Kyushu University
tumefaciens Ku7411, Erwini
a carotovora subsp. caroto
vora N7129, Clavibacterium mi
chiganense pv. michiganens
e N6601, Bacillus subtilis
ATCC 6633 , Pseudomonas yr
ingae pv. syringae I, and Pse
udomonas fluorescence p-1
5 was tested. Each of these bacteria was cultured on PSA slant medium at 30 ° C. for 48 hours, then transplanted to a Sakaguchi flask containing 200 ml of YPD liquid medium, cultured at 30 ° C. for 48 hours, and centrifuged (8,000 × g, 20 minutes). ), The bacterial cells were collected, suspended in sterilized water so as to have a concentration of about 10 ¹⁰ cfu / ml, used for the pretreatment, and the disease suppression test described above was performed.

As a result, the effect of suppressing the onset of rice seedling rot was shown only by the non-pathogenic rice bacterial blight fungus. It was revealed that the disease suppressive effect was not observed at all in the other tested bacteria.

3. In addition, the effect of disease control when rice seeds were treated with a culture filtrate of a non-pathogenic strain was examined.
That is, YPD liquid medium (200 ml) was inoculated with the non-pathogenic strain N7503, shake-cultured at 30 ° C. for 4 days, and then centrifuged (8,000 × g, 20 minutes), and the supernatant was added to a membrane having a pore size of 0.2 μm. A non-pathogenic strain culture filtrate was obtained by completely eliminating the bacteria through a filter. Surface-sterilized rice seeds were immersed in this culture filtrate for 24 hours, and the pathogenic strain So-1 contained 2.6 × 10 ⁷ cf per 1 g of soil.
The seeds were sown on the contaminated soil inoculated to give u, and the disease degree was investigated on the 15th day.

As a result, there was almost no difference in the degree of disease occurrence when the stock solution of the culture filtrate was used or when diluted 1/100, and the disease suppressive effect of the culture filtrate was not observed at all.

4. Furthermore, it was examined whether or not the killing effect of the non-pathogenic rice blast fungus also suppresses the onset of rice seedling rot. Non-pathogenic strain N7503 and pathogenic strain So-1 were used as test strains. The non-pathogenic strain N7503 cultured on PSA slant medium at 30 ° C. for 48 hours was suspended in sterile distilled water so as to have a concentration of about 10 ¹⁰ cfu / ml, and killed cells were prepared by the following three methods. went. Heat treatment at 100 ° C for 10 minutes, put 2 to 3 ml of bacterial suspension in a sterile petri dish, and use a 15W sterilization lamp (Toshiba GL-15) to obtain 30 cm.
Irradiation from the height of 4 hours, about 10 ml of bacterial suspension 50
Take in a beaker of ml, chloroform about 200m
It was placed in a 2,000 ml beaker containing 1 ml, sealed with aluminum foil, and subjected to steam treatment for 4 hours while stirring with a stirrer. The disease suppression test was carried out by the method described above.

In any of these cases, the disease suppressing effect was not observed at all. From this, it was clarified that the disease suppressive effect of the non-pathogenic strain is shown only by the viable bacteria.

5. Furthermore, the influence of the difference between the seed treatment method and the inoculation method on the disease control effect was examined. That is, a non-pathogenic strain N75 showing a high disease suppressive effect
03 and the pathogenic strain So-1 in combination. Mixture of non-pathogenic and pathogenic strains (concentration N750
3: 6.3 × 10 ⁹ cfu / ml, So-1: 2.3 ×
10 ⁷ cfu / ml) and non-pathogenic strains and pathogenic strains alone were added with methylcellulose to a concentration of 1.5%, and surface-sterilized rice seeds were dipped in these solutions,
Hold at 24 ° C for 24 hours. 60 seeds each of the treated seeds were sown in a container containing 100 ° C. cultivation soil, and kept in an inoculation box at 32 ° C. and 100% humidity for 48 hours, and then at 28 ° C. for 12 hours a day in a plant growing chamber. Place it on the ground and sow it 1
On the 5th day, the degree of disease was tested. Also, the concentration is 6.3 × 1
0 ⁹ cfu / nonpathogenic strains suspension and concentration of ml 2.7
1 container (1 container) of pathogenic strain suspension of × 10 ⁷ cfu / ml
After mixing 10 ml per 00 g of Kumiai culture soil and sowing seeds which had been surface sterilized in this soil, the degree of disease was investigated 15 days after sowing in the same manner as above. Furthermore, after the inoculation of the pathogenic strain, the disease-inhibiting effect when treated with the non-pathogenic strain was tested by the following method. Surface-sterilized rice seeds were used as a bacterial suspension of pathogenic strain So-1 (concentration: 2.3 ×
10 ⁸ cfu / ml) for various times (10 min, 24, 4
(8, 72, 96, 102 hours) After immersion inoculation, 60 seeds were sown in a container containing 100 g of the culture medium, and a bacterial suspension of non-pathogenic strain N7503 (concentration: about 10 ^9-10 cf)
u / ml) and irrigate 10 ml each, and the humidity is 100 at 32 ° C.
The seedlings were placed in an inoculation box kept at 48% for 48 hours and then placed in a plant growth chamber under illumination at 28 ° C. for 12 hours a day to raise seedlings. The test after the onset of disease was 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 section where the rice seed was coated with the pathogenic strain alone, all the solids were completely rotted and died, whereas the rice seed was mixed with the non-pathogenic strain and the pathogenic strain. When coated, the disease severity was 0.7, and a high disease-control effect was observed. In addition, the concentration is 2.3 × 10 ⁶ cf
When seeded in soil contaminated with u / g of the pathogenic strain, the disease level was 3.6, whereas the concentration of the non-pathogenic strain was 6.3 × 10 ⁸ cfu / g in this soil. When present, the disease degree was 0.6 and a remarkable inhibitory effect was exhibited. Further, as is clear from Table 9, the pathogenic strain So-1
When the rice seeds were dipped and inoculated into the seeds, and the nonpathogenic strain N7503 was sown in the soil in which the amount of the non-pathogenic strain N7503 was about 10 ⁹ cfu per 1 g of the soil, the disease was significantly suppressed. further,
It was revealed that even when rice seeds were immersed and inoculated in a bacterial suspension of a pathogenic strain for 120 hours, the disease was suppressed by sowing the seeds in the soil to which the nonpathogenic strain had been added. From this result, in order to prevent the disease from occurring, the effect can be sufficiently achieved by coating the rice seed with the non-pathogenic strain of the present invention or adding the non-pathogenic strain of the present invention to the soil in which the rice seed is sown. You can

[0041]

[Table 8]

[Table 9]

6. Furthermore, for the purpose of clarifying the mechanism of the disease suppressive effect of rice seedling rot by non-pathogenic strains, non-pathogenic strains and pathogenic strains in rice germination solution and in sterile water with extremely limited nutrients We examined the competitive relationship with. N7503 as a non-pathogenic strain,
Streptomycin-resistant So-1- as a pathogenic strain
SR was used. The rice germ solution was prepared as follows.
That is, seeds which had been surface sterilized with 3% sodium hypochlorite (antiformin) for 90 minutes were thoroughly washed with sterilized water, and then placed in 100 ml triangular Korben containing 50 ml of sterilized water.
Grain is put and placed in a plant growing chamber illuminated at 30 ° C for 12 hours a day for 7 days, and then the rice plant is removed with sterile tweezers, and a membrane filter (pore size 0.2 μm)
The solution completely sterilized by passing through was used as a rice germ solution. 28 ml of this germination solution in 100 ml of triangular Kolben
And put the test bacteria in a predetermined concentration (N7503: 1).
× 10 cfu / ml, So-1-SR: 3 × 10 cfu
/ Ml), static culture at 30 ° C., and YPDA and streptomycin 500 p
Bacteria were quantified by the dilution plate method using pm containing YPDA medium. A control using sterile distilled water instead of the rice germination solution was used as a control.

When the pathogenic strain So-1-SR was singly inoculated into the rice germination solution at a concentration of 3 × 10 ⁷ cfu / ml and cultured, the number of bacteria was 1 × 10 ⁷ cfu / day on the second day of the culture.
It reached 15 ml and then decreased slightly.
Up to the day 1, the cells survived with an almost constant bacterial load of about 10 ⁶ cfu / ml. When mixed-cultured with the non-pathogenic strain N7503, the growth of the pathogenic strain So-1-SR was remarkably suppressed, and about 1/100 of the number of single-cultured cells on the second day of the culture, the culture 5
Approximately 1/10 on the day, and then 1/15 until the 15th day
It changed at a concentration of 10. The growth of the non-pathogenic strain N7503 was not reduced even by the mixed culture with the pathogenic strain So-1-SR, and showed a growth pattern almost similar to that of the single culture. In addition, the same tendency as the growth pattern in the rice germination solution was observed in sterile distilled water. That is, the growth of the pathogenic strain So-1-SR was suppressed in the mixed culture, and the bacterial load was changed from the second to the 15th day of the culture at about 1/10 of that in the single culture.

7. Furthermore, by treating rice seeds with a non-pathogenic strain, infection and pathogenesis of rice seedling rot caused by the pathogenic strain can be suppressed. In this case, how the pathogenic and non-pathogenic strains fluctuated in rice was examined. As the strains, a non-pathogenic strain N7503 and a pathogenic strain 2-SR were tested as a combination showing a disease suppression effect, and a non-pathogenic strain YN7810 and a pathogenic strain 2-SR were tested as a combination showing no effect. The outer and inner spikes were removed from the seeds of rice variety Asominori and surface-sterilized with 3% sodium hypochlorite (antiformin) for 90 minutes, and thoroughly washed with sterile distilled water. 100 ml of this seed containing 20 ml of 0.5% plain agar
10 seeds are seeded on each triangular corben, and 2 ml of a mixed solution of a non-pathogenic strain (concentration: about 10 ¹⁰ cfu / ml) and a pathogenic strain (concentration: about 10 ⁸ cfu / ml) and each bacterial solution are doubled. 2 ml of the single solution diluted to 1 ml was inoculated. Then 2
The seedlings were raised at 5 ° C. under the conditions of 34,000-40,000 lux illumination, and the quantification of each bacterium was performed at 2-day intervals using a YPDA plate medium and a YPDA plate medium containing 100 ppm of streptomycin.

As a result, in the section in which the pathogenic strain 2-SR was inoculated alone, the cells grew over time and reached a bacterial amount of about 10 ⁷ cfu per seed 10 days after inoculation. However, a non-pathogenic strain N75 showing a disease suppressive effect
In mixed culture with 03, the pathogenic strain 2-SR was 1 × 10 ⁵ cfu per seed even on the 10th day after inoculation.
/ Ml value, which was almost the same as the bacterial amount at the time of inoculation. In addition, a non-pathogenic strain YN that does not show disease suppressive effect
In the mixed culture with 7810, the pathogenic strain 2-SR grew in the same manner as in the case of the single culture, and the bacterial amount per seed was 1 × 10 ⁷ cfu on the 10th day after inoculation. Both non-pathogenic strains are up to 10 days in both single and mixed cultures
It was constant with a bacterial load of about 10 ⁷ cfu per seed. As described above, it was confirmed that the combination of the strains showing the disease suppressive effect tended to suppress the growth of the pathogenic strain by the mixed culture with the non-pathogenic strain.

8. Furthermore, it has been reported that the rice blast fungus produces a toxin in the medium. However, it was clarified that toxic substances are produced in the medium regardless of the presence or absence of pathogenicity. On the other hand, since non-pathogenic strains do not show growth inhibition on rice in soil, the non-pathogenic strain neutralizes or detoxifies the toxin produced by the pathogenic strains under the conditions involving the rice and soil. It is fully conceivable that the disease will be suppressed. The following experiments were conducted for the purpose of clarifying this.

Detoxification of toxins by non-pathogenic strains was examined by the following method. That is, 1,000 ml of distilled water was added to 500 g of Kumiai soil, and 2 times with occasional stirring.
The mixture was allowed to stand for 4 hours, and suction filtration was performed to obtain a water extract of the contact culture medium. 18 ml of this was dispensed into 100 ml triangular Kolben and sterilized under pressure. 2 pieces of aseptic rice seed surface-sterilized with 3% sodium hypochlorite (antiformin)
Add 0 grain each and add pathogenic strain 2 (bacterial concentration: about 10 ⁹ cfu
/ Ml) bacterial suspension at 2 ° C and inoculated at 25 ° C for 3
Seedlings were grown for 10 days under 4,000-40,000 lux illumination. After that, the rice seeds are removed and 10,000 × g,
Centrifugation was carried out for 20 minutes, and the resulting supernatant was treated with a 0.2 μm membrane filter to obtain a culture filtrate of pathogenic strain 2 which was completely sterilized. 2 ml each of this culture filtrate was put into a sterilized test tube, and was placed in a YPDA slant medium at 30 ° C. for 48
About 10 ⁹ cfu of non-pathogenic strain N7503 cultured for a long time
The cells were inoculated so that the amount became / ml and cultured at 25 ° C. Culture 1
On day 0, the cells were removed from the culture filtrate by centrifugation at 10,000 xg for 20 minutes, completely sterilized by passing through a 0.2 μm membrane filter, and the obtained filtrate was sterilized in 2 ml increments. dispensing, this one by the surface sterilized rice seeds 2 grains input is the tube, 25 ℃, 34,000~40,0
The seedlings were raised under the condition of 00lux, and the seedling length and slenderness were measured on the 10th day to examine the presence or absence of detoxification.

As a result, in the culture filtrate of the pathogenic strain 2, strong growth inhibition was observed with a seedling length of 6.8 mm and a root length of 1.0 mm, whereas it was inoculated with the 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 in the control group, and it was revealed that the activity of the toxin was significantly reduced.

Next, examples of the present invention will be shown.

Example 1 Pseudomonas gourmet N7503 strain was suspended in sterilized distilled water so as to have a cell concentration of about 10 ¹⁰ cfu / ml, and methylcellulose was added thereto at 1.5%. Into 100 ml of this suspension, 60 seeds of rice seed asominori were immersed at 30 ° C. for 24 hours. This seed was sown in a container containing about 80 g of Kumiai Soil (manufactured by Mitsui Toatsu Co., Ltd.) sterilized at 120 ° C. for 30 minutes and covered with 20 g of soil, and then a suspension of pathogenic strain (about 10 ⁸ cfu / ml) was added. 1
0 ml was irrigated. After 15 days, no illness was observed. On the other hand, the same test was conducted on seeds that were not inoculated with the N7503 strain, and as a result, all of them became ill.

[0050]

Example 2 Pseudomonas gourmet N7503 strain 6.3 × 10 ⁸ c was added to soil sterilized at 120 ° C. for 30 minutes.
Fu / g-soil and pathogenic Pseudomonas gourmet were sprayed and mixed so as to be 2.3 × 10 ⁶ cfu / g-soil. This soil was sown with Noriminori seeds. The seed then germinated and grew, but no disease was observed. On the other hand, all the disease occurred in the group in which N7503 was not mixed and only pathogenic bacteria were mixed.

[0051]

Example 3 Rice variety Asominori was sown, and on the 26th day, 5 strains were transplanted into Wagner pots to grow, and at the flowering period 85 days after transplantation, Pseudomonas gourmet N7 was grown.
Suspension of 503 strain (concentration of bacterial cell 2.5 × 10 ¹⁰ cfu /
ml) and a suspension of pathogenic strain (So-1 cell concentration:
2.5 × 10 ⁸ cfu / ml, Ku8111 bacterial cell concentration:
1.7 × 10 ⁸ cfu / ml) mixed solution (1: 1), or a 2-fold dilution of the N7503 strain suspension or a 2-fold dilution of the pathogenic strain suspension per pot. 50 ml of each was spray-inoculated. After inoculation, it was covered with vinyl for 3 days, and on the 20th day, the presence or absence of disease in the ears was examined, and the diseased strain rate and the average disease rate of one ear were calculated. The results are shown in Table 10.
It was shown to.

[0052]

[Table 10] Inoculated strains Susceptible strain rate (%) 1 Two-fold diluted solution of average susceptibility to N7503 0 0 N7503: So-1 mixed solution 40.0 5 N7503: Ku8111 mixed solution 35.0 15 So-1 2 Double dilution 90.0 20 Ku8111 double dilution 90.0 38 Blank 0 0

[Brief description of drawings]

FIG. 1 is an index of rice seed diseases caused by Pseudomonas gourmet, 0 to 5N indicates necrosis, and C indicates chlorosis.

Claims (4)

[Claims] 1. A novel non-pathogenic Pseudomonas gourd ( Pseudomon) effective for disease control of gramineous crops.
as glumae ) N7503
05). 2. A novel non-pathogenic Pseudomonas glumae N750.
3. A method for controlling a disease of a gramineous crop, which comprises inoculating rice seeds with No. 3 (Microtechnology Research Institute No. 12105). 3. A novel non-pathogenic Pseudomonas glumae N750.
3. A method for controlling diseases of gramineous crops, which comprises spraying No. 3 (Microtechnology Research Institute No. 12105) on soil. 4. The disease control of a grass family crop is a control of rice seedling rot and rice bacterial blight disease (2).
The method according to any one of (3)