JP3209565B2 - Control of disease occurring in rice nursery by microbial rice seed treatment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[Object of the Invention]

[0002]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for controlling a disease occurring in a rice nursery with bacteria. More specifically,
Ps that antagonizes pathogens of disease that occurs in rice nursery
eudomonas bacteria, such as Pseudomonas cepacia AGF-1
By inoculating 58 strains into rice seeds, diseases occurring in rice seedbeds, such as rice stupid disease, rice seedling blight, rice blast, rice seedling blight bacterial disease, rice brown streak, etc. are suppressed. About the method.

[0003]

2. Description of the Related Art Diseases that occur in rice nurseries include stupid seedling disease, seedling wilt disease, blast, sesame leaf blight, bacterial seedling wilt disease, bacterial wilt disease (seedling rot) caused by fungi. There are diseases such as brown stripe disease. Regarding their control, idiot disease, blast disease, and sesame leaf blight are disinfected with seeds using fungicides. Seedling blight is treated with soil incorporation of fungicides. It is controlled by different means such as seed disinfection or soil admixture.

[0004]

However, there is no practical control method other than the above for the disease which occurs in rice nurseries. In addition, in the case of bacterial wilt disease of rice seedlings, which has recently been increasing, there is no registered pesticide at present, so it is difficult to take measures against this disease on site.

[0005] Furthermore, it is always feared that resistant (resistant) bacteria emerge with the continuous use of the fungicide, and that the fungicide has almost no effect. For example, bacteria resistant to benzimidazole fungicides have appeared in idiot disease, bacteria resistant to kasugamycin in brown streak, and bacteria resistant to kasugamycin and IBP in blast. Currently used triazole fungicides and metalaxyls also have the potential for the emergence of resistant bacteria.

In the case of using a disinfectant, depending on the type of the disinfectant, if the agent is directly attached to the skin, a rash may be caused by a person. Also, rice seed disinfection uses a thick chemical solution, but there are many fish toxic fungicides, so it is necessary to pay attention to disposal of the used chemical solution to prevent damage to marine animals.
Further, depending on the type of medicine, there is a combination that may cause chemical damage if the treatment or proximity treatment is performed at the same time, and it may be difficult to control different diseases by one chemical treatment.

The inventors of the present invention have conducted intensive studies to solve or alleviate the above problems when using chemical pesticides such as fungicides. As a result, bacteria having an antagonistic action against many pathogenic bacteria are directly inoculated into rice seeds. By doing so, the present inventors have found that it is possible to suppress and control the occurrence of a plurality of diseases that occur in rice nurseries without using chemical pesticides, and completed the present invention.

[Structure of the Invention]

[0009]

DISCLOSURE OF THE INVENTION The present invention relates to a Pseudomonas genus bacterium, preferably a Pseudomonas cepacia species bacterium, and more preferably a Pseudomonas cepacia AGF-158 strain having an antagonistic effect on a pathogen causing a disease occurring in a rice nursery. This is a method of inoculating seeds of S. cerevisiae to control and protect the disease that occurs in rice nurseries.

The genus Pseudomonas used in the present invention and
The Pseudomonas cepacia species bacterium may be any as long as it has an activity of suppressing a disease that occurs in a rice nursery, and a preferred example is Pseudomonas cepacia AGF-158 strain.

The Pseudomonas cepacia AGF-158 strain was isolated from the root surface of rice cultivated on soil collected from the suburbs of Sapporo, Hokkaido.

This Pseudomonas cepacia AGF-158 strain
It shows strong antagonistic activity against phytopathogenic fungi and phytopathogenic bacteria. The bacteriological properties are shown below.

Morphology Bacillus Size 0.8 to 1.0 × 1.8 to 3.0 microns Mobility Yes Flagella monopolar hair, 1 or more Water-soluble dyes are generated positive (yellow to green) Fluorescent dyes are generated Negative spores are not present Gram stain Sex negative Negative accumulation of poly-β-hydroxybutyric acid granules Positive Growth auxotrophy Negative Reaction to oxygen Aerobic Oxidase activity Positive Catalase activity Positive Levan production Negative Gelatin liquefaction Positive lecithinase (egg yolk) positive Starch hydrolysis Negative Tween 80 hydrolysis Positive casein hydrolysis Positive Esculin hydrolysis Negative Denitrification reaction Nitrate reduction Weak positive Arginine dehydrolase activity Negative Urease Positive Litmus milk Litmus is reduced, peptone production of hydrogen sulfide Negative VP test Negative MR test Negative OF test O-type gluconic acid Oxidation Negative Use of asparagine Positive Softening of potato slices Negative Softening of onion slices Positive Growth at 4 ° C Weak positive Growth at 41 ° C Weak positive Positive growth temperature 30-37 ° C Growth at pH 4.1 Negative Growth at pH 8.8 Negative Optimal growth pH 5 to 73 Growth on 3% sodium chloride positive Growth on 5% sodium chloride negative Production of acid from saccharides Positive: glucose, fructose, galactose, arabinose, mannitol, glycerin, sorbitol, sucrose, salicin, lactose, Maltose, cellobiose, D-xylose negative; raffinose, m-inositol, rhamnose availability test positive; D-xylose, D-ribose, D-arabinose, glucose, L-arabinose, D-mannose Galactose, fructose, trehalose, cellobiose, mannitol, m-inositol, sorbitol, glycerin, sucrose, levulinic acid, acetic acid, pyruvic acid, succinic acid, lactic acid, propionic acid, p-hydroxybenzoic acid, β-hydroxybutyric acid, butyric acid, Malonic acid, citric acid, 2,3-butylene glycol, L-tryptophan,
L-arginine, L-valine, L-lysine, β-alanine, betaine, putrescine, lactose, raffinose, glycolic acid, m-hydroxybenzoic acid, adnit, benzylamine, acetamide, salicin Weakly positive; L-rhamnose negative Of maltose, starch, isopropanol, ethanol, geraniol, mesaconic acid, D (-)-tartaric acid, nicotinic acid, phthalic acid, pantothenic acid, phenol, propylene glycol, ethylene glycol, tryptamine, glycine, naphthalene, oxalic acid, maleic acid acid.

From the above properties, Bergey's manual of syst
Referring to ematic bacteriology, Vol 1. (9th edition), this bacterium is a species very closely related to Pseudomonas cepacia. However, it is described in terms of the use of tryptamine in availability tests.
Different from Pseudomonas cepacia. Therefore, this bacterium was transformed into Pseudom
The strain was named onas cepacia AGF-158 strain. This Pseudomonas
The cepacia AGF-158 strain has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as "Peudomonasken No. 3834 (BP-3834)". Pseudomonas cepacia Polleroni e
t The same strain as the Holmes AGF-158 strain.

The Pseudomonas cepacia AGF-158 strain has an antagonistic activity against many of the pathogens of rice and various crops that occur in the nursery. Pseudomonas cepacia on agar
When the AGF-158 strain is cultured against each pathogen, rice germ fungus (Gibberella fujikuroi) and rice seedling take-all
(Fusarium solani, Rhizopus oryzae, Pythium gramini
cola, Pythium spinosum) and rice sesame leaf blight fungus (Cochliobo
lus miyabeanus), Pyricularia oryza
e), rice seedling bacterial wilt (Pseudomonas plantarii), rice brown streak (Pseudomonas avenae), rice wilt (Rhizo
ctonia solani) was significantly suppressed. Table 1 shows the results. The activity was displayed according to the following criteria.

(Criterion)-: No antagonistic activity +: Shows weak antagonistic activity (inhibition band width 3 mm or less) ++: Shows somewhat strong antagonistic activity (inhibition band width 3 to 8 m)
m) +++: shows strong antagonistic activity (inhibition zone width 8 mm or more)

[0017]

(Table 1) (Antagonistic activity of Pseudomonas cepacia AGF-158 against various pathogens)病原 Target pathogen name Disease name Activity ──────────────────────────────────── Pyricularia oryzae rice Rice blast ++++ Rhizoctonia solani Rice sheath blight ++ Cochliobolus miyabeanas Rice sesame leaf blight ++++ Gibberella fujikuroi Rice stupid disease ++++ Gibberella fujikuroi (drug resistant fungus) Rice stupid seedling ++ Rhizopus oryzae Rice seedling shoot up Blight +++ Pythium spinosum Rice seedling blight ++ Trichoderma viride Rice seedling blight + Fusarium solani Rice seedling blight +++ Fusarium nivale Wheat red snow rot +++ Fusarium oxysporum f.sp.lagenariae Fusarium porum spore + .sp.cepae Onion tofu Disease ++++ Fusarium oxysporum f.sp.cucumerinum cucumber vine disease ++++ Fusarium oxysporum f.sp.lycopersici tomato wilt ++++ Botrytis cinerea strawberry gray mold +++ Sclerotinia sclerotiorum kidney bean sclerotium + psy stomach bacillus aptata Sugar beet spot bacterial disease ++++ Pseudomonas avenae Rice brown streak ++ Pseudomonas plantarii Rice seedling blight bacterial disease ++ Agrobacterium tumefaciens処理 Treatment of rice seeds with a fungus-antagonizing activity can be carried out, for example, by immersion in rice seeds during the seed immersion period. By inoculating cells.

That is, in the seeding operation (dipping the seeds in water for several days) to germinate the rice seeds, a suitable amount of Pseudomonas bacterium having a disease-bacterial antagonistic activity is inoculated into the seeded water. is there. Before inoculation, the present cells may be sprayed on rice dry seeds for inoculation. The inoculated bacteria proliferate in the seeding water and adhere to the rice seeds, and the fungi colonize the rice seeds and roots even after sowing.

As a method for controlling a disease by directly using a bacterium having an antagonistic effect on a disease occurring in a rice nursery,
A method of culturing the bacteria in a solid medium and mixing it with rice seedling soil or artificial covering soil that is commercially available for seedling raising is also considered, but the method of the present invention has a higher effect of suppressing various diseases than this soil mixing treatment method. In particular, it is effectively exerted against stupid seedling disease, bacterial wilt disease, and brown streak disease caused by seed transmission (contaminated seeds).

[0020]

Industrial Applicability The present invention reduces the large economic burden on rice cultivation farmers when raising rice seedlings due to the occurrence of various diseases,
In addition, it is possible to omit many operations associated with disinfecting rice seeds with fungicides, reduce the amount of chemical treatment by half, or reduce the number of treated chemicals, and it is effective against resistant bacteria in which a specific chemical has lost its effect It is. One feature of the use of antagonistic microorganisms is that they do not cause environmental pollution unlike chemical pesticides.However, the antagonistic microorganisms used in the present invention are not bacteria that have been subjected to genetic manipulation, but are bacteria that exist in nature, and are present in the air. Since there is no need to apply large amounts of bacteria, the possibility of environmental pollution is small.

[0021]

[Test Examples] The present invention will be specifically described below with reference to Examples and Reference Examples.

In the following test examples, a solid medium,
The following liquid medium and artificial soil were used.

Solid medium-soy flour 4%, glycerin 4%,
A solution obtained by adding 60 ml of a solution in which 0.6% of sodium chloride is dissolved to 25 g of vermiculite.

Liquid medium-soy flour 2%, glycerin 2%,
A solution containing 0.3% sodium chloride (adjusted to pH 7 after sterilization).

Artificial soil-artificial soil commercially available for raising seedlings (trade name: Kumiai granular soil H)

[0026]

[Test Example 1] (Test for controlling rice stupid disease) Rice-contaminated seeds (variety: Mitsui Matsuyama) infected with stupid disease fungus were used.

The seeds were soaked at 20 ° C. for 3 days for 32 days.
° C and 24 hours.

Pseudomonas cepacia AGF-158 strain (hereinafter referred to as
AGF-158 strain) inoculated with inoculated water AGF-158 strain was cultured in a liquid medium at 27 ° C. for 2 days,
At the start of seeding, the cells collected by centrifugation are mixed with 4 × per liter of seeding water (soaked in 500 cc of seeds, the same applies hereinafter).
10 ¹¹ colony forming units (hereinafter abbreviated as cfu) were inoculated.

Soil-mixing treatment of AGF-158 strain The AGF-158 strain was cultured in a solid medium at 26 ° C. for 7 days.
This was mixed with artificial soil (volume ratio 1: 1), and the resulting soil was used for covering soil.

After raising the seedlings in a glass greenhouse for 16 days after sowing, the results of examination of the occurrence of stupid seedling disease are shown in Table 2.

[0031]

[Table 2] 苗 Treatment rate of seedlings of idiot disease ─浸 AGF-158 strain inoculated water inoculation 29% AGF-158 strain Soil mixing treatment (cover soil only) 40% No treatment 74% ────────────────────────────────────

[0032]

[Test Example 2] (Test for controlling rice seedling blight disease) Rice-contaminated seeds (variety: Yukihikari) artificially inoculated with seedling blight bacterial disease were used.

The seeds were soaked at 20 ° C. for 3 days for 32 days.
° C and 24 hours.

The AGF-158 strain was grown at 27 ° C.
After culturing for one day, the cells collected by centrifugation were separated at a rate of 2.4 × 10 ^{8 to} 2.4 × 1 per liter of seeding water at the start of seeding.
The inoculation was carried out while changing the bacterial amount to 0 ¹² cfu (5 stages).

After raising seedlings in a glass greenhouse for 21 days after sowing, Table 3 shows the results of examination of the onset of bacterial wilt.

[0036]

[Table 3] 処 Treatment Seedlings that cause bacterial blight of seedlings Rate 接種 Inoculation treatment at the time of immersion of AGF-158 strain 2.4 × 10 ¹² cfu / 1 liter immersion water 3.2% 2.4 × 10 ¹¹ cfu / 1 liter immersion water 2.4% 2.4 × 10 ¹⁰ cfu / 1 liter immersion water 19.1% 2.4 × 10 ⁹ cfu / 1 liter soaked water 31.6% 2.4 × 10 ⁸ cfu / 1 liter soaked water 34.3% untreated 99.2% ────────────────── ──────────────────

[0037]

[Test Example 3] (Test for controlling rice seedling blight disease) Rice-contaminated seeds (variety: Yukihikari) artificially inoculated with seedling blight bacterial disease were used.

The seeds were soaked at 15 ° C. for 4 days for 32 days.
° C and 24 hours.

AGF-158 strain inoculated with inoculated water AGF-158 strain was cultured in a liquid medium at 27 ° C. for 3 days.
The cells collected by centrifugation were inoculated at 1.4 × 10 ¹⁰ cfu per liter of seeding water at the start of seeding.

Dry seed inoculation treatment of AGF-158 strain Before inoculation, cells were cultivated at 1.4 × 10 ¹⁰ c per 10 g of dry seed.
fu was inoculated.

After raising the seedlings in a glass greenhouse for 20 days after sowing, the results of an examination of the onset of bacterial wilt of seedlings are shown in Table 4.

[0042]

[Table 4] 苗 Treatment Seedlings that cause bacterial blight of seedlings Rate 浸 AGF-158 strain inoculated water inoculation 10.2% Dry seed inoculation treatment of AGF-158 strain 20.9% No treatment 100.0% ─────────────────────────────── ─────

[0043]

[Test Example 4] (Rice control test for brown streak) A rice-contaminated seed (variety: Yukihikari) artificially inoculated with brown streak was used.

The seeds were soaked at 20 ° C. for 3 days for 32 days.
° C and 24 hours.

Inoculation of inoculated water with AGF-158 strain AGF-158 strain was cultured in a liquid medium at 27 ° C. for 2 days.
The cells collected by centrifugation were inoculated at the start of seeding with 2.9 × 10 ¹¹ cfu per liter of seeding water.

Soil-mixing treatment of AGF-158 strain The AGF-158 strain was cultured in a solid medium at 26 ° C. for 7 days.
This was mixed with artificial soil (volume ratio 1: 1), and the resulting soil was used for covering soil.

Table 5 shows the results of examining the onset of brown streak after raising seedlings in a glass greenhouse for 21 days after sowing.

[0048]

[Table 5] 苗 Treatment Browning disease incidence rate of seedlings ─浸 AGF-158 strain inoculated water inoculation 7.0% AGF- Soil admixture treatment of 158 strains (cover soil only) 20.5% No treatment 64.8% ────────────────────────────── ──────

[0049]

Test Example 5 (Rice Brown Stripe Control Test) Rice-contaminated seeds (variety: Yukihikari) artificially inoculated with brown stripe disease fungi were used.

The seeds were soaked at 20 ° C. for 3 days for 32 days.
° C and 24 hours.

The AGF-158 strain was incubated at 27 ° C.
After culturing for one day and centrifuging the collected cells, 2.4 × 10 ^{8 to} 2.4 × 10 ¹² cf per liter of seeding water at the start of seeding.
u (5 stages) and the amount of bacteria were changed and inoculated.

After raising seedlings in a glass greenhouse for 21 days after sowing, Table 6 shows the results of examination of the onset of brown streak.

[0053]

[Table 6] 率 Treatment Browning disease incidence rate of seedlings ─接種 Inoculation treatment of AGF-158 strain at the time of immersion 2.4 × 10 ¹² cfu / 1 liter soaked water 0.8% 2.4 × 10 ¹¹ cfu / 1 liter soaked water 10.0% 2.4 × 10 ¹⁰ cfu / 1 liter soaked water 60.0% 2.4 × 10 ⁹ cfu / 1 liter of immersion water 65.9% 2.4 × 10 ⁸ cfu / 1 liter of immersion water 80.9% untreated 81.3% ──────────────────── ────────────────

[0054]

[Test Example 6] (Test for controlling rice seedling blight) Using healthy rice seeds (variety: Kuiku No. 125), seedling damping fungi (Fusarium solani, Rhizoctonia solani) were used as pre-sterilized black soil.
Was used.

The seeds were soaked at 20 ° C. for 3 days for 32 days.
° C and 24 hours.

AGF-158 inoculated with inoculated water The AGF-158 strain was cultured in a liquid medium at 27 ° C. for 3 days.
At the start of seeding, the cells collected by centrifugation were inoculated at 3.4 × 10 ¹¹ cfu per liter of seeding water.

Soil-mixing treatment of AGF-158 strain The AGF-158 strain was cultured in a solid medium at 26 ° C. for 7 days.
This was mixed with artificial soil (volume ratio 1: 1), and the resulting soil was used for covering soil.

After raising seedlings in a glass greenhouse for 21 days after sowing, the results of examination of the onset of each seedling blight are shown in Table 7.

[0059]

[Table 7] 処理 Treatment Seedling incidence of seedling damping-off Fusarium Rhizoctonia 浸 AGF-158 inoculated water inoculation 1.2% 0 2.0% AGF-158 strain soil-mixing treatment (cover soil only) 2.9% 0.0% No treatment 10.3% 21.7% ───────────────── ───────────────────

[0060]

[Test Example 7] (Rice blast control test) Bacterial concentration 2.8 × 10 ⁹ cf
5 g of uGF / 158 AGF-158 strain was inoculated with 5 g of dry rice seed (cultivar: Norin 20), inoculated, placed in a 9 cm petri dish in a wet room, and kept at 25 ° C. for 12 hours under 12 hours of illumination. After that, the presence or absence of blast spore formation on the seeds was examined under a stereoscopic microscope, and the effect on rice blast (seedling blast) was examined. Table 8 shows the results.

[0061]

[Table 8] 率 Treated spore-forming seed rate ────イ AGF-158 rice seed inoculation treatment 0.0% Untreated 38.6% ────────────────────────────────────

[0062]

[Test Example 8] (Test for controlling rice foolish seedling disease) Rice-contaminated seeds (variety: Yukihikari) infected with idiot disease fungus (benomyl agent-resistant bacterium) were used.

The seeds were soaked at 20 ° C. for 4 days for 32 days.
° C and 24 hours.

Inoculation of inoculated water with AGF-158 strain AGF-158 strain was cultured in a liquid medium at 27 ° C. for 2 days.
The cells collected by centrifugation were inoculated with 2.5 × 10 ¹¹ cfu per liter of seeding water at the start of seeding or on the fourth day of seeding.

Dry seed inoculation treatment of AGF-158 strain Before inoculation, cells were washed with 2.5 × 10 ¹¹ cells per 100 g of dry seed.
Cfu (10 ml of a bacterial solution of 2.5 × 10 ¹⁰ cfu / ml) was dusted and inoculated.

After raising the seedlings in a glass greenhouse for 20 days after sowing, the results of examining the onset of idiot disease are shown in Table 9.

[0067]

[Table 9] 苗 Treatment rate of seedlings of idiot disease 馬処理 AGF-158 strain inoculated water inoculation treatment (at the start of inoculation) 5 0.7% AGF-158 strain inoculated water inoculation treatment (4 days after soaking) 9.8% AGF-158 strain dried seed inoculation treatment 7.7% No treatment 100.0% ────────── ──────────────────────────

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-4-295407 (JP, A) JP-A-2-35076 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 63/00

Claims (3)

(57) [Claims] 1. A method for controlling a disease occurring in a rice nursery, comprising treating a rice seed with Pseudomonas cepacia sp., A bacterium having an antagonistic effect on a pathogen causing a disease occurring in the rice nursery. 2. The method according to claim 1, wherein the bacterium is Pseudomonas cepacia AGF-158 strain. 3. A Pseudomonas cepacia AGF-158 strain.