WO2006109795A1 - 糸状菌胞子の製造方法及び植物病害防除方法 - Google Patents
糸状菌胞子の製造方法及び植物病害防除方法 Download PDFInfo
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- WO2006109795A1 WO2006109795A1 PCT/JP2006/307635 JP2006307635W WO2006109795A1 WO 2006109795 A1 WO2006109795 A1 WO 2006109795A1 JP 2006307635 W JP2006307635 W JP 2006307635W WO 2006109795 A1 WO2006109795 A1 WO 2006109795A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/10—Mycorrhiza; Mycorrhizal associations
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/36—Penicillium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
Definitions
- the present invention relates to a technique related to control of plant diseases. More specifically, the present invention relates to a technology that enables the formation of a sufficient amount of spores sufficient for practical use as a microbial pesticide or a material of the genus Pecilli.
- Chemical pesticides are indispensable means for controlling pests in plants and greatly contribute to stable food production.
- the emergence of resistant pests caused by multiple administrations of chemical pesticides and the problem of environmental burden have been taken up.
- filamentous fungi such as Penicillium are known as microorganisms expected to be used as the biopesticide.
- Talaromyces spp. The complete generation of the genus Pecilium, has already been used as an agricultural fungicide for strawberry anthracnose and powdery mildew (Talaromyces flavus (Talaromyces flavus)). flavus) wettable powder, Ministry of Agriculture, Forestry and Fisheries No. 20659).
- Patent Document 1 discloses a Talaromyces flavus having an antagonistic action against strawberry anthrax!
- Patent Document 2 discloses Penicillium expansum as a microorganism having an antagonistic action against mango anthracnose, and Patent Document 3 controls gray mold fungi. Pecillium-powered manmberti, which shows effects, is disclosed.
- a medium containing an antifoaming agent or a hardener such as agar at 0.0009 to 0.005 g / 1 has been reported. Furthermore, it is known that the addition of calcium salt is effective for sporulation of P. pyrium (see Non-Patent Document 1).
- Patent Document 1 Japanese Patent Laid-Open No. 10-229872.
- Patent Document 2 JP 2001-39810 A.
- Patent Document 3 Japanese Patent Application Laid-Open No. 2004-231626.
- Patent Document 4 JP-A-9 322759.
- Patent Document 5 JP-A-11 276158.
- Patent Document 6 JP 2000-201669 A.
- Patent Document 7 United State Patent 6593127.
- Non-patent literature l Trans.Br.Mycol.Soc., 80 (2), pp319-325, 1983.
- an object of the present invention is to provide a safe technique capable of forming a sufficient amount of filamentous fungal spores sufficient for practical use as a microbial pesticide or a material.
- the present invention provides a method for producing filamentous fungal spores. Since this manufacturing method can use many versatile culture devices, it is easy to sterilize, and has advantages such as easy control of the environment such as temperature during culture, oxygen supply, and pH. Select a liquid culture
- the liquid medium contains corn steep liquor or soybean-derived peptone as a carbon source and a nitrogen source, and the amount of inorganic components derived from the carbon source and the nitrogen source is a liquid of the target filamentous fungus. Judging from the number of spores formed during culture, if the amount is less than the required amount, first, calcium chloride, further magnesium sulfate if necessary, and dipotassium hydrogen phosphate if necessary. And cultivate filamentous fungi using this liquid medium to form spores.
- the corn steep liquor or the soybean-derived peptone as a carbon source and a nitrogen source is contained in a liquid medium in an amount of 0.1 to 10%, and further, the salt calcium as an inorganic component is 0.2 to 0.2%. It is contained at 5.0%, preferably 0.4 to 3.5%.
- magnesium sulfate and dipotassium hydrogen phosphate may be added as inorganic components so as not to be excessive. This is to compensate for the lack of the necessary amount of magnesium and potassium salts in the medium. In this case, it is desirable to add magnesium sulfate in an amount of 0.001 to 10%, preferably 0.005 to 5.0%. Further, it is desirable that dipotassium hydrogen phosphate is added so as to be contained in an amount of 0.001 to 0.3%, preferably 0.05 to 0.2%.
- the filamentous fungus is a fungus belonging to the genus Penicillium, and as a preferred example, Talaromyces sp. B-422 (FERM BP-08516) Talaromyces flavus can be employed.
- the number of spores formed during liquid culture should be about 10 8 or more. Effective in spore production. Therefore, if the number of spores formed in liquid culture is less than about 10 8 , the liquid medium must first contain calcium chloride as an inorganic component, magnesium sulfate if necessary, and hydrogen phosphate if necessary. It is desirable to capture potassium.
- the present invention cultivates filamentous fungi using a liquid medium containing corn steep liquor or peptone derived from soybean as a carbon source and nitrogen source, and containing at least calcium chloride calcium as an inorganic component.
- the present invention provides a method for controlling plant diseases, wherein the spores formed by the method are collected and the spores are brought into contact with a plant body.
- the term “contact” widely includes methods such as spraying, dipping, mixing, and application to plants, and after mixing and irrigating the spores with the soil, seeding the plant seeds to “contact” them. It is possible, and it should not be interpreted narrowly.
- dipotassium hydrogen phosphate and magnesium sulfate may be added to the liquid medium as inorganic components so as not to be excessive. This is to make up for the necessary amount of magnesium and potassium salts in the medium.
- magnesium sulfate so as to contain 0.001 to 10%, preferably 0.005 to 5.0%.
- dipotassium hydrogen phosphate in an amount of 0.001 to 0.3%, preferably 0.05 to 0.2%.
- Core Steep Liquor (abbreviated as CS L) is an organic by-product generated in the process of producing sugar (corn starch).
- Soy-derived peptone is obtained by partially hydrolyzing soy protein with a proteolytic enzyme or acid, and is mainly composed of oligopeptides and amino acids. These are all fungi Functions as an organic component for supplying a carbon source and a nitrogen source.
- filamentous fungal spores generally difficult to form by liquid culture can be industrially produced in large quantities and at low cost.
- the spores obtained by this method are useful as microbial pesticides and biological materials.
- it since it is cultured without using any animal organic components, it is safe to avoid BSE problems.
- corn steep liquor or soybean-derived peptone is used as the carbon source and nitrogen source used for liquid culture of filamentous fungal spores. In some cases, both of these may be used. Soy flour and kina flour can be used, but separation from spores becomes difficult after cultivation. In addition, sufficient amounts of spore formation cannot be obtained with animal peptone.
- "Corn steep liquor” is not particularly limited, but, for example, Showa Sangyo Co., Ltd., Hokkai Sugar Co., Ltd., Nippon Shokuhin Kako Co., Ltd., Oriental Yeast Co., Ltd. ) And the like can be used as appropriate.
- the “soybean-derived peptone” is not particularly limited, and for example, polypeptone-S manufactured by Nippon Pharmaceutical Co., Ltd. can be used.
- a suitable addition amount of corn steep liquor or soybean-derived peptone is 0.1% to 10%, preferably 0.5 to 10%, more preferably 1 to 5%. If an excessive amount is added, precipitation of insoluble matter increases and separation from spores becomes difficult.
- a sufficient amount of spores can be produced simply by adding corn steep liquor or soybean-derived peptone, but it is also possible to add a commonly used carbon source.
- a commonly used carbon source For example, glucose, sucrose, flatatose, galactose, glycerol, starch, sugarcane, and molasses derived from beet may be further added. Among them, it is desirable to use molasses mainly because it is inexpensive. For example, when beet molasses is used, the medium with 0.5% to 10% added can be shown in column f.
- carbonic acid in addition to the above carbon source and nitrogen source, as inorganic components, carbonic acid, phosphoric acid and the like Metal salts such as salts, potassium, sodium, iron and magnesium can be prepared.
- Metal salts such as salts, potassium, sodium, iron and magnesium can be prepared.
- dipotassium hydrogen phosphate and magnesium sulfate are preferred because they are necessary elements for the growth of P. pyrium.
- a calcium salt is added as a component for promoting sporulation.
- salty calcium is preferred mainly in terms of solubility.
- the calcium chloride concentration is 0.2 to 5.0%, preferably 0.4 to 3.5%. Addition of excess calcium chloride increases precipitation of insoluble matter and makes separation from spores difficult.
- various surfactants can be freely added to the medium as an antifoaming agent.
- the culture is performed at a culture temperature of about 20 ° C to 40 ° C, preferably 25 ° C force 35 ° C, and the initial pH of the medium is 4.0 force 8.0, preferably 5.0 to 7.0. It is carried out under aerobic conditions such as by aeration and agitation culture with a mentor.
- the culture is filtered with sashi, gauze, glass wool or the like to remove the mycelium.
- the obtained filtrate is centrifuged to separate spores. Add water to separate the centrifuge and wash. These steps are repeated several times to recover the washed spores.
- washing water ion-exchanged water, distilled water, pure water prepared using various filtration membranes, water, etc. can be used.
- the spore exhibits the same preservation and control effect as a spore obtained from a solid medium such as potato dextrose agar medium (PDA), and can be used as a biopesticide or a biological material.
- a solid medium such as potato dextrose agar medium (PDA)
- PDA potato dextrose agar medium
- the filamentous fungus that can be cultured in the present invention is, for example, the genus Persium.
- the Penicillium fungus is not particularly limited.For example, Eupenicillium sp. B-408 (FERM BP-08517), Talaromyces sp. B-422 (FERM BP-08516), Penicilli um sp. B-453 (FERM BP—08515), Talaromyces flavus, Penicillium expansum, Penicillium camemberti and the like.
- Eupenicillium sp.B-408 (FERM BP-08517), Talaro myces sp.B-422 (FERM BP-08516), Penicillium sp.B-453 (FERM BP-08515), Talaro myces flavus are preferred, but Talaromyces sp.B — 422 (FERM BP—08516) and Talaromyces flavus are more preferred.
- Eupenicillium sp. B-408 (FERM BP-08517), Talaromyces sp. B-422 (FERM BP- 0851) 6), Penicillium sp.B-453 (FERM BP-08515) is the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Postal Code: 305-8566, 1-chome Tsukuba, Ibaraki, Japan 1 Chuodai 6 ).
- Example 1 in the culture of fungal spores, the difference in the number of cultured spores when the composition of the liquid medium was changed was examined.
- Experimental Examples 1 to 3 are methods for producing filamentous fungal spores according to the present invention.
- Corn steep liquor (Oriental Yeast Co., Ltd.) 3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, calcium chloride dihydrate 1% ( ⁇ 7.0) medium 50 ml was dispensed into a 300 ml Erlenmeyer flask and sterilized (120 ° C, 20 minutes). Sterilized water was added to Talaromyces sp. B-422 (FERM BP-08516) or Talaromyces flavus pre-cultured in PDA medium to prepare a spore concentration of 1 ⁇ 10 6 cells / ml. The above liquid medium was inoculated with 0.5 ml of Talaromyces sp.
- Corn steep liquor Showa Sangyo 3%, Beet molasses (Hokkaido Sugar Industry) 0.5%, Dipotassium hydrogen phosphate 0.1%, Magnesium sulfate heptahydrate 0.05%, Calcium chloride dihydrate 1 50 ml of medium having a composition of% (pH 7.0) was dispensed into a 300 ml Erlenmeyer flask and sterilized (120 ° C., 20 minutes). Inoculate 0.5 ml of Talaromyces sp. B-422 (FERM BP-08516) spore solution or Talaromyces flavus spore solution prepared according to Experimental Example 1 and shake incubator (150 rpm, 25 ° C) for 7 days. Cultured. After completion of the culture, the spore count was measured with a hemocytometer.
- Experimental Example 1 0.5 ml of the adjusted Talaromyces sp. B-422 (FERM BP-08516) spore solution or Talaromyces flavus spore solution was inoculated and cultured in a shaking incubator (150 rpm, 25 ° C) for 7 days. After completion of the culture, the spore count was measured with a hemocytometer.
- B-422 (FERM BP-08516) spore solution or Talaromyces flavu s spore solution prepared according to Experimental Example 1 and shake incubator (150 rpm, 25 ° C) for 7 days. Cultured. After completion of the culture, the spore count was measured with a hemocytometer.
- 300 ml medium containing 50% galactose, calcium chloride dihydrate 2.5%, sodium nitrate 0.6%, potassium dihydrogen phosphate 0.15%, magnesium sulfate heptahydrate 0.05% (pH 7.0) Dispensed into a triangular flask and sterilized (120 ° C, 20 minutes). Inoculate 0.5 ml of Talaromyces sp. B-422 (FERM BP-08516) spore solution or Talaromyces flavus spore solution prepared according to Experimental Example 1, and then in a shaking incubator (150 rpm, 25 ° C) for 7 days. Cultured. After completion of the culture, the spore count was measured with a hemocytometer.
- Potato dextrose broth 50 ml of a medium having a composition of 2.4% (pH 7.0) was dispensed into a 300 ml Erlenmeyer flask and sterilized (120 ° C., 20 minutes). Apply 0.5 ml of Talarom yces sp. B-422 (FERM BP-08516) spore solution or Talaromyces flavus spore solution prepared according to Experimental Example 1. And seeds were cultured for 7 days in shaker (150 r P m, 25 ° C). After completion of the culture, the spore count was measured with a hemocytometer.
- Polypeptone (Nippon Pharmaceutical Co., Ltd.) 3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 7 hydrate 0.05%, calcium chloride dihydrate 1% ( PH 7.0) Dispensing into triangular flasks and sterilizing (120 ° C, 20 minutes).
- 0.5 ml of Talaromyces spB-422 (FERM BP-08516) spore solution or Talaromyces flavus spore solution prepared according to Experimental Example 1 was inoculated and cultured for 7 days in a shaking incubator (150 rpm, 25 ° C). After completion of the culture, the spore count was measured with a hemocytometer.
- Comparative experiment example 1 3. 2x 10 7 ⁇ 1.0. 0X 10 4 (mycelium) Comparative experiment example 2 1. 5 X 10 6 2. 3x 10 6 Comparative experiment example 3 1. 4 10 6 ⁇ 1.0 X 10 4 Comparative experiment example 4 ⁇ 1. 0X 10 4 (hyphae form) 1. 3 ⁇ 10 4 (hyphae form) Comparative experiment example 5 ⁇ 1. ⁇ 10 4 (hyphae form) ⁇ 1- 0X 10 4 (hyphae form) [0045] As is clear from the results of "Table 1" described above, the spore production in Experimental Examples 1 to 3 is significantly higher than the spore production in Comparative Experimental Examples 1 to 5 for any fungus. There were many.
- Example 2 the optimum concentration of calcium chloride used in the method for producing filamentous fungal spores according to the present invention was examined.
- Example 3 the transition of the number of spores for each culture time in the method for producing filamentous fungal spores according to the present invention was examined.
- composition of corn steep liquor (Nihon Shokuhin Kako) 3%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, calcium chloride dihydrate 1% ( ⁇ 7.0)
- the medium 2 1 was dispensed into 3 1 jar mentors and sterilized (120 ° C., 60 minutes).
- Figure 1 is a drawing substitute graph. As can be seen from the results shown in FIG. 1, the number of spores reached approximately 1 ⁇ 10 8 cells / ml in 3 days of culture.
- Example 4 spore viability was confirmed using the medium used in Example 3 above.
- Example 4 50 ml of the medium having the composition used in Example 4 was dispensed into a 300 ml Erlenmeyer flask and sterilized (120 ° C, 20 minutes). 0.5 ml of a spore solution of Talaromyces sp. B-422 (FERM BP-08516) pre-cultured in PDA medium was inoculated and cultured for 7 days in a shaking incubator (150 rpm, 25 ° C). After completion of the culture, the culture broth was filtered with a brush to remove the mycelium. The obtained filtrate was centrifuged to collect spores. Distilled water was added and the mixture was centrifuged and washed. This was repeated twice, and the washed spores were collected and prepared so that the number of spores counted under a microscope in tap water was about 2 ⁇ 10 8 Zml.
- the spores obtained by liquid culture showed the same viability as the spores obtained by the solid medium.
- Example 5 Talaromvces sp. B-422 (FERM BP-08516) liquid culture spore solution and solid solution The control effect of the body culture spore solution on rice seedling disease was verified.
- Talaromyces sp. B-422 (FERM BP-08516) liquid culture spore solution and solid culture spore solution obtained in the same manner as in Example 3 were stored at 5 ° C for 1 month. Used for this test.
- Talaromyces sp. B-422 (FERM BP-08516) spore solution prepared from rice-affected seeds (produced in 2001, varieties: short silver shaved) naturally infected in a field with abundant rice seedling disease at a bath ratio of 1: 1 Soaked for 24 hours, then seeded at 15 ° C for 4 days (bath ratio 1: 1), germinated for 1 day at 30 ° C, and then commercially available granular soil for seedling (trade name: Kumiai granular soil, stock A seedling box (10 ⁇ 15 cm) packed with Kureha Co., Ltd. was seeded with 5 g of dry rice per box (3 repeats in 1 section).
- Control value (1-1 (the ratio of the seedlings in the treated area ⁇ the ratio of the seedlings in the untreated area)) X 1 0 0 [0060] [Table 4]
- the present invention can be used as a technique for producing filamentous fungal spores that can be used in biological pesticides and materials for controlling plant diseases, or as a plant disease technique.
- FIG. 1 is a graph showing the results of Example 3, and is a drawing-substituting graph showing the transition of the number of spores per culture time.
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KR1020087024862A KR101108830B1 (ko) | 2005-04-11 | 2006-04-11 | 사상균 포자의 제조 방법 및 식물 병해 방제 방법 |
CN2006800542013A CN101415331B (zh) | 2005-04-11 | 2006-04-11 | 丝状菌孢子的制造方法及植物病害防治方法 |
JP2007513009A JP4340707B2 (ja) | 2005-04-11 | 2006-04-11 | 糸状菌胞子の製造方法 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2007031294A (ja) * | 2005-07-22 | 2007-02-08 | Idemitsu Kosan Co Ltd | イネの育苗時期に発生する病害に対する防除剤 |
JP2015193708A (ja) * | 2014-03-31 | 2015-11-05 | 栗田工業株式会社 | 植物の根圏改質資材、および植物の栽培方法 |
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JPH09322759A (ja) * | 1996-06-10 | 1997-12-16 | Snow Brand Milk Prod Co Ltd | 糸状菌の培養方法 |
JPH10229872A (ja) * | 1997-02-21 | 1998-09-02 | Tochigi Pref Gov | 炭そ病防除効果を示す新規微生物 |
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WO2003065812A1 (en) * | 2002-02-05 | 2003-08-14 | Council Of Scientific And Industrial Research | Novel composition for early and profuse sporulation in fungi and a method thereof |
JP2004231626A (ja) * | 2003-02-03 | 2004-08-19 | Nippon Soda Co Ltd | 植物病害防除剤組成物及び微生物 |
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CN1087903C (zh) * | 1996-01-19 | 2002-07-24 | 济南科贝尔生物工程有限公司 | 液体发酵法制备防治植物病毒的农药及其生产工艺 |
JP4346007B2 (ja) | 2002-07-08 | 2009-10-14 | 株式会社トナミエンテック | ベルトコンベヤの仕分け装置 |
CN1276713C (zh) * | 2003-04-14 | 2006-09-27 | 浙江省农业科学院 | 一种微生物农药及其制备方法 |
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- 2006-04-11 KR KR1020087024862A patent/KR101108830B1/ko not_active IP Right Cessation
- 2006-04-11 WO PCT/JP2006/307635 patent/WO2006109795A1/ja active Application Filing
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JPH09322759A (ja) * | 1996-06-10 | 1997-12-16 | Snow Brand Milk Prod Co Ltd | 糸状菌の培養方法 |
JPH10229872A (ja) * | 1997-02-21 | 1998-09-02 | Tochigi Pref Gov | 炭そ病防除効果を示す新規微生物 |
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WO2003065812A1 (en) * | 2002-02-05 | 2003-08-14 | Council Of Scientific And Industrial Research | Novel composition for early and profuse sporulation in fungi and a method thereof |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007031294A (ja) * | 2005-07-22 | 2007-02-08 | Idemitsu Kosan Co Ltd | イネの育苗時期に発生する病害に対する防除剤 |
JP2015193708A (ja) * | 2014-03-31 | 2015-11-05 | 栗田工業株式会社 | 植物の根圏改質資材、および植物の栽培方法 |
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KR20080112289A (ko) | 2008-12-24 |
CN101415331A (zh) | 2009-04-22 |
JP4340707B2 (ja) | 2009-10-07 |
CN101415331B (zh) | 2012-09-05 |
KR101108830B1 (ko) | 2012-01-31 |
JPWO2006109795A1 (ja) | 2008-11-20 |
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