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JP2827093B2 - Pest control material - Google Patents

Pest control material

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Publication number
JP2827093B2
JP2827093B2 JP7099628A JP9962895A JP2827093B2 JP 2827093 B2 JP2827093 B2 JP 2827093B2 JP 7099628 A JP7099628 A JP 7099628A JP 9962895 A JP9962895 A JP 9962895A JP 2827093 B2 JP2827093 B2 JP 2827093B2
Authority
JP
Japan
Prior art keywords
soil
bacteria
present
culture
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
JP7099628A
Other languages
Japanese (ja)
Other versions
JPH08268825A (en
Inventor
公孝 相野
浩之 牧
克彦 清水
義雄 前川
泰三 秋山
佳徳 林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
HYOGOKEN
TAKI KAGAKU KK
Original Assignee
HYOGOKEN
TAKI KAGAKU KK
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Filing date
Publication date
Application filed by HYOGOKEN, TAKI KAGAKU KK filed Critical HYOGOKEN
Priority to JP7099628A priority Critical patent/JP2827093B2/en
Publication of JPH08268825A publication Critical patent/JPH08268825A/en
Application granted granted Critical
Publication of JP2827093B2 publication Critical patent/JP2827093B2/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【0001】[0001]

【産業上の利用分野】本発明は、結晶性2,4-シ゛アセチルフロロク
゛ルシノール産生能を有し、かつ、抗生物質に対して耐性を示
さない蛍光性細菌からなる青枯病防除資材に関し、農作
物の青枯病による被害を軽減することにより環境保全下
における農業の生産性の向上を図ることを目的とするも
のである。
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bacterial wilt control material comprising a fluorescent bacterium which has the ability to produce crystalline 2,4-diacetylfluoroclucinol and has no resistance to antibiotics. The purpose of the present invention is to improve the productivity of agriculture under environmental protection by reducing the damage caused by bacterial wilt of crops.

【0002】[0002]

【従来の技術】土壌微生物病害の生物防除法として、こ
れまでに抗菌性物質あるいはシテ゛ロフォアを産生するシュート゛モナ
ス属の蛍光性細菌について数多くの検討が行われてきて
いる。最近ではシュート゛モナス・フ゜チタ゛(Pseudomonas putida)や
シュート゛モナス・フルオレッセンス(Pseudomonas fluorescens)を利用し
た生物防除に関する研究が注目され、シュート゛モナス・フルオレッセン
スに関する米国に於ける研究は、販売あるいは圃場での
効果確認試験の段階まで進展している。このシュート゛モナス・フ
ルオレッセンスは、古くからフェナシ゛ン系の抗菌性物質等を産生す
ることで知られていた。また、1953年には同菌による2,
4-シ゛アセチルフロロク゛ルシノールの産生が認められ抗生物質としての
作用が検定された。検定の結果、放線菌を含むク゛ラム陽性
菌及び糸状菌に対する抗生物質としての効果が認められ
た。
2. Description of the Related Art As a biological control method for soil microbial diseases, many studies have been made on fluorescent bacteria of the genus Aureus spp. Which produce antibacterial substances or cytophores. Recently, attention has been focused on research on biological control using Pseudomonas fluorescens (Pseudomonas putida) and Pseudomonas fluorescens. It has progressed to the stage. This shoot Demonas fluorescens has long been known for producing phenacidine-based antibacterial substances and the like. Also, in 1953,
Production of 4-diacetylfluorochlorocinol was observed, and the action as an antibiotic was tested. As a result of the assay, an effect as an antibiotic against quorum-positive bacteria including actinomycetes and filamentous fungi was recognized.

【0003】これらの知見から1980年代に入り、シュート゛モ
ナス・フルオレッセンスによる土壌伝染性糸状菌病害の生物防除へ
の利用が検討されるようになり1990年代に入って、in v
itroでの病原糸状菌に対する抗菌性からタハ゛コ黒根病、大
麦立枯病、苗立枯病、小麦葉枯病等の生物防除への利用
が提案された。一方、土壌伝染性細菌病害に対しては、
報告例は少ないが、E.Levyら(Plant Pathology,1992,4
1)により生菌体とその代謝物である2,4-シ゛アセチルフロロク゛ルシノ
ールの双方がin vitroで植物病原細菌及び植物病原糸状菌
に対して抗菌性を示すことが報告された。この報告の中
でE.Levyらは、糸状菌病害だけではなく青枯病及び軟腐
病等の土壌伝染性細菌病害に対する生物防除法としても
シュート゛モナス・フルオレッセンスが利用できることを提案している。
しかし、これらはいずれもin vitroでの抗菌性試験の結
果に基づくものであり実用化を裏付ける技術には至って
いない。
[0003] From these findings, in the 1980's, the use of shoot Pseudomonas fluorescens for the control of soil-borne fungal diseases in organisms was examined, and in the 1990's, in v.
Due to its antibacterial activity against fungal pathogens in itro, its use in biological control such as black rot, barley wilt, seedling wilt and wheat leaf wilt was proposed. On the other hand, for soil-borne bacterial diseases,
Although there are few reports, E. Levy et al. (Plant Pathology, 1992, 4
According to 1), it was reported that both viable cells and their metabolite 2,4-diacetylfluorochlorocinol have antibacterial activity against phytopathogenic bacteria and phytopathogenic fungi in vitro. In this report, E. Levy et al. Propose that Shoot Monas fluorescens can be used as a biological control method not only for fungal diseases but also for soil-borne bacterial diseases such as bacterial wilt and soft rot.
However, these are all based on the results of in vitro antibacterial tests, and have not yet reached a technology supporting practical application.

【0004】ところで、わが国農業の野菜栽培における
土壌伝染性病害は被害の程度において糸状菌病害と細菌
病害に二分され、細菌病害の代表が青枯病である。これ
までに、化学農薬等により種々の対策がとられれきたに
もかかわらず対応が困難な病害であり環境保全の面から
も生物防除に対する期待が高まってきている。しかし、
作用機構に関する研究の遅れもあり実用化には至ってい
ないのが現状である。
By the way, soil-borne diseases in vegetable cultivation in Japanese agriculture are divided into fungal diseases and bacterial diseases according to the degree of damage, and bacterial wilt is a representative of bacterial diseases. Until now, various measures have been taken with chemical pesticides and the like, but it is a disease that is difficult to deal with, and expectations for biological control are increasing from the viewpoint of environmental protection. But,
At present, it has not been put into practical use due to the delay in research on the mechanism of action.

【0005】[0005]

【発明が解決しようとする課題】そこで本発明者らは、
資材化が可能で、実圃場において再現性のある青枯病抑
制効果が発現し、かつ、効果の持続性を有する安全性の
確保された青枯病防除資材を開発するため種々検討した
結果、本発明を完成したものである。本発明者らは生物
防除資材への実用化が可能な微生物として、すでに開発
が行われた生物農薬に着目した。その中で一部の糸状菌
病に対する防除を目的に生物農薬として販売された実績
を有するシュート゛モナス・フルオレッセンスの青枯病防除資材としての
可能性について検討することとし、シュート゛モナス・フルオレッセンス
のなかでも青枯病菌に対して抗菌性を示す可能性のある
2,4-シ゛アセチルフロロク゛ルシノール産生菌を研究対象とした。
SUMMARY OF THE INVENTION Accordingly, the present inventors
As a result of various investigations to develop a material for controlling bacterial wilt which can be used as a material and has a reproducible bacterial wilt control effect in a real field, and has a long-lasting effect. The present invention has been completed. The present inventors have paid attention to biopesticides that have already been developed as microorganisms that can be practically used as biological control materials. Among them, the potential of Shoot 検 討 Monas fluorescens, which has been sold as a biopesticide for the purpose of controlling some fungal diseases, as a material for controlling bacterial wilt disease, was examined. May exhibit antibacterial properties against bacterial wilt
2,4-Diacetylfluorochlorocinol-producing bacteria were studied.

【0006】2,4-シ゛アセチルフロロク゛ルシノールを産生するシュート゛モナス
・フルオレッセンスについては、他の研究者らにより遺伝子レベ
ルでの研究も進められている。シュート゛モナス・フルオレッセンスを生
物的防除資材として利用するためには、少なくともin v
itroにおいて2,4-シ゛アセチルフロロク゛ルシノールを安定的に産生する
菌株を検索することが必要である。前述のE.Levyらの報
告では、シュート゛モナス・フルオレッセンスのハ゛イオハ゛ーのI(Bergeyらの
分類による)<Bergey'smanual of systematic bacteriol
ogy>を親株として抗生物質による耐性選抜菌を作出した
結果、選抜菌は最大で約70μg/mlの2,4-シ゛アセチルフロロク゛ルシノ
ールを産生したと述べられている。本発明者らは、種々の
分離源から検索した2,4-シ゛アセチルフロロク゛ルシノール産生菌の菌学
的特性と植物体を用いた発病抑制試験について詳細な検
討を行った。
[0006] Other researchers have studied the genetic level of Shoot Pmonas fluorescens, which produces 2,4-diacetylfluorochlorocinol. In order to use Shoots Monas fluorescens as a biological control material, at least in v
It is necessary to search for a strain that stably produces 2,4-diacetylfluorochlorocinol in itro. In the report of E. Levy et al. Mentioned above, the biomolecule I of Shooto Monas fluorescens (according to the classification of Bergey et al.) <Bergey'smanual of systematic bacteriol
It was stated that as a result of creating a resistant selection bacterium with an antibiotic using ogy> as the parent strain, the selected bacterium produced a maximum of about 70 μg / ml of 2,4-diacetylfluorochlorocinol. The present inventors have conducted detailed studies on the bacteriological properties of 2,4-diacetylfluoroclucinol-producing bacteria retrieved from various sources and disease suppression tests using plants.

【0007】一連の検討により、土壌及び根表面等から
分離した2,4-シ゛アセチルフロロク゛ルシノール産生菌は、E.Levyらの方
法に準じて検討した場合、培養液等の培養物から溶媒抽
出により2,4-シ゛アセチルフロロク゛ルシノールを確認せざるを得ない少
量産生菌株と、結晶性の2,4-シ゛アセチルフロロク゛ルシノールを産生す
る菌株とに分類されることを見出した。前者は根面及び
根圈土壌から多く分離され、2,4-シ゛アセチルフロロク゛ルシノール産生
量が最大で約100μg/mlとE.Levyらの耐性菌と同程度で
あった。後者は主に、作物根内から分離された。
[0007] According to a series of studies, 2,4-diacetylfluorochlorocinol-producing bacteria isolated from soil, root surface and the like were subjected to solvent extraction from a culture such as a culture solution when examined according to the method of E. Levy et al. As a result, it was found that the strain was classified into a small-producing strain that had to confirm 2,4-diacetylfluoroculocinol and a bacterial strain producing crystalline 2,4-diacetylfluoroclucinol. The former was largely isolated from the root surface and the rhizosphere soil, and the production of 2,4-diacetylfluorochlorocinol was up to about 100 μg / ml, which was comparable to the resistant bacteria of E. Levy et al. The latter was mainly isolated from within the crop roots.

【0008】分取した2,4-シ゛アセチルフロロク゛ルシノールは、共にin
vitroにおいて病原糸状菌、放線菌を含むク゛ラム陽性菌及
びク゛ラム陰性細菌に対して抗菌性を示した。しかし、産生
菌そのものの発病抑制効果には大きな相違が認められ
た。両者を個々に接種した植物体を育苗後、青枯病発病
土壌に定植し発病抑制試験を実施した結果、前者の菌株
は発病土壌及び作物根内での生育は殆ど認められず、発
病抑制効果も些少であった。しかし、後者の菌株は作物
根内での生育と顕著な発病抑制効果が認められた。しか
も前者の菌株は、2,4-シ゛アセチルフロロク゛ルシノールを比較的多く産
生する菌株であっても継代培養等による保存では、産生
能及び増殖能が極端に低下する菌株が多く認められ培養
及び品質管理上に大きな問題を残すものであった。
[0008] The fractionated 2,4-diacetylfluorochlorocinol is both in
In vitro, it exhibited antibacterial activity against quorum-positive and quorum-negative bacteria including actinomycetes and actinomycetes. However, a significant difference was observed in the disease-suppressing effects of the producing bacteria themselves. After raising seedlings of plants inoculated with both of them individually, the plants were planted on the bacterial blight disease-causing soil and a disease-inhibition test was carried out. Was also insignificant. However, the latter strain showed growth in the crop roots and a remarkable disease-control effect. Moreover, the former strain, even if it is a strain that produces relatively large amounts of 2,4-diacetylfluoroqulucinol, is greatly reduced in production capacity and growth capacity when preserved by subculturing, etc. This left a major problem in quality control.

【0009】[0009]

【課題を解決するための手段】かかる検討結果をもと
に、結晶性2,4−ジアセチルフロログルシノール産生
能を有する蛍光性細菌について、実圃場での効果確認試
験を実施し鋭意検討を重ねた結果、本発明に到達したも
のである。即ち、本発明は結晶性2,4−ジアセチルフ
ロログルシノール産生能を有し、かつ、抗生物質に対し
て耐性を示さない蛍光性細菌即ち、ストレプトマイシン
100μg/ml、ナリジキシン酸50μg/ml及び
リファンピシン50μg/mlを含有するキングB培地
に菌株を接種後、15〜40℃の温度で培養し、2週間
後においても生育の認められない蛍光性細菌からなる青
枯病防除資材に関する。
On the basis of the results of the study, a fluorescent bacteria having a crystalline 2,4-diacetylphloroglucinol-producing ability is subjected to an effect confirmation test in an actual field, and intensive studies are repeated. As a result, the present invention has been achieved. That is, the present invention relates to a fluorescent bacterium which has the ability to produce crystalline 2,4-diacetylphloroglucinol and does not exhibit resistance to antibiotics, ie, streptomycin.
100 μg / ml, nalidixic acid 50 μg / ml and
King B medium containing 50 μg / ml rifampicin
After inoculation of the strain, the cells are cultured at a temperature of 15 to 40 ° C. for 2 weeks.
The present invention relates to a bacterial wilt control material comprising fluorescent bacteria that cannot be grown even afterwards .

【0010】[0010]

【作用】以下に本発明について更に詳記する。先ず本発
明に於いて、結晶性2,4-シ゛アセチルフロロク゛ルシノール産生能を有す
る蛍光性細菌とは、キングB寒天平板培地あるいはポテ
トデキストロース寒天平板培地に、菌株を接種後、15〜
35℃の温度で培養し、紫外線(365nm)を照射した場合、
肉眼で確認できる結晶性の2,4-シ゛アセチルフロロク゛ルシノールを、培
養後4週間以内に産生する菌を云う。また、抗生物質に
対して耐性を示さない菌とは、ストレプトマイシン100
μg/ml、ナリジキシン酸50μg/ml及びリファンピシン50
μg/mlを含有するキングB培地に菌株を接種後、15〜40
℃の温度で培養し、2週間後においても生育の認められ
ない菌を云う。このような本発明の菌株は、特にトマ
ト、ナス、キュウリ、ピーマン等の蔬菜の根内に多く生
育する。従って、本発明菌株はこれら蔬菜の根を良く水
洗し、これをホモジナイズして培養し、培養物に紫外線
を照射することにより容易に検索することができる。
The present invention will be described below in more detail. First, in the present invention, a fluorescent bacterium having the ability to produce crystalline 2,4-diacetylfluorculocinol is defined as 15 to 15 μm after inoculating the strain on a King B agar plate medium or a potato dextrose agar plate medium.
When cultured at a temperature of 35 ° C and irradiated with ultraviolet light (365 nm),
It refers to a bacterium that produces crystalline 2,4-diacetylfluorochlorocinol, which can be visually confirmed, within 4 weeks after culture. Bacteria that do not show resistance to antibiotics include streptomycin 100
μg / ml, nalidixic acid 50 μg / ml and rifampicin 50
After inoculation of the strain into King B medium containing μg / ml, 15-40
A bacterium that is cultured at a temperature of ° C. and does not grow even after 2 weeks. Such a strain of the present invention grows particularly in the roots of vegetables such as tomato, eggplant, cucumber, and pepper. Therefore, the strain of the present invention can be easily searched for by washing the roots of these vegetables well, homogenizing and culturing the roots, and irradiating the culture with ultraviolet rays.

【0011】このようにして検索された本発明の菌株
は、E.Levyらの作出した菌株とは根本的に相違する。即
ち、E.Levyらの作出した菌株は、ストレプトマイシン(2
50μg/ml)、ナリジキシン酸(250μg/ml)及びリファンピ
シン酸(75μg/ml)含有培地中で生育するのに対し、本発
明の菌株はこのような高濃度の抗生物質培地中では到底
生育せず、この濃度の約1/10以下で僅かに増殖が認めら
れる点である。本発明の結晶性2,4-シ゛アセチルフロロク゛ルシノール産
生能を有し、抗生物質に対して耐性を示さない蛍光性細
菌は、これを継代培養した場合に於いても結晶性2,4-シ゛
アセチルフロロク゛ルシノールの産生能の極度の低下は認められず、保
存あるいは培養が可能であり、青枯病防除資材として利
用可能なることを確認した。
[0011] The strain of the present invention searched in this way is fundamentally different from the strain created by E. Levy et al. That is, the strain created by E. Levy et al. Is streptomycin (2
(50 μg / ml), nalidixic acid (250 μg / ml) and rifampicic acid (75 μg / ml), whereas the strain of the present invention never grows in such a high concentration of antibiotic medium. The point is that proliferation is slightly observed at about 1/10 or less of this concentration. The fluorescent bacterium of the present invention, which has the ability to produce crystalline 2,4-diacetylfluorochlorocinol and does not show resistance to antibiotics, is capable of producing a crystalline 2,4-digestion even when this is subcultured. No extreme decrease in the production ability of diacetylfluorochlorocinol was observed, and it was confirmed that storage or cultivation was possible and that it could be used as a material for controlling bacterial wilt.

【0012】本発明の青枯病防除資材について述べれ
ば、前述の如くして検索した菌株を液体培養あるいは固
体培養により大量培養を行った培養菌体を含む培養液を
資材として使用することもできるし、培養菌体のみを集
菌してこれを資材として使用することもできる。またこ
れらを液状で使用しても良いし、乾燥して使用しても良
い。更に別の態様としては、菌体含有液状物あるいは乾
燥物を培土、ロックウール資材、シリカ、ゼオライト、
バーミキュライト、珪藻土、砂等の土壌改良剤と混合し
て使用することもできる。このように本発明の菌体を土
壌改良剤等他の担体と混合して使用する場合、菌体安定
性あるいは効果の維持継続の点から106cfu/g以上菌体を
含有させることが必要である。
With respect to the material for controlling bacterial wilt of the present invention, a culture solution containing cultured cells obtained by subjecting the strains searched as described above to large-scale culture by liquid culture or solid culture may be used as the material. Alternatively, only the cultured cells may be collected and used as a material. These may be used in liquid form or may be used after drying. As still another embodiment, the cultivation of a cell-containing liquid or dried material, rock wool material, silica, zeolite,
It can be used in combination with a soil conditioner such as vermiculite, diatomaceous earth, and sand. As described above, when the cells of the present invention are used in combination with other carriers such as a soil conditioner, it is necessary to contain 10 6 cfu / g or more of cells from the viewpoint of maintaining cell stability or maintaining the effect. It is.

【0013】本発明の青枯病防除資材は、菌体の活性低
下を防止するため、調製後脱酸素を行った窒素等の不活
性ガスで置換して密封保存するか、10℃以下の低温で保
存することが望ましい。本発明青枯病防除資材の適用方
法について云えば、例えば、本発明資材を種子に適用す
る場合、菌体含有液に種子を浸漬する浸種法、菌を担体
と混合した微粉末を種子と攪拌混合する種皮磨傷法ある
いは減圧下に浸種法を行う浸種減圧法等を利用すること
ができる。またプラグ苗への適用方法としては、本発明
資材である菌体含有液あるいは微粉末をプラグ培土に添
加混合して利用することができる。更にまた、植物定植
前にあっては本発明資材を予め土壌に添加混合しても良
いし、定植後にあっては本発明資材を散布しても良い。
The material for controlling bacterial wilt of the present invention is preferably prepared by replacing it with an inert gas such as nitrogen which has been deoxygenated after its preparation, and storing it tightly, or keeping it at a low temperature of 10 ° C. or less in order to prevent a decrease in the activity of the cells. It is desirable to save with. Regarding the method for applying the material for controlling bacterial wilt of the present invention, for example, when the material of the present invention is applied to seeds, a seeding method in which the seeds are immersed in a cell-containing liquid, a fine powder obtained by mixing the bacteria with a carrier and stirring the seeds A seed coat abrasion method of mixing, a soaking pressure reduction method of performing a soaking method under reduced pressure, or the like can be used. As a method of applying to the plug seedlings, the cell-containing solution or the fine powder, which is the material of the present invention, can be added to and mixed with the plug culture medium. Further, the material of the present invention may be added to and mixed with soil before planting the plant, or the material of the present invention may be sprayed after planting.

【0014】本発明青枯病防除資材に於ける菌体濃度に
関して云えば、担体と混合して使用する場合106cfu/g以
上が必要であり、液状物にあっては106cells/ml以上が
望ましい。また本発明資材の使用量は、作物の種類、使
用時期、資材の形態等により異なるが、種子処理の場
合、種子1mlに対して本発明菌体108cells/mlの菌体懸濁
液10ml程度に浸種処理することが望ましく、プラグ苗培
土に添加混合する場合、培土1g当たり本発明菌体106cfu
以上になるように添加することが効果の点から望まし
い。また定植後の土壌に添加する場合は、根圏土壌1g当
たり本発明菌体104cfu以上が効果持続の点から望まし
い。
The bacterial concentration in the material for controlling bacterial wilt of the present invention requires at least 10 6 cfu / g when used in a mixture with a carrier, and 10 6 cells / ml for a liquid material. The above is desirable. The amount of the material of the present invention varies depending on the type of crop, the time of use, the form of the material, and the like.In the case of seed treatment, the bacterial cell suspension of the present invention 10 8 cells / ml per 1 ml of seed is 10 ml. It is desirable to perform seeding treatment to a degree, and when added to and mixed with the plug seedling culture soil, the bacterial cells of the present invention per 10 g of culture soil 10 6 cfu
It is desirable to add so that it becomes above from the point of effect. When added to the soil after planting, the bacterial cells of the present invention are preferably 10 4 cfu or more per g of rhizosphere soil from the viewpoint of maintaining the effect.

【0015】[0015]

【実施例】以下、実施例により更に詳記するが、本発明
はこれらに限定されるものではない。また、実施例に於
いて%は特に断らない限り全て重量%を示す。
EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. In the examples, all percentages are by weight unless otherwise specified.

【0016】(実施例1)兵庫県下の施設栽培圃場より
栽培中のトマト(A)、ナス(B)、キュウリ(C)、ピーマン
(D)と、露地栽培のチンゲンサイ(E)、ネギ(F)及びゴル
フ場の芝(G)について、根圈土壌及び植物体を採取し
た。採取した植物体は根部を水洗後、約5cmに裁断し、
約10gの裁断根を各50mlの滅菌水及び0.005%エアロソ゛ル OT
(アメリカンサイアナミット゛社製)液を用いてスクリューミキサーで
撹拌洗浄を行った。洗液は、混合して根面微生物分離源
とした。撹拌洗浄根を80%エタノール溶液に1分間浸漬
し、さらに1%次亜塩素酸ナトリウム水溶液に10分間浸漬し根
表面の殺菌処理を行った後、滅菌水で洗浄し根内微生物
分離源とした。
(Example 1) Tomatoes (A), eggplants (B), cucumber (C), peppers cultivated from a facility cultivation field in Hyogo Prefecture
The root zone soil and the plant were collected from (D), the bok choy (E), the green onion (F), and the turf (G) at the golf course. The collected plant body is cut into about 5 cm after washing the roots with water,
Approximately 10 g of the cut roots are each 50 ml of sterilized water and 0.005% aerosol OT
(American Cyanamit Co., Ltd.) was used to perform stirring and washing with a screw mixer. The washing liquid was mixed to obtain a root microorganism separation source. The agitated and washed roots were immersed in an 80% ethanol solution for 1 minute, further immersed in a 1% aqueous sodium hypochlorite solution for 10 minutes to sterilize the root surface, washed with sterile water, and used as a root microbial isolation source. .

【0017】根圈土壌、混合洗液、表面殺菌根を微生物
分離源として分離操作を行った。根圈土壌は、100倍量
の滅菌水で10分間振盪、1分間静置後の上澄液をさらに1
00倍希釈し、その1mlをポテトデキストロース培地(寒天
1.5%)9mlで混釈合しペトリ皿に流し込んだ(混釈法)。
植物体採取時の地温に応じて15℃から40℃で培養を行っ
た。混合洗液についても、その1mlを同混釈法により同
様に培養を行った。表面殺菌根は、その1gを5mm程度に
さらに裁断後、20mlの滅菌水に入れ、10000rpmで15分間
ホモジナイズを行い、磨砕液の1mlを同混釈法により同
様に培養を行った。各培養共に72時間後にキングB寒天
培地及びシュードモナス選択分離P-1寒天培地の各平板
培養基にレプリカし、15℃から40℃で72時間培養後、36
5nmの紫外線を照射して、蛍光性色素を産生するコロニ
ーを識別した。蛍光性色素を産生するコロニーを再びキ
ングB液体培地及びポテトデキストロース液体培地に接
種し、3週間25℃で静置培養を行った。
Separation operation was performed using the rhizosphere soil, the mixed washing solution, and the surface sterilized root as a microorganism separation source. The rhizosphere soil was shaken with 100 times the volume of sterile water for 10 minutes, and allowed to stand for 1 minute.
Dilute 00-fold and add 1 ml of the potato dextrose medium (agar
The mixture was pulverized with 9 ml (1.5%) and poured into a Petri dish (pour method).
Culturing was carried out at 15 ° C to 40 ° C depending on the soil temperature at the time of plant collection. 1 ml of the mixed washing solution was similarly cultured by the same pour method. 1 g of the surface-sterilized root was further cut into about 5 mm, placed in 20 ml of sterilized water, homogenized at 10,000 rpm for 15 minutes, and cultured with 1 ml of the ground solution by the same pour method. After 72 hours, each culture was replicated on each plate medium of King B agar medium and Pseudomonas selective separation P-1 agar medium, and cultured at 15 to 40 ° C. for 72 hours.
By irradiating with 5 nm ultraviolet rays, colonies producing a fluorescent dye were identified. The colony producing the fluorescent dye was again inoculated into King B liquid medium and potato dextrose liquid medium, and incubated at 25 ° C. for 3 weeks.

【0018】培養終了後、培養液に365nmの紫外線を照
射し、黄緑色から黄色の蛍光性物質の産生が認められた
培養液と青白色蛍光を有する結晶の産生が認められた培
養液について以下の操作を行った。結晶の産生が認めら
れた培養液については、5A濾紙(Toyo製)で濾別した。そ
の他の培養液については凍結乾燥し、乾燥物からクロロ
ホルム−アセトン(1:1)混合溶媒に可溶な成分を抽出し
た。結晶物及び抽出物を乾燥後、少量の水及びメタノー
ルに懸濁させて、不溶物を濾別後乾燥し秤量した。乾燥
物を、薄層クロマト(シリカケ゛ルフ゜レート,ヘ゛ンセン:クロロホルム:メタノール=
4:2:1)分析に供し、Rf値が0.5〜0.53のスポットを有す
る乾燥物について、核磁気共鳴装置及び質量分析装置に
よる測定を行った。その結果、抽出物については2,4-シ゛
アセチルフロロク゛ルシノールが主成分であり、結晶物は2,4-シ゛アセチルフロ
ロク゛ルシノールであることを確認した。
After completion of the culture, the culture is irradiated with ultraviolet light of 365 nm, and the culture in which the production of a yellow-green to yellow fluorescent substance is observed and the culture in which the production of crystals having blue-white fluorescence is observed are described below. Was performed. The culture solution in which the production of crystals was observed was separated by filtration with 5A filter paper (manufactured by Toyo). Other culture solutions were freeze-dried, and components soluble in a mixed solvent of chloroform-acetone (1: 1) were extracted from the dried product. The crystals and the extract were dried, suspended in a small amount of water and methanol, and the insolubles were separated by filtration, dried and weighed. The dried product was subjected to thin-layer chromatography (silica gel plate, benzene: chloroform: methanol =
4: 2: 1) The sample was subjected to analysis, and the dried product having spots with Rf values of 0.5 to 0.53 was measured by a nuclear magnetic resonance apparatus and a mass spectrometer. As a result, it was confirmed that the extract was mainly composed of 2,4-diacetylfluorochlorocinol, and the crystal was 2,4-diacetylfluorochlorocinol.

【0019】次に、2,4-シ゛アセチルフロロク゛ルシノールの産生の認め
られた菌株の抗生物質に対する耐性をストレプトマイシ
ン、ナリジキシン酸、リファンピシンを用いて検定し
た。(以下、三重耐性という)ストレプトマイシンを100
μg/ml、ナリジキシン酸を50μg/ml及びリファンピシン
を50μg/mlとなるようにキングB寒天培地に混合後、ペ
トリ皿に流し込んだ。寒天表面の余剰水を除いてから産
生菌を画線接種し、25℃で3週間培養を行い、生育の有
無を調査した。表1に2,4-シ゛アセチルフロロク゛ルシノール産生菌の産
生量及び三重耐性を示した。
Next, the resistance to antibiotics of the strain in which the production of 2,4-diacetylfluorochlorocinol was recognized was examined using streptomycin, nalidixic acid and rifampicin. Streptomycin (100%)
μg / ml, nalidixic acid at 50 μg / ml, and rifampicin at 50 μg / ml were mixed in a King B agar medium and poured into a Petri dish. After removing excess water on the agar surface, the produced bacteria were streaked, cultured at 25 ° C for 3 weeks, and examined for growth. Table 1 shows the production amount and the triple resistance of the 2,4-diacetylfluorochlorocinol-producing bacteria.

【0020】[0020]

【表1】 [Table 1]

【0021】(実施例2)表1に示した2,4-シ゛アセチルフロロク
゛ルシノール産生菌について、青枯病菌に対する抗菌性を検定
した。トマト青枯病菌としてシュート゛モナス・ソラナセアラム(Pseudom
onas solanacearum,MAFF-03-01485)を、ポテトデキスト
ロース斜面培養基で35℃で72時間培養し指示菌とした。
2,4-シ゛アセチルフロロク゛ルシノール産生菌は、ポテトデキストロース
斜面培養基で25℃で96時間培養し検定菌とした。指示菌
を1白金耳100mlの滅菌水に懸濁させ、その1mlをポテト
デキストロース寒天培地9mlで混釈してペトリ皿に流し
込んだ。ペトリ皿の表面の余剰水を除いた後、検定菌を
約1cmに画線接種し30℃で48時間培養し、検定菌の周縁
で形成した阻止円径(画線に対して垂直方向の径)を測定
した。その結果、表1に示した検定菌はすべて青枯病菌
に対して生育阻止円を形成し、R-3,7,8,9,RS-16では12m
m以下、その他の検定菌の阻止円径は35mmから49mmの範
囲であった。
Example 2 Antibacterial activity against bacterial wilt was tested on 2,4-diacetylfluorochlorocinol-producing bacteria shown in Table 1. Shoots as tomato bacterial wilt fungus: Monas solanacearum (Pseudom
onas solanacearum, MAFF-03-01485) was cultured in a potato dextrose slant culture medium at 35 ° C. for 72 hours to obtain indicator bacteria.
2,4-Diacetylfluorochlorocinol-producing bacteria were cultured in a potato dextrose slant culture medium at 25 ° C. for 96 hours to obtain test bacteria. One indicator loop was suspended in 100 ml of sterile water, and 1 ml of the suspension was mixed with 9 ml of potato dextrose agar medium and poured into a petri dish. After removing excess water from the surface of the Petri dish, streak the test bacteria to about 1 cm in streaks and incubate at 30 ° C for 48 hours.The diameter of the inhibition circle formed at the periphery of the test bacteria (diameter perpendicular to the streaks) ) Was measured. As a result, all the test bacteria shown in Table 1 formed a growth inhibition circle against the bacterial wilt fungus, and 12 m in R-3, 7, 8, 9, and RS-16.
m or less, and the inhibition circle diameter of other test bacteria ranged from 35 mm to 49 mm.

【0022】(実施例3)表1に示した2,4-シ゛アセチルフロロク
゛ルシノール産生菌のうち、R-3,7,8,9,RS-16を除く菌株を実
施例2と同操作により検定菌とした。検定菌の青枯病発
病抑制試験を上層が0.8%寒天5ml、中層が海砂3ml、下
層が蔗糖を除くホワイト寒天培地15mlの三層からなる外
径2.5cm、高さ15cmの栓付き培養瓶で行った。先ずこの
培養瓶に、80%エタノール溶液と1%次亜塩素酸ナトリ
ウム水溶液により殺菌したトマト種子(品種:大型福寿)
を播種した。28℃で4日間、暗所で発芽させた後、人工
気象器に移し30℃で栽培を3日間行った。
Example 3 Among the 2,4-diacetylfluorochlorocinol-producing bacteria shown in Table 1, those strains excluding R-3,7,8,9, RS-16 were subjected to the same operation as in Example 2. Test bacteria were used. Inhibition test for bacterial wilt disease of the test bacteria. Culture bottle with outer diameter 2.5cm, height 15cm, consisting of three layers: upper layer: 5% 0.8% agar, middle layer: 3ml sea sand, lower layer: 15ml white agar medium excluding sucrose. I went in. First, in this culture bottle, tomato seeds sterilized with 80% ethanol solution and 1% sodium hypochlorite aqueous solution (variety: Large Fukuju)
Was sown. After germination at 28 ° C for 4 days in a dark place, the seeds were transferred to an artificial weather device and cultivated at 30 ° C for 3 days.

【0023】この培養瓶に検定菌を107cells/mlに調製
した菌体懸濁液0.5mlを接種した。接種後、30℃で栽培
を3日間行った後に、実施例2の青枯病菌を108cells/ml
に調製した菌体懸濁液0.5mlを接種した。接種後は35℃
に変更し、人工気象器中で栽培を継続した。青枯病菌接
種後30日の罹病程度を測定し防除価を算出し、表2に示
した。対照区として、青枯病菌のみを接種した区を設け
た。各試験区は1区3株の3反復で行った。また、各区の
苗の根部を種子殺菌と同方法により表面殺菌し、根部を
約2cmの長さに裁断してキングB寒天平板及びクリスタ
ルバイオレットを5mg/l含有するポテトデキストロース
寒天平板に置床した。25℃で7日間培養を行い、キング
B寒天平板では、365nmの紫外線照射下でコロニー周辺
の蛍光性物質産生で検定菌の生息部位を検定し、ポテト
デキストロース寒天平板では、紫外線照射下で蛍光性結
晶産生で生息部位を検定した。検定菌の根内定着状態を
ライン交差法による全根長に対する生息部位の根長比を
根内定着度として表示した。結果を表2に示した。尚、
罹病程度及び防除価の算出は以下の方法で行った。 罹病程度=[(Σ罹病指数×株数)/(10×全株数)]×100 罹病指数:枯死,10;全身萎凋,5;部分萎凋,3;黄変,2 防除価=[(対照区罹病程度−検定菌接種区罹病程度)/対
照区罹病程度]×100
The culture bottle was inoculated with 0.5 ml of a cell suspension prepared at 10 7 cells / ml of the test bacteria. After inoculation, cultivation was performed at 30 ° C. for 3 days, and then the bacterial wilt of Example 2 was cultured at 10 8 cells / ml.
Was inoculated with 0.5 ml of the prepared cell suspension. 35 ℃ after inoculation
And continued cultivation in an artificial weather vessel. The disease control was measured 30 days after the inoculation of the bacterial wilt fungus, and the control value was calculated. As a control section, a section inoculated with only the bacterial wilt was provided. Each test group was performed in triplicate with 3 strains per group. The roots of the seedlings in each section were surface-sterilized by the same method as for seed sterilization, and the roots were cut to a length of about 2 cm and placed on a King B agar plate and a potato dextrose agar plate containing 5 mg / l of crystal violet. After culturing at 25 ° C for 7 days, King B agar plate was tested for the presence of the test bacteria by the production of fluorescent material around the colonies under 365 nm UV irradiation, and potato dextrose agar plate was fluorescent under UV irradiation. Habitats were assessed by crystal production. The root colonization state of the test bacteria was indicated by the root length ratio of the habitat to the total root length by the line crossing method as the root colonization degree. The results are shown in Table 2. still,
The degree of disease and the control value were calculated by the following methods. Disease degree = [(Σ disease index x number of strains) / (10 x total number of strains)] x 100 disease index: withering, 10; whole body withering, 5; partial withering, 3; yellowing, 2 control value = [(control group disease Degree-degree of disease inoculated with test bacteria) / degree of disease in control group] x 100

【0024】[0024]

【表2】 注) 対照区の罹病程度は93であり、0.0は試験区におい
て対照区よりも罹病程度 の高い区を表示した。
[Table 2] Note) The degree of disease in the control plot was 93, and 0.0 indicates the plot with higher disease in the test plot than the control plot.

【0025】尚、以下の実施例に於いて、実施例で使用
する本発明菌及び対照菌は、ポテトデキストロース斜面
培養基で25℃、96時間培養した菌体を用いた。
In the following examples, the cells of the present invention and the control cells used in the examples were cells cultured on a potato dextrose slant culture medium at 25 ° C. for 96 hours.

【0026】(実施例4)実施例3の表2中の本発明の
結晶性2,4-シ゛アセチルフロロク゛ルシノール産生菌としてER-32、対照
菌としてRS-19を含有する資材を調製し、青枯病発病土
壌における発病抑制試験を行った。市販の水稲用育苗培
土と園芸用培土を4:1に混合し、180℃で1時間乾熱殺菌
を行い室温に冷却後、直ちに本発明菌及び対照菌の108c
ells/ml、107cells/ml、106cells/mlの菌体懸濁液を熱
処理培土に対して25v/v%混合した。混合後、25℃で2週
間静置した。静置後、キングB寒天培地及び5mg/lのク
リスタルバイオレットを含有するポテトデキストロース
寒天培地を用いて、培土中の本発明菌及び対照菌の菌数
を測定した。本発明菌を使用した資材は、1g中に107cf
u、106cfu、105cfuの本発明菌を含有しており、対照菌
を使用した資材は1g中に108cfu、107cfu、106cfuの対照
菌を含有していた。また、培土を4:1に混合した培土(無
処理1)資材と、さらに熱処理を行った培土に滅菌水を2
5v/v%の割合で添加混合した培土(無処理2)を調製し
た。これらの培土をプラグ苗用トレイ(136穴)に充填
し、トマト種子(品種:桃太郎)を播種し、カ゛ラス温室(30℃
〜40℃)で2週間育苗した。尚、育苗期間中は自動灌水を
行った。育苗後、各プラグ苗を青枯病菌が土壌1gあたり
107cfu分離される青枯病発病土を充填したコンテナ(ハウス
桃太郎のフ゜ラク゛苗では、定植後2週間で全株が枯死する状
態)に定植した。定植後の罹病程度を調査した。調査方
法は、実施例3と同方法で行った。試験株数は、1区12
株の3反復で行った。結果を表3に示した。
(Example 4) A material containing ER-32 as a crystalline 2,4-diacetylfluorochlorocinol-producing bacterium of the present invention in Table 2 of Example 3 and RS-19 as a control bacterium was prepared. A disease suppression test was carried out on a bacterial wilt disease soil. Commercial Rice nursery soil and horticultural soil 4: 1 mixture, after cooling to room temperature for 1 hour dry heat sterilized at 180 ° C., immediately present invention bacterium and 10 8 c of the control fungus
Cell suspensions of ells / ml, 10 7 cells / ml, and 10 6 cells / ml were mixed at 25 v / v% to the heat-treated soil. After mixing, the mixture was allowed to stand at 25 ° C. for 2 weeks. After standing, the numbers of the bacteria of the present invention and the control bacteria in the soil were measured using King B agar medium and potato dextrose agar medium containing 5 mg / l crystal violet. The material using the bacterium of the present invention is 10 7 cf / g.
u, 10 6 cfu, and 10 5 cfu of the present bacterium, and the material using the control bacterium contained 10 8 cfu, 10 7 cfu, and 10 6 cfu of the control bacterium in 1 g. Also, sterile water was added to the cultivated soil (untreated 1) mixed with cultivated soil 4: 1 and further heat-treated cultivated soil.
Cultivated soil (no treatment 2) was added and mixed at a ratio of 5 v / v%. Filling these cultivated soil into plug seedling trays (136 holes), sowing tomato seeds (variety: Momotaro), crow greenhouse (30 ° C)
-40 ° C) for 2 weeks. In addition, automatic watering was performed during the seedling raising period. After raising the seedlings, each plug seedling was treated with bacterial wilt per gram of soil.
The plant was planted in a container filled with 10 7 cfu-isolated bacterial wilt diseased soil (in the case of house seedlings of Momotaro, all strains died two weeks after planting). The degree of disease after planting was investigated. The investigation method was the same as in Example 3. The number of test strains is 12 in 1 ward
Three replicates of the strain were performed. The results are shown in Table 3.

【0027】[0027]

【表3】 [Table 3]

【0028】(実施例5)実施例3の表2中のER-26を
使用して、108cells/mlの菌体懸濁液を調製した。この
菌体懸濁液10mlに1%次亜塩素酸ナトリウムと80%エタ
ノール水溶液により殺菌したトマト種子(品種:ハウス桃太
郎)1mlを浸漬し、室温で4時間浸種処理を行った。実施
例4で使用した熱処理培土をプラグ苗用トレイに充填し
滅菌水で充分に灌水後、浸種処理を行った処理種子を播
種し、28℃の人工気象器内で3週間育苗した。対照区と
して殺菌したトマト種子を同様に育苗した。育苗終了
後、実施例4と同じ青枯病発病土を充填したコンテナに
定植した。定植後の罹病程度を調査した。調査方法は、
実施例3と同方法で行った。試験株数は、1区6株の3反
復で行った。結果を表4に示した。
Example 5 A cell suspension of 10 8 cells / ml was prepared using ER-26 in Table 2 of Example 3. To 10 ml of this cell suspension, 1 ml of tomato seeds (variety: House Momotaro) sterilized with 1% sodium hypochlorite and an 80% aqueous ethanol solution was immersed, and immersed at room temperature for 4 hours. The heat-treated soil used in Example 4 was filled in a tray for plug seedlings, sufficiently irrigated with sterilized water, and then immersed in seeds. The treated seeds were sown and grown in an artificial weather device at 28 ° C for 3 weeks. A sterilized tomato seed was similarly raised as a control. After the completion of the seedling raising, the seedlings were planted in containers filled with the same bacterial wilt disease soil as in Example 4. The degree of disease after planting was investigated. The survey method is
Performed in the same manner as in Example 3. The number of test strains was 3 in 6 replicates per section. The results are shown in Table 4.

【0029】[0029]

【表4】 [Table 4]

【0030】(実施例6)実施例3の表2中の本発明の
ER-71を使用して、5倍に希釈したポテトデキストロース
液体培地に接種し、28℃で2週間静置培養を行った。培
養後、滅菌水で100倍に希釈した。市販の育苗用培土を
用いて28℃の人工気象器内で4週間プラグ育苗したトマ
ト苗(品種:ハウス桃太郎)を実施例4と同じ青枯病発病土を
充填したコンテナにプラグ苗を定植する際の植穴に1穴
あたり50mlのER-71希釈培養液を灌注した。対照区とし
て滅菌水50mlを灌注した(1)区と5倍に希釈したポテトデ
キストロース液体培地50mlを灌注した(2)区を設けた。
灌注後、トマト苗を定植し定植後の罹病程度を調査し
た。調査方法は、実施例3と同方法で行った。試験株数
は、1区6株の3反復で行った。結果を表5に示した。
(Example 6) Table 3 of Example 3
The potato dextrose liquid medium diluted 5-fold was inoculated using ER-71 and allowed to stand at 28 ° C. for 2 weeks. After the culture, the cells were diluted 100-fold with sterile water. Tomato seedlings (cultivar: House Momotaro) which were plug-raised for 4 weeks in a 28 ° C artificial weathering vessel using commercially available seedling culture soil are planted with plugs in the same container filled with the bacterial wilt disease as in Example 4. The wells at the end were perfused with 50 ml of ER-71 diluted culture solution per well. As a control, there were provided a section (1) in which 50 ml of sterilized water was perfused and a section (2) in which 50 ml of a 5-fold diluted potato dextrose liquid medium was perfused.
After irrigation, tomato seedlings were planted and the degree of disease after planting was investigated. The investigation method was the same as in Example 3. The number of test strains was 3 in 6 replicates per section. Table 5 shows the results.

【0031】[0031]

【表5】 [Table 5]

【0032】[0032]

【発明の効果】本発明の青枯病防除資材は、結晶性2,4-
シ゛アセチルフロロク゛ルシノール産生能を有し、かつ、抗生物質に対し
て耐性を示さない蛍光性細菌からなる青枯病防除資材で
あって、このような資材の使用によって農作物の青枯病
による被害を軽減することにより、環境保全下における
農業の生産性の向上を図ることができる。
EFFECT OF THE INVENTION The material for controlling bacterial wilt of the present invention comprises crystalline 2,4-
A bacterial wilt control material composed of fluorescent bacteria having a diacetylfluoroclucinol-producing ability and exhibiting no resistance to antibiotics. Use of such a material reduces damage caused by bacterial wilt of agricultural crops. By reducing, it is possible to improve the productivity of agriculture under environmental protection.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 前川 義雄 兵庫県三木市志染町東自由が丘3−491 番地 (72)発明者 秋山 泰三 兵庫県高砂市米田町神爪331−9番地 (72)発明者 林 佳徳 兵庫県加古郡稲美町国北2−50番地 審査官 大久保 元浩 (58)調査した分野(Int.Cl.6,DB名) A01N 63/00──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yoshio Maekawa 3-491 Higashi-Jiyugaoka, Shizen-cho, Miki-shi, Hyogo (72) Inventor Taizo Akiyama 331-9 Shinzume, Yoneda-cho, Takasago-shi, Hyogo (72) Yoshinori Hayashi, Inventor 2-50 Kokukita, Inami-cho, Kako-gun, Hyogo Examiner Motohiro Okubo (58) Field surveyed (Int. Cl. 6 , DB name) A01N 63/00

Claims (1)

(57)【特許請求の範囲】(57) [Claims] 【請求項1】 結晶性2,4−ジアセチルフロログルシ
ノール産生能を有し、かつ、抗生物質に対して耐性を示
さない蛍光性細菌即ち、ストレプトマイシン100μg
/ml、ナリジキシン酸50μg/ml及びリファンピ
シン50μg/mlを含有するキングB培地に菌株を接
種後、15〜40℃の温度で培養し、2週間後において
も生育の認められない蛍光性細菌からなる青枯病防除資
材。
1. A fluorescent bacterium having an ability to produce crystalline 2,4-diacetylphloroglucinol and showing no resistance to antibiotics, ie, 100 μg of streptomycin.
/ Ml, nalidixic acid 50 μg / ml and rifampi
The strain was placed in King B medium containing 50 μg / ml of syn.
After seeding, culture at a temperature of 15 to 40 ° C, and after 2 weeks
Bacterial wilt control material consisting of fluorescent bacteria that do not grow .
JP7099628A 1995-03-31 1995-03-31 Pest control material Expired - Fee Related JP2827093B2 (en)

Priority Applications (1)

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JP7099628A JP2827093B2 (en) 1995-03-31 1995-03-31 Pest control material

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Application Number Priority Date Filing Date Title
JP7099628A JP2827093B2 (en) 1995-03-31 1995-03-31 Pest control material

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JP2827093B2 true JP2827093B2 (en) 1998-11-18

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Country Link
JP (1) JP2827093B2 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2835598B2 (en) * 1996-05-20 1998-12-14 多木化学株式会社 Seedling cultivation soil, method for producing the same, and method for growing disease-resistant seedlings
JP2016057794A (en) 2014-09-09 2016-04-21 村田機械株式会社 Management device, control device, textile machine management system, and management method for textile machine

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