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JPH07163334A - Activity maintenance and storage of fluorescent bacterium and microorganism material comprising its culture product - Google Patents

Activity maintenance and storage of fluorescent bacterium and microorganism material comprising its culture product

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Publication number
JPH07163334A
JPH07163334A JP34166893A JP34166893A JPH07163334A JP H07163334 A JPH07163334 A JP H07163334A JP 34166893 A JP34166893 A JP 34166893A JP 34166893 A JP34166893 A JP 34166893A JP H07163334 A JPH07163334 A JP H07163334A
Authority
JP
Japan
Prior art keywords
culture
plant
root
bacteria
fluorescent
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.)
Granted
Application number
JP34166893A
Other languages
Japanese (ja)
Other versions
JP2660317B2 (en
Inventor
Kimitaka Aino
公孝 相野
Hiroyuki Maki
浩之 牧
Katsuhiko Shimizu
克彦 清水
Akihiro Ushio
昭浩 牛尾
Takeo Kuwana
健夫 桑名
Tamotsu Kobayashi
保 小林
Shoji Kobayashi
尚司 小林
Minoru Matsuyama
稔 松山
Yoshio Maekawa
義雄 前川
Taizo Akiyama
泰三 秋山
Keitoku Hayashi
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.)
Hyogo Prefectural Government
Taki Chemical Co Ltd
Original Assignee
Hyogo Prefectural Government
Taki Chemical Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Hyogo Prefectural Government, Taki Chemical Co Ltd filed Critical Hyogo Prefectural Government
Priority to JP34166893A priority Critical patent/JP2660317B2/en
Publication of JPH07163334A publication Critical patent/JPH07163334A/en
Application granted granted Critical
Publication of JP2660317B2 publication Critical patent/JP2660317B2/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

PURPOSE:To maintain the activities of fluorescent bacteria living in the roots of a plant, and to perform the growth stimulation, yield increase and disease control of agricultural crops with the activated microorganisms or their culture product, thus improving the productivity of agriculture. CONSTITUTION:Fluorescent bacteria separated from the roots of a plant are subjected to a symbiotic culture together with the culture roots of the same kind of a plant as that of the separating source to maintain the activities of the fluorescent bacteria, and the culture product obtained by culturing the fluorescent bacteria is used as a microorganism material. The fluorescent bacteria subjected to the symbiotic culture can be immobilized and dried for storage in a state maintaining the activities of the fluorescent bacteria. The fluorescent bacteria can massively cultured by culturing the immobilized fluorescent bacteria. The application of the culture product of such fluorescent bacteria to plants such as agricultural crops as a microorganism material exerts remarkable effects on the control of soil diseases the growth stimulation of the plants.

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、根圏、殊に植物体根内
に生息する蛍光性細菌の活性維持法及び保存法並びにこ
の培養物からなる微生物資材に関し、この活性化された
微生物並びにその培養物によって農業作物の生育の促
進、増収並びに病害の防除を行い、以て農業の生産性向
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 method for maintaining and preserving the activity of a fluorescent bacterium inhabiting the rhizosphere, particularly a plant root, and a microbial material consisting of this culture. The purpose of the culture is to promote the growth of agricultural crops, increase the yield, and control diseases, thereby improving agricultural productivity.

【0002】[0002]

【従来の技術】近年、農業分野での有用微生物の利用に
ついては、多くの研究並びに報告が成されている。これ
らは主に土壌病害の生物的防除に関するものであり、有
用微生物の病原菌に対する拮抗作用は殊に重要な機能と
して考えられている。また、このような有用微生物が生
産する各種抗生物質等について、拮抗物質としての作用
メカニズムについても多くの検討がされている。中で
も、シュードモナス属細菌は、このような拮抗物質産生
能が高く、また植物の根面に容易に定着することが知ら
れている。従って、この細菌を植物体に接種することに
より植物の生育を促進させ、以てその植物の増収を図る
研究が行われ、このような細菌がPGPR(Plant Growt
h Promoting Rhizobacteria:植物生育促進性根圏細菌)
と称されるまでに至っている。しかし、このようなPG
PRによる作用については、この細菌が生産する物質で
あるシデロフォアを介した作用機作での理論的説明に留
まり、その細菌による土壌病害の生物的防除作用並びに
植物の生育促進作用についての実証的解明は勿論、実用
的効果の実現も未だ見られないのが現状である。
2. Description of the Related Art In recent years, many studies and reports have been made on the utilization of useful microorganisms in the agricultural field. These are mainly related to biological control of soil diseases, and the antagonistic action of useful microorganisms against pathogens is considered to be a particularly important function. In addition, with regard to various antibiotics produced by such useful microorganisms, many studies have been conducted on the action mechanism as an antagonist. Among them, Pseudomonas bacteria are known to have high ability to produce such antagonists and to easily settle on the root surface of plants. Therefore, studies have been carried out to promote the growth of plants by inoculating the plants with this bacterium and thereby increase the yield of the plant.
h Promoting Rhizobacteria: Plant growth promoting rhizobacteria)
Has been called. But such a PG
Regarding the action by PR, only a theoretical explanation of the mechanism of action via the siderophore, which is a substance produced by this bacterium, and empirical elucidation of the biological control action of soil diseases and the growth promotion action of plants by the bacterium Of course, the reality is that practical effects have not yet been realized.

【0003】[0003]

【発明が解決しようとする課題】上述のPGPRのよう
な細菌を、一般に農作物等植物体の土壌病害防除並びに
植物の生育促進に利用するためには数多くの問題があ
る。即ち、先ず自然生態系から検索された有用微生物の
取り扱いの問題がある。自然生態系に於いて微生物が保
有していた有用な機能を、如何にして失活させることな
く維持するかの問題である。更に、このような微生物を
如何にして実用化のために保存するか、また大量培養に
移行させるかの問題である。次に、培養微生物を土壌に
再び施用する際の問題もある。即ち、例え植物体あるい
は根圏土壌等から分離した微生物であっても、人為的な
分離、培養を受けた微生物を再び自然の土壌に施用し、
これを土壌中あるいは植物体内で安定に定着させること
は、現在のところ極めて困難な技術とされている。即
ち、土壌に施用した微生物が、既に土壌中に生息する多
くの土着菌との競合によって更に劣性化し、植物の根圏
環境下で安定に定着することが困難となる。
There are many problems in using the above-mentioned bacteria such as PGPR for controlling soil diseases of plants such as agricultural crops and promoting the growth of plants. That is, there is a problem in handling useful microorganisms retrieved from natural ecosystems. The question is how to maintain the useful functions of microorganisms in natural ecosystems without inactivating them. Furthermore, there is a problem of how to store such microorganisms for practical use and to transfer them to mass culture. Next, there is a problem when the cultured microorganisms are applied to the soil again. That is, even if it is a microorganism isolated from a plant or rhizosphere soil, artificial separation and culturing are applied to the natural soil again,
Stable establishment of this in soil or in plants is currently considered extremely difficult technology. That is, the microorganisms applied to the soil become more inferior by competition with many indigenous bacteria already existing in the soil, and it becomes difficult to stably establish in the rhizosphere environment of the plant.

【0004】従って、PGPRのような微生物を、植物
体の土壌病害防除並びに生育促進に利用するためにはこ
のような問題を解決する必要がある。具体的には、植物
根との親和性の高いシュードモナス属細菌を根圏土壌或
いは植物体の根面より探索し、これを種子バクテリゼー
ション等によって植物体に定着させる方法、あるいは植
物体から分離した微生物を再度植物体に接種し、植物体
内で生育、定着させる方法が検討されている。例えば、
微生物を胚軸にトラップすることにより病害防除に有用
な共生微生物を分離選抜する方法、あるいは無病徴組織
内に共生する微生物を分離培養して、これらの微生物と
親和性のある菌にこの微生物を接種する方法等である。
しかしながら、これらは何れも研究段階に留まり、未だ
このような問題を解決するまでには至っていない。本発
明者らはこのような問題を解決すべく、殊に植物体の栽
培時に於ける作物根に着眼し、その植物根内に棲息、定
着している微生物を利用する方法について、鋭意検討を
重ねた結果本発明に到達したものである。
Therefore, in order to utilize microorganisms such as PGPR for controlling soil diseases and promoting growth of plants, it is necessary to solve such problems. Specifically, a Pseudomonas bacterium with a high affinity for plant roots was searched from the rhizosphere soil or the root surface of the plant, and a method of fixing the Pseudomonas bacteria to the plant by seed bacteriosis or the like was isolated from the plant. A method of inoculating a plant again with a microorganism to grow and settle in the plant is being studied. For example,
A method of separating and selecting a symbiotic microorganism useful for disease control by trapping the microorganism in the hypocotyl, or separating and culturing a symbiotic microorganism in an asymptomatic tissue, and selecting this microorganism as a bacterium having an affinity for these microorganisms. For example, the method of inoculation.
However, all of these remain in the research stage and have not yet solved such problems. In order to solve such problems, the present inventors have paid particular attention to a method of utilizing a microorganism that focuses on a crop root at the time of cultivating a plant body, inhabits in the plant root, and is colonized. As a result of the repetition, the present invention has been reached.

【0005】[0005]

【課題を解決するための手段】即ち、本発明は植物体根
内から分離した蛍光性細菌を、分離源の植物体と同種の
植物体の培養根と共生培養することを特徴とする蛍光性
細菌の活性維持方法及びこのような共生培養によって得
られる培養物からなる微生物資材に関する。更に、本発
明はこのような培養物から培養根を分取し、これを固定
化細胞とした後、乾燥することを特徴とする蛍光性細菌
の保存法及びこのような保存菌を培養した培養物からな
る微生物資材に関する。尚、本発明は、植物根内に棲
息、定着している蛍光性細菌を利用するものであって、
このような蛍光性細菌を分離し、これを共生培養するこ
とによって植物根内に棲息している状態を再現すると共
に活性ある細菌を選抜し、この活性を低下させることな
く利用することに特徴を有するものである。
[Means for Solving the Problems] That is, the present invention is characterized in that a fluorescent bacterium isolated from the inside of a plant root is co-cultivated with a culture root of a plant of the same species as the plant of the source of separation. The present invention relates to a method for maintaining bacterial activity and a microbial material comprising a culture obtained by such co-cultivation. Further, the present invention is a method for preserving fluorescent bacteria, characterized in that a culture root is collected from such a culture, and the cells are fixed cells, and then dried, and a culture in which such a preservation bacterium is cultured. It relates to a microbial material consisting of things. The present invention utilizes a fluorescent bacterium that is inhabited and settled in a plant root,
It is characterized by separating such fluorescent bacteria and reproducing the state inhabiting the plant roots by co-culturing them and selecting active bacteria and using them without reducing this activity. I have.

【0006】[0006]

【作用】以下に本発明の蛍光性細菌の活性維持法及び保
存法並びにこの培養物からなる微生物資材について更に
詳記する。本発明の蛍光性細菌は、植物体内とりわけ植
物体根内から分離した細菌である。また、このような蛍
光性細菌としては、シュードモナス・フルオレッセンス
の各種バイオタイプの細菌、シュードモナス・プチダ等
の根内定着型の蛍光性細菌が挙げられる。このような根
内定着型の蛍光性細菌を分離する手段は、植物根の表面
を殺菌処理した後、細菌を分離する方法によればよい。
しかし、この細菌の分離培養に際しては、細菌の活性維
持のために培養時の栄養条件として貧栄養条件下で培養
することが望ましい。例えば、植物根の表面を洗浄しこ
れをエタノール等で殺菌処理した後、その植物根をホモ
ジナイズし、これをそのまま素寒天と混釈培養する方法
でよい。このような方法で分離培養を行い、次いで生成
したコロニーの中から蛍光性を発するコロニーを選抜す
る。分離した蛍光性細菌からなるコロニーは、次にこれ
を本発明の活性維持方法に供する。
The method for maintaining and preserving the activity of the fluorescent bacterium of the present invention and the microbial material comprising this culture will be described in more detail below. The fluorescent bacterium of the present invention is a bacterium isolated from within a plant, particularly within a plant root. Examples of such fluorescent bacteria include various biotypes of Pseudomonas fluorescens and root-root-fixing fluorescent bacteria such as Pseudomonas putida. The means for separating such root-root-fixing fluorescent bacteria may be a method of sterilizing the surface of the plant root and then separating the bacteria.
However, in separating and culturing the bacteria, it is desirable to culture them under oligotrophic conditions as nutrient conditions during the culture in order to maintain the activity of the bacteria. For example, a method may be used in which the surface of the plant root is washed and sterilized with ethanol or the like, and then the plant root is homogenized and then directly pour-cultured with bare agar. Separation culture is performed by such a method, and then colonies that emit fluorescence are selected from the generated colonies. The colonies composed of the separated fluorescent bacteria are then subjected to the activity maintaining method of the present invention.

【0007】本発明の活性維持方法は、根内に定着し、
且つ植物根と共生状態にあった蛍光性細菌を、分離前と
類似の環境下で増殖させるため、分離源の植物体と同種
の植物体の培養根と共生培養することに特徴を有するも
のであり、その方法は次のような手段によって行う。
The activity maintaining method of the present invention is established in roots,
And, in order to grow the fluorescent bacteria that were in a symbiotic state with the plant roots in an environment similar to that before separation, it is characterized by being co-cultivated with the culture roots of the plant of the same species as the plant of the separation source. Yes, the method is as follows.

【0008】先ず、予め上記の蛍光性細菌を分離した植
物体と同種の植物体の種子を使用し、これを殺菌処理し
た後、素寒天培地等で発芽生育させる。尚、この場合に
於いて殊に重要なことは、発芽生育に使用する種子は、
蛍光性細菌を分離した植物体と同種の植物体の種子を使
用することであり、この場合に異なった植物体の種子を
使用しても本発明の目的を達成することができない。ま
た、この場合の同種の植物体とは、トマト、ナス、キュ
ウリ等の種別を云い、同一植物体種であればいかなる品
種であってもよい。更に、本発明では種子の発芽によっ
て生育した根端のみを使用するが、この場合に生育した
植物体の根端以外の部位の使用では本発明の目的を達成
することができない。また、本発明に於いては発芽生育
した植物体根端以外に、カルス等の未分化植物細胞や毛
状根培養によって得られる不定根を使用しても、同様に
本発明の目的を達成することができない。本発明では、
この発芽生育した苗の根端部を分離し、これをホワイト
培地等の培地中で根端培養する。このようにして得られ
た根端培養根は、次いで行う共生培養に供する。
First, seeds of a plant of the same species as the plant in which the above-mentioned fluorescent bacteria have been separated in advance are used, sterilized and then germinated and grown on agar agar medium or the like. In this case, the seeds used for germination and growth are especially important.
The purpose is to use seeds of a plant of the same species as the plant from which the fluorescent bacteria have been separated, and in this case, the use of seeds of different plants cannot achieve the object of the present invention. In addition, the same type of plant body in this case refers to types such as tomato, eggplant, and cucumber, and may be any variety as long as it is the same plant body type. Furthermore, in the present invention, only the root tips grown by germination of seeds are used, but the use of a portion other than the root tips of the plant body grown in this case cannot achieve the object of the present invention. Further, in the present invention, in addition to the germinated and grown plant root tip, even if an adventitious root obtained by culturing undifferentiated plant cells such as callus or hairy root is used, the same object of the present invention can be achieved. I can't. In the present invention,
The root tips of the germinated and grown seedlings are separated, and the root tips are cultured in a medium such as white medium. The root tip culture root thus obtained is subjected to the subsequent co-cultivation.

【0009】共生培養法は、ホワイト培地等を使用し、
これに根端培養根を移し、前述の分離した蛍光性細菌か
らなるコロニーを接種して約10日程度の培養を行う。こ
の場合に蛍光性細菌は、培養液中の植物根内あるいは根
面において増殖するが、このように蛍光性細菌を根内あ
るいは根面で増殖させることにより、細菌の活性低下は
極めて抑制され、この菌を微生物資材として使用すると
きは、蛍光性細菌の植物体根内での活性を維持できる。
このように活性が維持された蛍光性細菌は、その培養物
をそのまま後述する微生物資材として使用することもで
きるが、より望ましくは培養液中の細菌について更に機
能性の検定を行い、スクリーニングした細菌について更
に上記の共生培養を行うことにより、蛍光性細菌の効果
はより確実となる。
The co-cultivation method uses white medium or the like,
The root tip culture roots are transferred to this, and the colonies consisting of the above-mentioned separated fluorescent bacteria are inoculated and cultured for about 10 days. In this case, the fluorescent bacterium grows in the root or the root surface of the plant in the culture solution, but by growing the fluorescent bacterium in the root or the root surface in this way, the decrease in the activity of the bacterium is extremely suppressed, When this bacterium is used as a microbial material, the activity of fluorescent bacteria in plant roots can be maintained.
Fluorescent bacteria whose activity has been maintained in this way can be used as the microbial material described below as it is as a culture, but more desirably, the bacteria screened by performing a further functional assay for the bacteria in the culture solution. By further performing the co-cultivation as described above, the effect of the fluorescent bacteria becomes more certain.

【0010】次に、本発明の微生物資材について詳記す
るが、本発明ではこのように共生培養を行った培養物を
そのまま使用しても良いし、或いは蛍光性細菌を含む培
養根と培養液とを分離し、各々を個々に使用することも
可能である。微生物資材の利用形態としては、培養物の
プラグ苗、種子、培土へのバクテリゼーション方法があ
る。より具体的には、培養液またはその分離培養根を殺
菌した種子にコーティングして使用する方法、あるいは
培養液をそのまま播種土、床土等と混合して使用する方
法、更には培養根を適当な担体を使用して固定化し、固
定化微生物として使用する方法等がある。尚、このよう
な場合に於いて、培養液をそのまま利用する場合の培養
液中の菌濃度は概ね105cells/ml以上で使用することが
望ましい。
Next, the microbial material of the present invention will be described in detail. In the present invention, the culture product thus co-cultivated may be used as it is, or a culture root and a culture solution containing fluorescent bacteria may be used. It is also possible to separate and to use each individually. As a utilization form of the microbial material, there is a bacterification method for plug seedlings, seeds, and soil of a culture. More specifically, a method in which the culture solution or its isolated culture root is used by coating it on sterilized seeds, or a method in which the culture solution is directly mixed with sowing soil, bed soil, etc., and further, a culture root is suitable. There is a method of immobilizing using a different carrier and using it as an immobilized microorganism. In such a case, it is desirable that the bacterial concentration in the culture medium when the culture medium is used as it is is approximately 10 5 cells / ml or more.

【0011】次に、本発明の更に望ましい形態について
詳記する。本発明は、上述したように植物体根内から分
離した蛍光性細菌を、分離源の植物体と同種の植物体の
培養根と共生培養することに主たる特徴を有するが、こ
のような培養によって得られた蛍光性細菌は、この活性
を維持させながら長期間保存することは困難である。即
ち、共生培養後の培養物をそのまま保存する場合1〜2ケ
月が限度である。また、共生培養後の細菌を分離し、こ
れを既知の保存法により保存すると、蛍光性細菌の活性
は著しく低下し、また細菌の変異等によって共生培養時
の細菌の形態学的、生理学的特性を維持することができ
ない。従って更なる本発明の目的は、上述した共生培養
後の蛍光性細菌の活性を低下させることなく保存するこ
とであり、また共生培養後の蛍光性細菌を微生物資材と
してより有効に利用するために、この蛍光性細菌を大量
培養することである。
Next, a more preferable mode of the present invention will be described in detail. The present invention, as described above, the fluorescent bacteria isolated from the inside of the plant root, the main feature in co-cultivating with the culture root of the plant of the same species as the plant of the source of separation, but by such culture The obtained fluorescent bacteria are difficult to store for a long period of time while maintaining this activity. That is, when the culture after co-cultivation is stored as it is, it is limited to 1 to 2 months. In addition, when the bacteria after co-cultivation are separated and stored by a known preservation method, the activity of fluorescent bacteria is remarkably reduced, and the morphological and physiological characteristics of the bacteria during co-cultivation due to mutation of the bacteria. Can't be maintained. Therefore, a further object of the present invention is to preserve the activity of the fluorescent bacterium after co-cultivation described above without lowering the activity, and to utilize the fluorescent bacterium after co-cultivation more effectively as a microbial material. It is to mass-culture this fluorescent bacterium.

【0012】本発明の蛍光性細菌の保存法について詳記
すると、先ず前述の共生培養によって得られた培養物中
の培養根のみをろ過等の手段により分取する。次いで、
この培養根を固定化細胞とするが、この方法は一般に用
いられる細胞の固定化手法によって行うことができる。
例えば、先ず培養根をホモジナイズした後、これにアル
ギン酸ナトリウム溶液を加えて攪拌する。この培養根分
散液を塩化カルシウム等の液中に滴下し、約3〜6mm径の
蛍光性細菌を含有する植物体細胞の固定化細胞とする。
このような固定化細胞は、これをシリカゲルあるいは五
酸化リン等の乾燥剤を使用し、40℃以下で短時間に乾燥
することにより、蛍光性細菌の活性を低下させることな
く長期間の保存が可能となる。
The method for preserving the fluorescent bacteria of the present invention will be described in detail. First, only the culture roots in the culture obtained by the above co-cultivation are separated by a means such as filtration. Then
This cultured root is used as an immobilized cell, and this method can be performed by a commonly used cell immobilization technique.
For example, first, the culture root is homogenized, and then a sodium alginate solution is added thereto and stirred. This culture root dispersion is added dropwise to a liquid such as calcium chloride to give fixed cells of plant somatic cells containing fluorescent bacteria having a diameter of about 3 to 6 mm.
Such fixed cells can be stored for a long period of time without decreasing the activity of fluorescent bacteria by using a desiccant such as silica gel or phosphorus pentoxide and drying them at 40 ° C or lower for a short time. It will be possible.

【0013】また、前述の固定化細胞あるいは乾燥固定
化細胞は、これを培養することにより固定化した蛍光性
細菌は増殖し、前述のような共生培養後の培養液中の菌
濃度は、概ね105〜106cells/mlであったものからこの固
定化細胞培養によって菌濃度は109〜1010cells/ml程度
まで増加する。培養方法は、例えばポテト・デキストロ
ース培地等で約2週間培養する。培養初期には固定化細
胞内の菌は植物細胞の周辺にコロニーを形成し、植物細
胞に親和性のある蛍光性細菌が増殖し、培養中期から後
期にかけては、細胞外部からの栄養源の供給と共に細菌
は固定化細胞の表面を通過し、培養液中へと移行しなが
ら増殖する。
Further, in the above-mentioned fixed cells or dry fixed cells, the immobilized fluorescent bacteria grow by culturing the cells, and the bacterial concentration in the culture solution after co-cultivation as described above is generally From 10 5 to 10 6 cells / ml, this fixed cell culture increases the bacterial concentration to about 10 9 to 10 10 cells / ml. The culture method is, for example, about 2 weeks of culture in a potato-dextrose medium or the like. At the beginning of the culture, the bacteria in the immobilized cells form colonies around the plant cells, and fluorescent bacteria that have an affinity for the plant cells grow, and from the middle to the second half of the culture, the nutrient source is supplied from outside the cell. At the same time, the bacteria pass through the surface of the immobilized cells and grow while transferring into the culture medium.

【0014】このようにして蛍光性細菌の増殖した培養
物を使用し、これを前述の微生物資材として使用するこ
とにより、本発明の微生物資材はより実用的なものとな
る。尚、このような固定化細胞を利用した微生物資材へ
の適用手段については前述の通りである。
By using the culture in which the fluorescent bacteria are proliferated as described above and using this as the above-mentioned microbial material, the microbial material of the present invention becomes more practical. The means for applying such a fixed cell to a microbial material is as described above.

【0015】[0015]

【実施例】以下に本発明の実施例を掲げ更に説明を行う
が、本発明はこれらに限定されるものではない。尚、本
実施例に於いて、%は特に断らない限り全て重量%を示
す。
The present invention will be further described below with reference to examples of the present invention, but the present invention is not limited thereto. In this example,% means% by weight unless otherwise specified.

【0016】(実施例1)1.蛍光性細菌の分離、培養 兵庫県内の施設栽培作物を中心に、幼苗期から収穫終了
期に至るまでの栽培中の作物を採取し、これより根内の
蛍光性細菌の分離を行った。分離方法は、採取した作物
の根を洗浄し、これをエタノールで殺菌処理した後、根
をホモジナイズしこれをそのまま素寒天と混釈培養し
た。次いで培養物中で生成したコロニーの中から蛍光性
を発するコロニーを選抜した。表1に兵庫県神戸市西区
および姫路市網干区の施設栽培圃場のトマト(品種:瑞
健、瑞栄、ハウス桃太郎)、チンゲン菜およびネギの根
より分離した菌株を示した。また、これらの菌株につい
て、各種の病原菌に対する抗菌活性について評価し、そ
の結果を併せて表1に示した。また、表1の代表菌株に
ついての菌学的特性を表2に示した。更に、本発明実施
例に使用する菌株は、菌の分離後、5倍希釈ポテト・デ
キストロース寒天斜面培地を使用し、23〜25℃で5〜7日
間継代培養を行った後、4〜6℃で保存したものを使用し
た。
(Example 1) 1. Separation and culture of fluorescent bacteria Centering on in-house-grown crops in Hyogo prefecture, crops under cultivation from the seedling stage to the end of harvest are collected and Isolation of fluorescent bacteria was performed. The method of separation was to wash the roots of the collected crops, sterilize the roots with ethanol, homogenize the roots, and pour the homogenized mixture into pure agar. Then, colonies that emit fluorescence were selected from the colonies formed in the culture. Table 1 shows the strains isolated from tomatoes (variety: Mizuken, Mizuei, House Momotaro), bok choy, and leek roots in facility-grown fields in Nishi-ku, Kobe-shi and Himeji-shi, Aboshi-ku, Hyogo Prefecture. In addition, these bacterial strains were evaluated for antibacterial activity against various pathogenic bacteria, and the results are also shown in Table 1. The bacteriological characteristics of the representative strains in Table 1 are shown in Table 2. Furthermore, the strains used in the examples of the present invention, after isolation of the bacteria, using a 5-fold diluted potato dextrose agar slant medium, after subculture at 23-25 ° C. for 5-7 days, 4-6 The one stored at ℃ was used.

【0017】2.根端培養 上記保存菌株を共生培養に供するため、分離源の植物と
同種の種子を使用して培養根を得た。先ず、トマトの各
品種である瑞健、瑞栄、ハウス桃太郎及びファーストメ
モリーの種子を使用し、この種子を1%次亜塩素酸ナト
リウム水溶液と80%エタノール水溶液により表面殺菌を
行った後、これを0.8%素寒天に播種し、23℃、暗好気
下で5日間保持し種子を発芽させた。発芽した種子根の
根端部を約1cm切り取り、これをホワイト液体培地中に
接種した。この培地を25℃で約10日間保持して根端を培
養した後、生長した培養根を新しいホワイト液体培地に
植え継ぎ、更に25℃で約10日間培養を行った。
2. Root tip culture In order to subject the above-mentioned preserved strains to co-cultivation, seeds of the same species as the plant of the separation source were used to obtain culture roots. First, the seeds of each of the varieties of tomatoes, Zuiken, Ruiei, Hausomoro, and First Memory are used, and the seeds are surface sterilized with a 1% sodium hypochlorite aqueous solution and an 80% ethanol aqueous solution. Was sown on 0.8% bare agar and kept at 23 ° C. under dark aerobic condition for 5 days to germinate seeds. Approximately 1 cm of the germinated seed root was cut off and inoculated into a white liquid medium. This medium was kept at 25 ° C for about 10 days to culture root tips, and then the grown cultured roots were subcultured to a new white liquid medium, and further cultured at 25 ° C for about 10 days.

【0018】3.共生培養 共生培養法は、先ず10倍希釈ホワイト液体培地に上記の
培養根を加え、25℃で1〜2日間培養した。次いで、これ
に前記1.の継代培養菌を種菌として接種し、25℃で約2
週間の共生培養を行った。培養終了後、培養根を取り出
し、これを滅菌水で洗浄した。このように共生培養を行
った培養菌の活性を評価するため、培養根の根端部を除
く部位から約1cm長の根を数根採取し、これを前記培養
前の継代培養菌と同一培養条件とするため、培養根を5
倍希釈ポテト・デキストロース寒天斜面培地に接種し、
23〜25℃で5〜7日間培養を行った。そして共生培養後の
供試菌株の共生の可否を調べた。その結果を表3に示し
た。継代培養菌と共生培養菌との活性の比較は、以下に
記載した根内定着能評価試験、病原菌に対する抗菌性物
質産生能評価試験、植物生育促進能評価試験、病害防除
活性評価試験によって比較した。
3. Co-cultivation method In the co-cultivation method, first, the above-mentioned culture roots were added to a 10-fold diluted white liquid medium and cultured at 25 ° C. for 1 to 2 days. Then, inoculate this with the subculture of the above 1.
Weekly co-cultivation was performed. After completion of the culture, the culture root was taken out and washed with sterilized water. In order to evaluate the activity of the cultivated bacteria co-cultivated in this manner, several roots of about 1 cm long were collected from the site excluding the root end of the cultured roots, which were the same as the subcultured bacteria before the culturing. To obtain the culture conditions, 5 culture roots
Inoculate the double-diluted potato dextrose agar slant medium,
Culture was performed at 23 to 25 ° C for 5 to 7 days. Then, it was investigated whether or not the test strains after symbiotic culture could coexist. The results are shown in Table 3. The comparison of the activity between the subculture bacteria and the co-cultivation bacteria is made by the following root root fixing capacity evaluation test, antibacterial substance production capacity evaluation test against pathogenic bacteria, plant growth promoting capacity evaluation test, disease control activity evaluation test. did.

【0019】A.根内定着能評価試験 上記2.の培養根に付着している培地成分を滅菌水で洗
浄除去した。この培養根を約1cm間隔に切断し、これを
滅菌水に105cells/ml濃度に調製した供試菌液中に24時
間浸漬した。浸漬後、根を滅菌水で洗浄し、これを滅菌
水の入った試験管中で25℃で2日間保持した。次いで、
試験管より根を取り出し、滅菌水で洗浄後、根の中央部
を切断して切断面をスライドガラスにレプリカした。レ
プリカしたガラスを風乾した後、火炎固定を行い、グラ
ム染色によって接種菌の根内増殖状態を観察した。評価
試験結果を表4に示した。
A. Intra-root colonization ability evaluation test The medium components adhering to the culture root in the above 2. were washed off with sterilized water. Cutting the cultured roots about 1cm intervals, it was immersed for 24 hours in test bacteria liquid prepared in 10 5 cells / ml concentration with sterilized water. After soaking, the roots were washed with sterile water and kept in a test tube containing sterile water at 25 ° C for 2 days. Then
The root was taken out from the test tube, washed with sterilized water, the center part of the root was cut, and the cut surface was replicated on a slide glass. After air-drying the replicated glass, flame fixation was performed, and the inoculum growth state in the roots was observed by Gram staining. The evaluation test results are shown in Table 4.

【0020】B.病原菌に対する抗菌性物質産生能評価
試験 病原菌として、トマト青枯病菌、トマト萎凋病菌、トマ
ト根腐萎凋病菌等を使用し、各供試菌株の病原菌に対す
る抗菌性物質産生能を評価した。尚、方法はトマト青枯
病菌についてはプレートクロロホルム法と混釈培養法に
より、またその他の病原糸状菌については対峙培養法に
より評価試験を行った。供試菌No.F-16の継代培養菌及
び共生培養菌について、プレートクロロホルム法による
評価試験結果を表5に示した。また、供試菌No.T-32とT
-33の継代培養菌及び共生培養菌を使用し、青枯病菌に
対しては混釈培養法で、その他の病原菌に対しては対峙
培養法で抗菌性物質生産能の評価試験を行った。その結
果を表6に示した。
B. Evaluation of antibacterial substance production ability against pathogenic bacteria
As test pathogens, tomato wilt disease fungus, tomato wilt disease fungus, tomato root rot wilt disease fungus and the like were used, and the ability of each test strain to produce an antibacterial substance against the pathogenic fungus was evaluated. The evaluation test was carried out by the plate chloroform method and the pour-in culture method for the bacterial wilt disease of tomato, and by the confrontation culture method for other pathogenic filamentous fungi. Table 5 shows the evaluation test results by the plate chloroform method for the subcultured bacteria and co-cultivated bacteria of the test bacterium No. F-16. Also, test bacteria No. T-32 and T
-33 subculture and co-cultivation were used to evaluate the antibacterial substance-producing ability by the pour-in culture method for bacterial wilt disease and the confrontation culture method for other pathogens. . The results are shown in Table 6.

【0021】C.植物生育促進能評価試験 前述の根端培養に使用した各品種のトマト種子を使用
し、この種子を発芽させた後、明好気条件下で更に1週
間寒天培地で生育させた。生育した幼苗の根部を切断
し、穂木部をホワイト寒天培地(庶糖無添加)に挿し木し
た。この挿し木を28〜30℃の明好気条件下で24時間生育
させ、これに滅菌水で105cells/ml濃度となるように調
製した供試菌液を、培地容量の約1v/v%となるように挿
し木部に接種した。菌を接種後も栽培を継続し、挿し木
の発根作用について評価検定を行った。評価試験結果と
して、各種供試菌のトマト・ハウス桃太郎に対する発根
作用を表7に、供試菌No.F-16の各種トマトの品種に対
する発根作用を表8に示した。
C. Plant Growth Promoting Ability Evaluation Test Using the tomato seeds of each variety used in the root tip culture described above, after germination of the seeds, the seeds were allowed to grow on an agar medium for 1 week under light and aerobic conditions. It was The roots of the grown seedlings were cut, and the saplings were cut into white agar medium (without sucrose added). The cuttings were grown for 24 hours under aerobic conditions at 28 to 30 ° C, and a test bacterial solution prepared to have a concentration of 10 5 cells / ml with sterile water was added to the medium at a volume of about 1 v / v%. The cuttings were inoculated so that Cultivation was continued even after inoculation with the fungus, and an evaluation test was performed for rooting action of the cuttings. As an evaluation test result, the rooting action of various test bacteria on tomato house Momotaro is shown in Table 7, and the rooting action of various test bacteria No. F-16 on various tomato varieties is shown in Table 8.

【0022】D.病害防除活性評価試験 前述の根端培養に使用した各品種のトマト種子を使用
し、この種子を発芽させた。種子の発芽後、直ちにこれ
を予め下層部にホワイト寒天培地(庶糖無添加)、中層部
に3〜5mmの海砂層、上層部に寒天培地を形成させた培地
上に置床し、28〜30℃で明好気条件下で生育させた。発
芽根の先端部が寒天層より海砂層に達した時点で、これ
に滅菌水で105cells/ml濃度に調製した供試菌液を培地
容量の約1v/v%となるように培地表面に接種した。菌の
接種後24時間発芽種子の生育を継続させた後、これに滅
菌水で107cells/ml濃度に調製した青枯病菌液を、培地
容量の約2v/v%となるように培地表面に接種した。この
病原菌接種後も同条件で生育を継続し罹病調査を行い、
病害防除活性について評価検定を行った。評価試験結果
を表9に示した。
D. Disease Control Activity Evaluation Test Tomato seeds of each variety used in the above-mentioned root tip culture were used, and this seed was germinated. Immediately after germination of the seed, this was placed on a medium in which a white agar medium (without sucrose added) was previously formed in the lower layer, a sea sand layer of 3 to 5 mm was formed in the middle layer, and an agar medium was formed in the upper layer, and 28 to 30 ° C. Were grown under light and aerobic conditions. When the tip of the germinated root reaches the sea sand layer from the agar layer, the test bacterial solution prepared to a concentration of 10 5 cells / ml with sterilized water is added to the surface of the medium so that it is about 1 v / v% of the medium volume. Was inoculated. After continuing the growth of germinated seeds for 24 hours after inoculation of the fungus, add bacterial wilt disease solution prepared to a concentration of 10 7 cells / ml with sterile water to the surface of the medium so that the volume is about 2 v / v% of the medium volume. Was inoculated. After the inoculation of this pathogen, we continue to grow under the same conditions and carry out morbidity surveys.
An evaluation test was performed for disease control activity. The evaluation test results are shown in Table 9.

【0023】[0023]

【表1】 [Table 1]

【0024】[0024]

【表2】 [Table 2]

【0025】[0025]

【表3】 [Table 3]

【0026】[0026]

【表4】 [Table 4]

【0027】[0027]

【表5】 [Table 5]

【0028】[0028]

【表6】 [Table 6]

【0029】[0029]

【表7】 [Table 7]

【0030】[0030]

【表8】 [Table 8]

【0031】[0031]

【表9】 [Table 9]

【0032】(実施例2)共生培養にトマト(品種:ハウス
桃太郎)の分離根を使用して培養を行った表3のNo.F-16
の培養物(300ml)を共生根と培養液に濾別した。分離し
た共生根を1%アルギン酸ナトリウム水溶液の300mlに入
れ、これをホモジナイザーを使用して回転数8000〜1000
0rpmの条件下、約10分間の混合粉砕に供し、ゲル状の資
材No.1の微生物資材を得た。
(Example 2) No. F-16 of Table 3 in which co-cultivation was carried out using isolated roots of tomato (variety: House Momotaro)
The culture (300 ml) was separated into symbiotic roots and a culture solution by filtration. Put the separated symbiotic root in 300 ml of 1% sodium alginate aqueous solution, and use a homogenizer to rotate at 8,000-1000 rpm.
The mixture was subjected to mixed pulverization for about 10 minutes under the condition of 0 rpm to obtain a gel material No. 1 microbial material.

【0033】また、共生培養にトマト(品種:ファーストメモリー)
の分離根を使用して培養を行った表3のNo.T-32の培養
物(300ml)を共生根と培養液に濾別した。分離した培養
液に2%アルギン酸ナトリウム水溶液の300mlを添加し、
攪拌を行いながらこれにシリカ粉末の240gを添加し、粉
状の資材No.2の微生物資材を得た。尚、分離した共生根
は、これを1%アルギン酸ナトリウム水溶液の300mlに3g
の活性炭を添加した溶液中に入れ、ホモジナイザーを使
用して回転数8000〜10000rpmの条件下、約10分間の混合
粉砕に供し、ゲル状の微生物資材を得た。次に、このゲ
ル状微生物資材の100mlに、40gのシリカ粉末を添加して
粉状の資材No.3の微生物資材を得た。
In addition, tomatoes (cultivar: First Memory) are used for co-cultivation.
The culture (300 ml) of No. T-32 in Table 3, which was cultivated using the isolated root of No. 3, was separated by filtration into a symbiotic root and a culture solution. Add 300 ml of 2% sodium alginate aqueous solution to the separated culture solution,
While stirring, 240 g of silica powder was added to this to obtain a powdery material No. 2 of the microbial material. The separated symbiotic roots are 3 g in 300 ml of 1% sodium alginate aqueous solution.
The activated carbon was added to the solution, and the mixture was subjected to mixed pulverization for about 10 minutes under the condition of a rotation speed of 8,000 to 10,000 rpm using a homogenizer to obtain a gel-like microbial material. Next, 40 g of silica powder was added to 100 ml of this gel-like microbial material to obtain powdery microbial material of Material No. 3.

【0034】これら調製した資材No.1〜3の微生物資材
を使用して、種子のバクテリゼーション処理及び培土処
理を行った。先ず、種子のバクテリゼーション処理は、
トマト(品種:大型福寿、ハウス桃太郎)の種子を使用し、こ
の種子を殺菌処理した後、資材No.1の微生物資材に浸漬
し、次に1%塩化カルシウム水溶液に浸漬した後、直ち
に滅菌水で種子表面を洗浄し、これを4〜6℃で保存し
た。また、資材No.1の微生物資材で処理を行った種子の
一部は、これに更に資材No.3の微生物資材をコーティン
グ処理し、二種の蛍光性細菌によるバクテリゼーション
処理種子を得た。
Using the prepared microbial materials Nos. 1 to 3, seeds were subjected to bacterification treatment and soil treatment. First, the seed bacteriolysis
Tomato (variety: large Fukuju, House Momotaro) seeds are used, and after the seeds are sterilized, they are immersed in the microbial material of Material No. 1, then in a 1% calcium chloride aqueous solution, and immediately sterilized water. The seed surface was washed with and stored at 4-6 ° C. Further, a part of the seeds treated with the microbial material of Material No. 1 was further coated with the microbial material of Material No. 3 to obtain seeds treated with bacteria of two types of fluorescent bacteria.

【0035】処理培土に使用した培土は、トマト青枯病
菌を菌密度105〜106(cfu/g乾土)とした汚染土壌であ
る。この培土に資材No.2の微生物資材を約10v/v%とな
るように混合し、また資材No.3の微生物資材については
約5v/v%となるように混合し、それぞれを播種用の処理
培土とした。
The soil used as the treated soil is a contaminated soil having a bacterial density of tomato wilt disease of 10 5 to 10 6 (cfu / g dry soil). To this soil, mix the microbial material of material No. 2 to about 10 v / v%, and mix the microbial material of material No. 3 to about 5 v / v%, and mix them for seeding. It was treated soil.

【0036】各種の微生物資材、バクテリゼーション処
理種子及び処理培土を用い、トマトの栽培試験を行っ
た。尚、トマト青枯病菌の菌密度105〜106(cfu/g乾土)
の土壌を汚染土壌とした。試験方法は、先ず1穴の容量
が約15mlのプラグ苗用のトレイに、汚染土壌及び処理培
土を充填し、このトレイを汚染土壌を敷設した土壌表面
に埋め込んだ。次いで、これにトマト種子を播種した
が、汚染土壌を充填したトレイにはバクテリゼーション
種子を播種し、処理培土を充填したトレイには未処理種
子(トマト品種:大型福寿、ハウス桃太郎)を播種した。種子の播
種後、ハウス内で自動灌水等による栽培管理を行いなが
らトマトの栽培を行い、その苗の生育状態の観察によっ
て罹病調査を行った。結果を表10に示した。
A tomato cultivation test was carried out using various microbial materials, bacterification-treated seeds and treated soil. In addition, the bacterial density of tomato wilt fungus 10 5 to 10 6 (cfu / g dry soil)
The soil was designated as contaminated soil. The test method was as follows. First, a tray for plug seedlings having a capacity of about 15 ml per hole was filled with contaminated soil and treated soil, and this tray was embedded in the soil surface on which the contaminated soil was laid. Then, tomato seeds were sown on this, but the bacterium seeds were sown on the tray filled with the contaminated soil, and the untreated seeds (tomato varieties: large Fukuju, House Momotaro) were sown on the tray filled with the treated soil. . After sowing the seeds, tomatoes were cultivated in the house while the cultivation was controlled by automatic irrigation, etc., and the disease state was investigated by observing the growth condition of the seedlings. The results are shown in Table 10.

【0037】[0037]

【表10】 [Table 10]

【0038】(実施例3)実施例1と同様にして施設栽
培のトマトより分離した5菌株と5品種の供試根を使用
し、実施例1と同様に共生培養を行い共生根を得た。こ
の共生根を、活性炭1%を添加した1%アルギン酸ナトリ
ウム水溶液中でホモジナイズしゲル状溶液とした後、こ
のゲル溶液を1%塩化カルシウム水溶液中に滴下するこ
とにより、外径が約5mmの固定化細胞を得た。次に、シ
リカゲルを充填した密閉容器内に濾紙を置き、この濾紙
上に固定化細胞を広げ、これを30℃で1時間保持し固定
化細胞の乾燥を行った。乾燥した固定化細胞は、これを
アンプル中で真空溶封し、2ケ月間冷蔵保存した。
(Example 3) In the same manner as in Example 1, using 5 strains and 5 kinds of test roots isolated from tomatoes grown in-house, co-cultivation was carried out in the same manner as in Example 1 to obtain symbiotic roots. . This symbiotic root was homogenized in a 1% sodium alginate aqueous solution containing 1% activated carbon to form a gel solution, and this gel solution was dropped into a 1% calcium chloride aqueous solution to fix the outer diameter of about 5 mm. The activated cells were obtained. Next, a filter paper was placed in a closed container filled with silica gel, the immobilized cells were spread on the filter paper, and this was maintained at 30 ° C. for 1 hour to dry the immobilized cells. The dried fixed cells were vacuum-sealed in ampoules and refrigerated for 2 months.

【0039】保存した乾燥固定化細胞の活性評価のた
め、この固定化細胞を5倍希釈ポテト・デキストロース
寒天平板上に置き、25〜28℃で5日間培養を行った。こ
の培養物について実施例1と同様に根内定着能評価試験
及び病原菌に対する抗菌性物質産生能評価試験を行っ
た。保存菌の復元性を表11に、定着性及び抗菌活性を
表12並びに表13に示した。
In order to evaluate the activity of the dried and fixed cells stored, the fixed cells were placed on a 5-fold diluted potato-dextrose agar plate and cultured at 25 to 28 ° C. for 5 days. In the same manner as in Example 1, this culture was subjected to the root root colonization ability evaluation test and the antibacterial substance production ability evaluation test against pathogenic bacteria. Restorability of the preserved bacteria is shown in Table 11, and fixing ability and antibacterial activity are shown in Tables 12 and 13.

【0040】[0040]

【表11】 [Table 11]

【0041】[0041]

【表12】 [Table 12]

【0042】[0042]

【表13】 [Table 13]

【0043】(実施例4)実施例1の分離蛍光性細菌の
表1菌株No.T-33を用い、トマト(品種:ハウス桃太郎)の分
離根との共生培養によって得られた共生根を分離した。
この共生根を1%アルギン酸ナトリウム水溶液中でホモ
ジナイズしてゲル状溶液とした後、このゲル溶液を1%
塩化カルシウム水溶液中に滴下することにより、外径が
約5mmの固定化細胞を得た。次いで、この固定化細胞の1
0粒を採り、これを5倍希釈ポテト・デキストロース溶液
の200mlに接種した後、30℃で10日間の静置培養を行っ
た。10日間の培養後、固定化細胞を除く培養物を抜き出
した。抜き出した培養物を5倍希釈ポテト・デキストロ
ース寒天斜面培地に接種し、23〜25℃で5〜7日間培養を
行った。培養後の菌株について、実施例1と同様に根内
定着能評価試験及び病原菌に対する抗菌性物質産生能評
価試験を行った。評価試験結果を表14及び表15に示
した。
(Example 4) Isolation of Example 1 Using the fluorescent bacteria Table 1 strain No. T-33, a symbiotic root obtained by symbiotic culture with an isolated root of tomato (variety: House Momotaro) was isolated. did.
This symbiotic root was homogenized in a 1% sodium alginate aqueous solution to give a gel solution, and the gel solution was
Immobilized cells with an outer diameter of about 5 mm were obtained by dropping into an aqueous solution of calcium chloride. Then one of the fixed cells
After taking 0 grain, inoculating this with 200 ml of a 5-fold diluted potato dextrose solution, static culture was performed at 30 ° C. for 10 days. After culturing for 10 days, the culture excluding the fixed cells was extracted. The extracted culture was inoculated on a 5-fold diluted potato-dextrose agar slant medium and cultured at 23 to 25 ° C for 5 to 7 days. With respect to the strains after the culture, as in Example 1, the root-root colonization ability evaluation test and the antibacterial substance production ability evaluation test against pathogenic bacteria were performed. The evaluation test results are shown in Tables 14 and 15.

【0044】また、抜き出した培養物を使用し、実施例
2の資材No.2の微生物資材の調製法と同様の方法により
微生物資材を調製した。この資材を使用し、資材の施用
によるトマト育苗期に於ける苗の生育促進作用の検定
と、病原菌汚染土壌に定植後のトマト青枯病に対する抵
抗性を検定するため、以下の方法によってトマトの栽培
試験を行った。
Using the extracted culture, a microbial material was prepared by the same method as the method for preparing the microbial material of Material No. 2 of Example 2. Using this material, in order to test the growth promoting action of seedlings in the tomato seedling raising period by applying the material and to test the resistance to bacterial wilt disease after planting in pathogen-contaminated soil, A cultivation test was conducted.

【0045】栽培試験方法は、先ず1穴の容量が約15ml
のプラグ苗用のトレイに、上記微生物資材を市販の園芸
培土に対して5v/v%となるように混合した培土を充填
し、これにトマト(品種:ハウス桃太郎)の種子を播種して14
日間育成した。次に、鉢上げ用培土を上記と同様に調製
し、上記の生育したトマト苗をこの調製培土を充填した
鉢に移植した後、16日間栽培を継続した。16日間の栽培
後に苗の生育調査を行い、結果を表16に示した。生育
調査後、苗をトマト青枯病菌の汚染した圃場(病原菌密
度:105〜107cfu/g乾土)に定植し、発病苗数の調査によ
る病害防除効果の検定を行った。結果を表17に示し
た。
As for the cultivation test method, first, the volume of one hole is about 15 ml.
The plug seedling tray was filled with a soil containing the above-mentioned microbial materials mixed at 5 v / v% with respect to the commercially available horticultural soil, and the seeds of tomato (variety: House Momotaro) were sown in this 14
Raised for a day. Next, a potting medium was prepared in the same manner as above, and the above-grown tomato seedlings were transplanted into pots filled with this prepared medium, and then cultivation was continued for 16 days. After 16 days of cultivation, the seedlings were examined for growth and the results are shown in Table 16. After the growth survey, the seedlings were planted in a field (pathogen density: 10 5 to 10 7 cfu / g dry soil) contaminated with tomato wilt fungus, and the disease control effect was tested by investigating the number of diseased seedlings. The results are shown in Table 17.

【0046】[0046]

【表14】 [Table 14]

【0047】[0047]

【表15】 [Table 15]

【0048】[0048]

【表16】 [Table 16]

【0049】[0049]

【表17】 [Table 17]

【0050】[0050]

【発明の効果】本発明の蛍光性細菌の活性維持法は、蛍
光性細菌を分離、培養するに際して植物根内に棲息して
いる状態を再現することで、この活性を低下させること
なく利用することができる。また、このような方法によ
って取得、培養された蛍光性細菌を含む培養物は、これ
を農作物等の植物体に施用することにより、土壌病害の
防除並びに植物の生育促進に顕著な効果を発現し優れた
微生物資材となる。
INDUSTRIAL APPLICABILITY The method for maintaining the activity of a fluorescent bacterium of the present invention is utilized without reducing the activity of the fluorescent bacterium by reproducing the state inhabiting the plant root when the fluorescent bacterium is separated and cultured. be able to. Further, the culture containing the fluorescent bacteria obtained by such a method and cultivated, by applying this to plants such as agricultural crops, a remarkable effect is exhibited in controlling soil diseases and promoting the growth of plants. It becomes an excellent microbial material.

【0051】更に、本発明の効果をより有効なものとす
るため、上記培養物中の共生微生物を含有する植物体細
胞を分取し、これを固定化細胞とした後、短時間に乾燥
することにより、蛍光性細菌はその活性を低下させるこ
となく長期間の保存が可能となり、またこのような固定
化細胞を培養することにより、蛍光性細菌の大量培養が
可能となる。従って、このような培養物を微生物資材と
して利用することにより、本発明の効果はより実用的な
ものとなる。
Further, in order to make the effect of the present invention more effective, the plant cells containing the symbiotic microorganisms in the above culture are fractionated, made into fixed cells, and then dried in a short time. As a result, fluorescent bacteria can be stored for a long period of time without lowering their activity, and by culturing such fixed cells, large-scale cultivation of fluorescent bacteria becomes possible. Therefore, by using such a culture as a microbial material, the effect of the present invention becomes more practical.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 清水 克彦 兵庫県神戸市垂水区下畑町西下代161番地 の3 (72)発明者 牛尾 昭浩 兵庫県加西市朝妻町1220番地の4 (72)発明者 桑名 健夫 兵庫県加西市朝妻町1220番地の4 (72)発明者 小林 保 兵庫県宝塚市旭町3丁目7番1−606号 (72)発明者 小林 尚司 兵庫県加古川市山手2丁目2番地 (72)発明者 松山 稔 兵庫県明石市大久保町大窪1338番地 (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岡2689−1番地 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Katsuhiko Shimizu 3 at 161 Nishishitshiro, Shimohata-cho, Tarumi-ku, Kobe-shi, Hyogo (72) Inventor Akihiro Ushio 4 at 1220 Asazuma-cho, Kasai-shi, Hyogo (72) Invention Takeo Kuwana 4 1220 Asazuma-cho, Kasai-shi, Hyogo Prefecture (72) Inventor Kobayashi 3-7-606 Asahi-cho, Takarazuka-shi Hyogo (72) Inventor Shoji Kobayashi 2-chome Yamate, Kakogawa-shi Hyogo (72) Minoru Matsuyama 1338 Okubo, Okubo-cho, Akashi-shi, Hyogo Prefecture (72) Yoshio Maekawa 3-491 Higashi Jiyugaoka, Shizome-cho, Miki-shi, Hyogo (72) Taizo Akiyama Kamizume 331, Yoneda-cho, Takasago-shi, Hyogo Address-9 (72) Inventor Yoshinori Hayashi 2689-1 Oka, Inami-cho, Kako-gun, Hyogo Prefecture

Claims (4)

【特許請求の範囲】[Claims] 【請求項1】 植物体根内から分離した蛍光性細菌を、
分離源の植物体と同種の植物体の培養根と共生培養する
ことを特徴とする蛍光性細菌の活性維持法。
1. A fluorescent bacterium isolated from the root of a plant,
A method for maintaining the activity of a fluorescent bacterium, which comprises co-culturing with a culture root of a plant of the same species as a plant of a separation source.
【請求項2】 植物体根内から分離した蛍光性細菌を、
分離源の植物体と同種の植物体の培養根と共生培養した
培養物からなる微生物資材。
2. A fluorescent bacterium isolated from the inside of a plant root,
A microbial material comprising a culture that is co-cultivated with a culture root of a plant of the same species as the plant of the separation source.
【請求項3】 植物体根内から分離した蛍光性細菌を、
分離源の植物体と同種の植物体の培養根と共生培養し、
次いでその培養根を分取し、これを固定化細胞とした
後、乾燥することを特徴とする蛍光性細菌の保存法。
3. A fluorescent bacterium isolated from the inside of a plant root,
Co-cultivated with a culture root of a plant of the same species as the plant of the separation source,
Next, a method for preserving fluorescent bacteria, which comprises collecting the cultured roots, making them fixed cells, and then drying.
【請求項4】 植物体根内から分離した蛍光性細菌を、
分離源の植物体と同種の植物体の培養根と共生培養し、
次いでその培養根を分取し、これを固定化細胞とした
後、更にこれを培養した培養物からなる微生物資材。
4. A fluorescent bacterium isolated from the root of a plant,
Co-cultivated with a culture root of a plant of the same species as the plant of the separation source,
Then, the cultivated root is separated, and the cultivated material is used as a fixed cell, and the cultivated material is further cultivated.
JP34166893A 1993-12-10 1993-12-10 Method for maintaining and preserving the activity of fluorescent bacteria and microbial material comprising the culture Expired - Fee Related JP2660317B2 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0808571A1 (en) * 1996-05-20 1997-11-26 Taki Chemical Co., Ltd. Plug mixture for raising seedlings and method for producing it, and method for raising disease tolerant seedlings
EP0872182A3 (en) * 1997-04-15 1999-04-21 Taki Chemical Co., Ltd. Plant growth regulator
WO2003034807A1 (en) * 2001-10-22 2003-05-01 Taki Chemical Co., Ltd. Rooted cutting and method of inoculating rooted cutting with microbial strain
JP2008525022A (en) * 2004-12-23 2008-07-17 ベッカー アンダーウッド インコーポレイテッド Improved shelf life and stability on seeds of liquid bacterial inoculum
JP2021510296A (en) * 2018-01-10 2021-04-22 バイエル クロップサイエンス エルピーBayer Cropscience Lp Improved microorganisms and their manufacturing methods

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0808571A1 (en) * 1996-05-20 1997-11-26 Taki Chemical Co., Ltd. Plug mixture for raising seedlings and method for producing it, and method for raising disease tolerant seedlings
US5935571A (en) * 1996-05-20 1999-08-10 Taki Chemical Co., Ltd. Plug mixture for raising seedlings and method for producing it, and method for raising disease tolerant seedlings
EP0872182A3 (en) * 1997-04-15 1999-04-21 Taki Chemical Co., Ltd. Plant growth regulator
WO2003034807A1 (en) * 2001-10-22 2003-05-01 Taki Chemical Co., Ltd. Rooted cutting and method of inoculating rooted cutting with microbial strain
JP2008525022A (en) * 2004-12-23 2008-07-17 ベッカー アンダーウッド インコーポレイテッド Improved shelf life and stability on seeds of liquid bacterial inoculum
JP2021510296A (en) * 2018-01-10 2021-04-22 バイエル クロップサイエンス エルピーBayer Cropscience Lp Improved microorganisms and their manufacturing methods

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