JP2019180361A - Method for increasing yield of grains, method for cultivating plants for grain cultivation, and composition and microorganism for increasing yield of grains - Google Patents
Method for increasing yield of grains, method for cultivating plants for grain cultivation, and composition and microorganism for increasing yield of grains Download PDFInfo
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Abstract
Description
本発明は、穀物類の収量を増加させる方法、穀物類の栽培用植物体を栽培する方法、穀物類の収量増加用組成物及び微生物に関する。 The present invention relates to a method for increasing the yield of cereals, a method for cultivating plants for cultivating cereals, a composition for increasing the yield of cereals, and a microorganism.
イネ科植物等の穀物類の収量を増加させることは、非常に重要であり、従来、多量の肥料を使用したり、肥料を栄養価の高いものにしたり、植物成長調節剤が使われたり、遺伝子導入などによる品種改良が行われたりしてきた。
しかしながら、これらの技術では、合成品を用いることによる土壌に対する影響の問題や、組換え植物は一般に受け入れ難いという問題などがあり、新たな技術が求められている。
Increasing the yield of cereals such as gramineous plants is very important. Conventionally, a large amount of fertilizer is used, fertilizer is made highly nutritious, plant growth regulators are used, Breeding has been improved by gene transfer.
However, in these techniques, there are a problem of influence on soil due to the use of synthetic products, a problem that recombinant plants are generally unacceptable, and new techniques are required.
これまでに、イネの生育促進と種子増量効果を得るために、窒素固定細菌をイネに感染させる技術が提案されている（例えば、特許文献１参照）。しかしながら、前記提案では、生きた菌を使用しなければならないため、製剤化や品質管理が難しく、また、使用した際の環境安全面でのリスクもあるという問題がある。 Until now, in order to obtain the growth promotion and seed increase effect of rice, a technique for infecting rice with nitrogen-fixing bacteria has been proposed (see, for example, Patent Document 1). However, in the above proposal, since live bacteria must be used, formulation and quality control are difficult, and there are also problems in terms of environmental safety when used.
したがって、簡易、かつ安全に穀物類の収量を増加させることができる新たな技術の速やかな提供が強く求められている。 Therefore, there is a strong demand for prompt provision of a new technology that can easily and safely increase the yield of grains.
本発明は、前記従来における諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、簡易、かつ安全に穀物類の収量を増加させることができる穀物類の収量を増加させる方法及び穀物類の栽培用植物体を栽培する方法、並びに穀物類の収量増加用組成物及び新規微生物を提供することを目的とする。 An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention provides a method for increasing the yield of cereals that can easily and safely increase the yield of cereals, a method for cultivating plants for cultivating cereals, and a composition for increasing the yield of cereals. The object is to provide products and new microorganisms.
本発明者らは、前記目的を達成するべく鋭意検討を行った結果、メタノール資化性細菌の細胞壁を含有する組成物を穀物類の栽培用植物体に接種することにより、不稔籾を減少させ、穀物類の収量を増加させることができることを知見した。 As a result of intensive studies to achieve the above object, the present inventors reduced sterility by inoculating a plant for cultivating cereals with a composition containing cell walls of methanol-assimilating bacteria. And found that the yield of cereals can be increased.
本発明は、本発明者らの前記知見等に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ メタノール資化性細菌の細胞壁含有物を穀物類の栽培用植物体に接種することを含むことを特徴とする穀物類の収量を増加させる方法である。
＜２＞ メタノール資化性細菌の細胞壁含有物を穀物類の栽培用植物体に接種することを含むことを特徴とする穀物類の栽培用植物体を栽培する方法である。
＜３＞ 前記接種が、出穂前３０日から出穂後３０日の間に行われる前記＜１＞から＜２＞のいずれかに記載の方法である。
＜４＞ 前記接種が出穂の前と後に行われる前記＜１＞から＜３＞のいずれかに記載の方法である。
＜５＞ 前記メタノール資化性細菌が植物体の表面から単離された前記＜１＞から＜４＞のいずれかに記載の方法である。
＜６＞ 前記メタノール資化性細菌の単離が、前記メタノール資化性細菌を前記植物体の表面から洗い流して得た洗浄液又は前記植物体を液体培地に入れて培養して得られた液を、培地に接種し、増殖してきたコロニーを同定し、前記メタノール資化性細菌のコロニーを単離することにより行われる前記＜５＞に記載の方法である。
＜７＞ 前記メタノール資化性細菌の単離に用いる植物体が、茎及び籾の少なくともいずれかである前記＜５＞から＜６＞のいずれかに記載の方法である。
＜８＞ 前記接種が、前記メタノール資化性細菌の細胞壁含有物を含む液を前記栽培用植物体に噴霧することにより行われる前記＜１＞から＜７＞のいずれかに記載の方法である。
＜９＞ 前記メタノール資化性細菌が、受託番号ＮＩＴＥ Ｐ−０２６２８のメチロバクテリウム エスピー（Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ ｓｐ．）Ｒｓｔ株である前記＜１＞から＜８＞のいずれかに記載の方法である。
＜１０＞ 前記メタノール資化性細菌の細胞壁含有物が、死菌体及び菌体細胞壁の少なくともいずれかを含む前記＜１＞から＜９＞のいずれかに記載の方法である。
＜１１＞ 前記死菌体が破砕菌体を含む前記＜１＞から＜１０＞のいずれかに記載の方法である。
＜１２＞ 前記穀物類が米である前記＜１＞から＜１１＞のいずれかに記載の方法である。
＜１３＞ 前記米が酒米である前記＜１２＞に記載の方法である。
＜１４＞ 前記酒米が白鶴錦である前記＜１３＞に記載の方法である。
＜１５＞ メタノール資化性細菌の細胞壁含有物を含むことを特徴とする穀物類の収量増加用組成物である。
＜１６＞ 前記メタノール資化性細菌が、受託番号ＮＩＴＥ Ｐ−０２６２８のメチロバクテリウム エスピー（Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ ｓｐ．）Ｒｓｔ株である前記＜１５＞に記載の組成物である。
＜１７＞ 前記メタノール資化性細菌の細胞壁含有物が、死菌体及び菌体細胞壁の少なくともいずれかを含む前記＜１５＞から＜１６＞のいずれかに記載の組成物である。
＜１８＞ 受託番号ＮＩＴＥ Ｐ−０２６２８のメチロバクテリウム エスピー（Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ ｓｐ．）Ｒｓｔ株であることを特徴とする微生物である。
The present invention is based on the above knowledge and the like of the present inventors, and means for solving the problems are as follows. That is,
<1> A method for increasing the yield of cereals, comprising inoculating a plant body for cultivating cereals with a cell wall-containing substance of methanol-assimilating bacteria.
<2> A method of cultivating a plant for cultivating cereals, comprising inoculating a cell cultivated plant for cereals with a cell wall containing substance of methanol-assimilating bacteria.
<3> The method according to any one of <1> to <2>, wherein the inoculation is performed between 30 days before heading and 30 days after heading.
<4> The method according to any one of <1> to <3>, wherein the inoculation is performed before and after heading.
<5> The method according to any one of <1> to <4>, wherein the methanol-assimilating bacterium is isolated from a surface of a plant body.
<6> Isolation of the methanol-assimilating bacterium is obtained by washing the methanol-assimilating bacterium from the surface of the plant body or a liquid obtained by culturing the plant body in a liquid medium. The method according to <5>, wherein the method is carried out by inoculating the medium, identifying colonies that have grown, and isolating the methanol-utilizing bacterial colonies.
<7> The method according to any one of <5> to <6>, wherein the plant used for isolation of the methanol-assimilating bacterium is at least one of a stem and a cocoon.
<8> The method according to any one of <1> to <7>, wherein the inoculation is performed by spraying a liquid containing a cell wall-containing material of the methanol-assimilating bacterium onto the plant for cultivation. .
<9> The method according to any one of <1> to <8>, wherein the methanol-assimilating bacterium is a Methylobacterium sp. Rst strain having an accession number of NITE P-02628.
<10> The method according to any one of <1> to <9>, wherein the cell wall-containing substance of the methanol-assimilating bacterium includes at least one of dead cells and cell walls.
<11> The method according to any one of <1> to <10>, wherein the dead cells include crushed cells.
<12> The method according to any one of <1> to <11>, wherein the cereal is rice.
<13> The method according to <12>, wherein the rice is sake rice.
<14> The method according to <13>, wherein the sake rice is white crane Nishiki.
<15> A composition for increasing the yield of cereals, comprising a cell wall-containing substance of methanol-assimilating bacteria.
<16> The composition according to <15>, wherein the methanol-assimilating bacterium is a Methylobacterium sp. Rst strain having an accession number of NITE P-02628.
<17> The composition according to any one of <15> to <16>, wherein the cell wall-containing substance of the methanol-assimilating bacterium includes at least one of dead cells and cell walls.
<18> A microorganism characterized in that it is a Metylobacterium sp. Rst strain having an accession number of NITE P-02628.
本発明によると、従来における前記諸問題を解決し、前記目的を達成することができ、簡易、かつ安全に穀物類の収量を増加させることができる穀物類の収量を増加させる方法及び穀物類の栽培用植物体を栽培する方法、並びに穀物類の収量増加用組成物及び新規微生物を提供することができる。 According to the present invention, it is possible to solve the conventional problems, achieve the object, and increase the yield of cereals which can easily and safely increase the yield of cereals. A method for cultivating a plant for cultivation, a composition for increasing the yield of grains, and a novel microorganism can be provided.
本発明の穀物類の収量を増加させる方法（以下、「穀物類の増収方法」と称することがある。）及び本発明の穀物類の栽培用植物体を栽培する方法（以下、「穀物類の栽培方法」と称することがある。）は、いずれもメタノール資化性細菌の細胞壁含有物を穀物類の栽培用植物体に接種する工程（以下、「接種工程」と称することがある。）を少なくとも含み、必要に応じて更にその他の工程を含む。そのため、以下では、穀物類の増収方法及び穀物類の栽培方法について、まとめて説明する。
また、本発明の微生物は本発明の穀物類の収量増加用組成物に好適に用いることができ、本発明の穀物類の収量増加用組成物は本発明の穀物類の増収方法及び穀物類の栽培方法に好適に用いることができる。そのため、本発明の微生物及び本発明の穀物類の収量増加用組成物についても、本発明の穀物類の増収方法及び穀物類の栽培方法の説明と併せて説明する。
A method for increasing the yield of cereals of the present invention (hereinafter sometimes referred to as “a method for increasing the yield of cereals”) and a method for cultivating plants for cultivating cereals of the present invention (hereinafter referred to as “cereals of cereals”). In some cases, the term “cultivation method” refers to a step of inoculating a plant for cultivating cereals with a cell wall-containing substance of methanol-assimilating bacteria (hereinafter, sometimes referred to as “inoculation step”). Including at least, and further including other steps as necessary. Therefore, in the following, a method for increasing grain yield and a method for cultivating grain will be described together.
In addition, the microorganism of the present invention can be suitably used in the composition for increasing the yield of cereals of the present invention, and the composition for increasing the yield of cereals of the present invention is a method for increasing the yield of cereals of the present invention and It can use suitably for a cultivation method. Therefore, the microorganisms of the present invention and the composition for increasing the yield of cereals of the present invention will be described together with the description of the method for increasing the yield of cereals and the method for cultivating the cereals of the present invention.
（穀物類の増収方法及び穀物類の栽培方法）
前記穀物類の増収方法及び穀物類の栽培方法は、接種工程を含む限り、特に制限はなく、通常の穀物類の栽培方法を目的に応じて適宜選択することができる。
(How to increase grain yield and how to grow grains)
The grain yield increasing method and the grain cultivation method are not particularly limited as long as they include the inoculation step, and a normal grain cultivation method can be appropriately selected according to the purpose.
＜接種工程＞
前記接種工程は、メタノール資化性細菌の細胞壁含有物を穀物類の栽培用植物体に接種する工程である。
<Inoculation process>
The inoculation step is a step of inoculating a plant body for cultivating cereals with a cell wall-containing substance of methanol-assimilating bacteria.
＜＜メタノール資化性細菌の細胞壁含有物＞＞
前記メタノール資化性細菌の細胞壁含有物は、メタノール資化性細菌の細胞壁を少なくとも含み、必要に応じて更にその他の成分を含む。
<< Methanol-utilizing bacteria cell wall content >>
The cell wall-containing material of methanol-assimilating bacteria includes at least the cell wall of methanol-assimilating bacteria, and further includes other components as necessary.
−メタノール資化性細菌−
前記メタノール資化性細菌は、メチロバクテリウム（Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ）属に属する細菌であり、シソやイネ等の植物表面上に生育し、植物由来のメタノールを利用するとともに、植物へはビタミンや植物ホルモンなどを供給し、共生関係にあることが知られている。
-Methanol-utilizing bacteria-
The methanol-assimilating bacterium is a bacterium belonging to the genus Metylobacterium, grows on the surface of plants such as perilla and rice, uses plant-derived methanol, and gives vitamins and plant hormones to plants. It is known that there is a symbiotic relationship.
前記メタノール資化性細菌としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、植物体の表面から単離されたものを用いることができる。 There is no restriction | limiting in particular as said methanol assimilation bacterium, According to the objective, it can select suitably, For example, what was isolated from the surface of the plant body can be used.
前記メタノール資化性細菌の単離の方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、培地の炭素源としてメタノールのみを使用する集積培養などが挙げられる。
前記単離の方法の具体例としては、例えば、前記メタノール資化性細菌を前記植物体の表面から洗い流して得た洗浄液を、培地に接種し、増殖してきたコロニーを同定し、前記メタノール資化性細菌のコロニーを単離することにより行う方法、前記植物体を液体培地に入れて培養して得られた液を、培地に接種し、増殖してきたコロニーを同定し、前記メタノール資化性細菌のコロニーを単離することにより行う方法などが挙げられる。
The method for isolating the methanol-assimilating bacterium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include enrichment culture using only methanol as the carbon source of the medium.
As a specific example of the isolation method, for example, a washing solution obtained by washing the methanol-assimilating bacterium from the surface of the plant body is inoculated on a medium, a colony that has grown is identified, and the methanol assimilation is performed. A method for isolating colonies of bacterial bacteria, inoculating the medium with a liquid obtained by culturing the plant in a liquid medium, identifying the colonies that have proliferated, and the methanol-utilizing bacteria The method performed by isolating these colonies is mentioned.
前記メタノール資化性細菌の単離に用いる植物体の部位としては、メタノール資化性細菌が生育している部位であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、穀物類の茎、籾などが挙げられる。前記部位は、１箇所を用いてもよいし、２箇所以上を用いてもよい。 The plant part used for isolation of the methanol-assimilating bacterium is not particularly limited as long as it is a part where the methanol-assimilating bacterium is growing, and can be appropriately selected according to the purpose. Cereal stalks and straws. One part may be used as the part, or two or more parts may be used.
前記培地の組成、培養の条件としては、特に制限はなく、目的に応じて適宜選択することができる。 There is no restriction | limiting in particular as a composition of the said culture medium and culture conditions, According to the objective, it can select suitably.
前記メタノール資化性細菌の中でも、本発明の新規微生物であるメチロバクテリウム エスピー（Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ ｓｐ．）Ｒｓｔ株が好適に挙げられる。前記Ｒｓｔ株は、独立行政法人製品評価技術基盤機構 特許微生物寄託センター（〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８）に寄託申請し、ＮＩＴＥ Ｐ−０２６２８として、２０１８年２月７日に受託されている。 Among the methanol-assimilating bacteria, a methylobacterium sp. Rst strain, which is a novel microorganism of the present invention, is preferable. The Rst strain was filed with NPE P-02628 in February 2018 as an application for deposit at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture 292-0818). Commissioned on the 7th.
−菌体細胞壁−
前記メタノール資化性細菌の細胞壁を調製する方法としては、特に制限はなく、公知の方法を適宜選択することができる。例えば、培養し、回収した培養菌体を５０ｍＭ ＥＤＴＡと２％ ＳＤＳを含有する水溶液に懸濁し、３７℃程度で６０分間程度インキュベートした後遠心分離し、その上清に３Ｍ酢酸ナトリウムとエタノールを加え混和し、遠心分離を行い、ペレット状の菌体細胞壁を調製する方法などが挙げられる。
前記菌体細胞壁成分の有無を確認する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、フェノール−硫酸法を用い、多糖を指標として確認する方法などが挙げられる。
-Bacterial cell wall-
There is no restriction | limiting in particular as a method of preparing the cell wall of the said methanol assimilation bacterium, A well-known method can be selected suitably. For example, the cultured cells recovered after culturing are suspended in an aqueous solution containing 50 mM EDTA and 2% SDS, incubated at about 37 ° C. for about 60 minutes, centrifuged, and 3 M sodium acetate and ethanol are added to the supernatant. Examples of the method include mixing and centrifuging to prepare a pellet-like cell wall.
The method for confirming the presence or absence of the bacterial cell wall component is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method of confirming a polysaccharide using a phenol-sulfuric acid method as an index. .
前記菌体細胞壁のメタノール資化性細菌の細胞壁含有物における含有量としては、特に制限はなく、植物体への接種量などに応じて適宜選択することができる。 There is no restriction | limiting in particular as content in the cell wall containing substance of the methanol assimilation bacterium of the said cell body wall, According to the inoculation amount etc. to a plant body, it can select suitably.
−その他の成分−
前記メタノール資化性細菌の細胞壁含有物におけるその他の成分としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、賦形剤、乳化剤、湿潤剤、分散剤、可溶化剤、懸濁剤、展着剤、浸透剤、安定剤等の公知の製剤用添加剤などが挙げられる。前記その他の成分は、１種単独で使用してもよいし、２種以上を併用してもよい。また、殺菌剤、殺虫剤、植物生長調節剤などを配合してもよい。
前記その他の成分のメタノール資化性細菌の細胞壁含有物における含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
-Other ingredients-
The other components in the cell-wall-containing product of the methanol-assimilating bacterium are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. For example, excipients, emulsifiers, Known additives for pharmaceutical preparations such as a wetting agent, a dispersing agent, a solubilizer, a suspending agent, a spreading agent, a penetrating agent, and a stabilizer can be used. The said other component may be used individually by 1 type, and may use 2 or more types together. Moreover, you may mix | blend a fungicide, an insecticide, a plant growth regulator, etc.
There is no restriction | limiting in particular as content in the cell wall containing substance of methanol assimilation bacteria of said other component, According to the objective, it can select suitably.
前記メタノール資化性細菌の細胞壁含有物の態様としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記メタノール資化性細菌の生菌体を含む態様、前記メタノール資化性細菌の死菌体を含む態様、前記メタノール資化性細菌の菌体細胞壁を含む態様などが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。これらの中でも、前記メタノール資化性細菌の死菌体及び菌体細胞壁の少なくともいずれかを含む態様が、製剤化や品質管理が容易であり、使用した際の環境安全面でのリスクも少ない点で、好ましい。 The aspect of the cell-wall-containing substance of the methanol-assimilating bacterium is not particularly limited and can be appropriately selected according to the purpose. For example, the aspect containing viable cells of the methanol-assimilating bacterium, Examples include a dead cell of an assimilating bacterium, an embodiment including a cell wall of the methanol-assimilating bacterium, and the like. These may be used individually by 1 type and may use 2 or more types together. Among these, the aspect containing at least one of the dead cells and cell walls of the methanol-assimilating bacterium is easy to formulate and quality control, and has few risks in terms of environmental safety when used. It is preferable.
前記メタノール資化性細菌の生菌体の調製方法としては、特に制限はなく、公知の方法を目的に応じて適宜選択することができ、例えば、培養した菌体を回収する方法などが挙げられる。 The method for preparing viable cells of the methanol-assimilating bacterium is not particularly limited, and a known method can be appropriately selected according to the purpose. Examples thereof include a method for recovering cultured cells. .
前記メタノール資化性細菌の死菌体の調製方法としては、特に制限はなく、公知の方法を目的に応じて適宜選択することができ、例えば、培養し、回収した培養菌体をオートクレーブで滅菌する方法などが挙げられる。前記死菌体は、破砕処理された破砕菌体であってもよい。
前記破砕処理の方法としては、特に制限はなく、公知の方法を目的に応じて適宜選択することができる。前記破砕の程度としても、特に制限はなく、目的に応じて適宜選択することができる。
The method for preparing the dead cell of the methanol-assimilating bacterium is not particularly limited, and a known method can be appropriately selected according to the purpose. For example, the cultured cell body that has been cultured and collected is sterilized by autoclaving. The method of doing is mentioned. The dead cells may be crushed cells that have been crushed.
There is no restriction | limiting in particular as the method of the said crushing process, A well-known method can be suitably selected according to the objective. There is no restriction | limiting in particular also as a grade of the said crushing, According to the objective, it can select suitably.
前記メタノール資化性細菌の菌体細胞壁の調製方法の例は、上述したとおりである。 An example of the method for preparing the cell wall of the methanol-assimilating bacterium is as described above.
前記メタノール資化性細菌の細胞壁含有物の形態としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、粉状、液体状などが挙げられる。
前記メタノール資化性細菌の細胞壁含有物の剤型としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、液剤、乳剤、油剤、水溶液、懸濁液、水和剤、フロアブル、粉剤、微粒剤、粒剤、エアゾール、ペースト剤などが挙げられる。
There is no restriction | limiting in particular as a form of the cell wall containing substance of the said methanol assimilation bacterium, According to the objective, it can select suitably, For example, a powder form, a liquid form, etc. are mentioned.
The dosage form of the cell-containing substance of methanol-assimilating bacteria is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a liquid, an emulsion, an oil, an aqueous solution, a suspension, a wettable powder, Flowable, powder, fine granule, granule, aerosol, paste and the like can be mentioned.
＜＜穀物類の栽培用植物体＞＞
前記穀物類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、イネ科植物、マメ科植物などが挙げられる。
前記イネ科植物の具体例としては、例えば、コムギ、オオムギ、ライムギ、エンバク、ハダカムギ等のムギ類、イネ、モロコシ、トウモロコシ、アワ、キビ、ヒエ、シコクビエ、トウジンビエなどが挙げられる。
前記マメ科植物の具体例としては、例えば、ダイズ、アズキ、リョクトウ、ササゲ、インゲンマメ、ライマメ、ラッカセイ、エンドウ、ソラマメなどが挙げられる。
<< Plant for cereal cultivation >>
There is no restriction | limiting in particular as said cereals, According to the objective, it can select suitably, For example, Gramineae plant, leguminous plant etc. are mentioned.
Specific examples of the grass family include, for example, wheat such as wheat, barley, rye, oat, and humpback, rice, sorghum, corn, millet, millet, millet, millet, pearl millet and the like.
Specific examples of the leguminous plant include soybean, azuki bean, mung bean, cowpea, kidney bean, lima bean, groundnut, pea, and broad bean.
前記穀物類の中でも、本発明の方法は、イネ（米）に好適に用いることができる。 Among the cereals, the method of the present invention can be suitably used for rice (rice).
前記米の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、白鶴錦、山田錦、五百万石、美山錦、雄町、八反、八反錦、吟風、ゆめさんさ、若水、夢の香等の酒米、日本晴、コシヒカリ、ひとめぼれ、ヒノヒカリ、あきたこまち、キヌヒカリ、ななつぼし、はえぬき等の食用米などが挙げられる。これらの中でも、本発明の方法は、酒米に好適に用いることができる。 The type of rice is not particularly limited and may be appropriately selected depending on the purpose. For example, Hakutsuru Nishiki, Yamada Nishiki, Hyakumangoku, Miyama Nishiki, Omachi, Hachiman, Hachimannishiki, Ginfu, Yumesansa, Wakamizu, Yume nocense and other sake rice, Nihonbare, Koshihikari, Hitomebore, Hinohikari, Akitakomachi, Kinuhikari, Nanatsuboshi, Haenuki and other edible rice. Among these, the method of the present invention can be suitably used for sake rice.
前記穀物類の栽培用植物体の態様としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、播種前の種子、発芽又は定植などにより栽培場所で生育するときの通常の態様などが挙げられる。 The aspect of the plant body for cultivating the cereals is not particularly limited and can be appropriately selected according to the purpose. For example, a normal plant when growing in a cultivated place by seeds before seeding, germination or planting, etc. An aspect etc. are mentioned.
＜＜接種＞＞
前記接種の時期としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、播種前の種子の段階、出穂前の段階、出穂後の段階などが挙げられる。前記接種の時期は、１つの時期であってもよいし、２つ以上の時期であってもよい。これらの中でも、出穂前３０日から出穂後３０日の間が好ましく、出穂の前と後の両方で行われることがより好ましく、出穂前１日〜３０日の間と、出穂後１日〜３０日の間とに行われることが更に好ましく、出穂前１５日〜３０日の間と、出穂後５日〜２５日の間とに行われることが特に好ましい。前記好ましい時期に接種を行うと、穀物類の収量をより増加させることができる点で、有利である。
<< Inoculation >>
There is no restriction | limiting in particular as the time of the said inoculation, According to the objective, it can select suitably, For example, the stage of the seed before sowing, the stage before heading, the stage after heading, etc. are mentioned. The time of inoculation may be one time or two or more times. Among these, the period between 30 days before heading and 30 days after heading is preferable, and it is more preferable to carry out both before and after heading, between 1 day to 30 days before heading and 1 day to 30 after heading. More preferably, it is carried out during the day, particularly preferably between 15 and 30 days before heading and between 5 and 25 days after heading. Inoculation at the preferred time is advantageous in that the yield of cereals can be further increased.
前記接種の回数としては、１回であってもよいし、複数回であってもよい。また、接種時期が複数の場合における、各接種時期における接種の回数としても、１回であってもよいし、複数回であってもよい。 The number of inoculations may be one time or multiple times. Moreover, as the frequency | count of inoculation in each inoculation time in the case where there are a plurality of inoculation times, it may be one time or a plurality of times.
前記メタノール資化性細菌の細胞壁含有物の前記穀物類の栽培用植物体への接種量としては、特に制限はなく、目的に応じて適宜選択することができるが、ＯＤ６００換算でＯＤが０．５相当になるように調製したメタノール資化性細菌の細胞壁含有物の栽培用植物体１株あたりの接種量として、１ｍＬ以上が好ましく、２ｍＬ以上がより好ましく、３ｍＬ以上が特に好ましい。前記好ましい接種量であると、穀物類の収量をより増加させることができる点で、有利である。なお、前記接種量は、各接種時において、等量を接種してもよいし、異なる量を接種してもよい。 There is no restriction | limiting in particular as an inoculation amount to the plant body for cultivation of the said grain by the cell wall containing substance of the said methanol assimilation bacterium, Although it can select suitably according to the objective, OD is OD600 conversion. As an inoculation amount per plant plant for cultivation of a cell wall-containing product of methanol-assimilating bacteria prepared to be equivalent to 5, 1 mL or more is preferable, 2 mL or more is more preferable, and 3 mL or more is particularly preferable. The preferable inoculation amount is advantageous in that the yield of cereals can be further increased. In addition, the said inoculation amount may inoculate equal amount at the time of each inoculation, and may inoculate a different amount.
前記接種の方法としては、前記メタノール資化性細菌の細胞壁含有物を前記穀物類の栽培用植物体の少なくとも一部分に接触させ得る限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、噴霧、塗布、浸漬などが挙げられる。これらは、１種単独で行ってもよいし、２種以上を組み合わせて行ってもよい。これらの中でも、前記メタノール資化性細菌の細胞壁含有物を含む液を前記栽培用植物体に噴霧することにより行われることが好ましい。 The method of inoculation is not particularly limited as long as the cell wall-containing substance of the methanol-assimilating bacterium can be brought into contact with at least a part of the plant for cultivating cereals, and can be appropriately selected according to the purpose. For example, spraying, application | coating, immersion, etc. are mentioned. These may be performed singly or in combination of two or more. Among these, it is preferable to perform by spraying the liquid containing the cell wall-containing substance of the methanol-assimilating bacterium onto the plant for cultivation.
前記メタノール資化性細菌の細胞壁含有物を接種する前記穀物類の栽培用植物体の部位としては、特に制限はなく、接種する時期に応じて適宜選択することができ、例えば、種子に接種したり、出穂の前に接種する場合は栽培用植物体の地上部に接種したり、出穂の後に接種する場合は栽培用植物体の地上部に接種したりするなどが挙げられる。本発明によれば、前記メタノール資化性細菌の細胞壁含有物の接種が、根圏まで行き渡らなくても、栽培用植物体の地上部に接種することで、穀物類の増収効果を得ることもできる。そのため、肥料として加えるよりも簡便で、農薬との併用も可能な点で、有利である。 The part of the plant for cultivating the cereals to be inoculated with the cell wall content of the methanol-assimilating bacteria is not particularly limited and can be appropriately selected according to the time of inoculation. Or when inoculating before heading, inoculate the above-ground part of the plant for cultivation, or inoculating above-ground part of the plant for cultivation when inoculating after heading. According to the present invention, even if the inoculation of the cell wall-containing substance of the methanol-assimilating bacterium does not reach the rhizosphere, it is possible to obtain an increase in the yield of cereals by inoculating the above-ground part of the plant for cultivation. it can. Therefore, it is advantageous in that it is simpler than adding as a fertilizer and can be used in combination with agricultural chemicals.
前記接種では、殺菌剤、殺虫剤、植物生長調節剤などを併用してもよい。 In the inoculation, a fungicide, an insecticide, a plant growth regulator and the like may be used in combination.
＜その他の工程＞
前記その他の工程としては、本発明の効果を損なわない限り、特に制限はなく、通常の穀物類の栽培における工程を目的に応じて適宜選択することができる。
前記栽培は、水耕であってもよいし、土耕であってもよい。
<Other processes>
There is no restriction | limiting in particular as said other process, unless the effect of this invention is impaired, The process in cultivation of normal grain can be suitably selected according to the objective.
The cultivation may be hydroponics or soil cultivation.
＜穀物類の収量増加用組成物＞
本発明の穀物類の収量増加用組成物は、前記メタノール資化性細菌の細胞壁含有物を少なくとも含み、必要に応じて更にその他の成分を含む。
<Composition for increasing the yield of cereals>
The composition for increasing the yield of cereals according to the present invention contains at least the cell wall-containing substance of methanol-assimilating bacteria, and further contains other components as necessary.
前記穀物類の収量増加用組成物における前記メタノール資化性細菌の細胞壁含有物の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。前記穀物類の収量増加用組成物は、前記メタノール資化性細菌の細胞壁含有物のみからなるものであってもよい。 There is no restriction | limiting in particular as content of the cell wall containing substance of the said methanol assimilation bacterium in the said composition for the yield increase of cereals, According to the objective, it can select suitably. The composition for increasing the yield of cereals may consist only of the cell wall-containing material of the methanol-assimilating bacteria.
前記穀物類の収量増加用組成物におけるその他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、上記したメタノール資化性細菌の細胞壁含有物におけるその他の成分と同様のものなどが挙げられる。前記穀物類の収量増加用組成物におけるその他の成分の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。 The other components in the composition for increasing the yield of cereals are not particularly limited and can be appropriately selected according to the purpose, and are the same as the other components in the cell-wall-containing product of methanol-assimilating bacteria described above. Things. There is no restriction | limiting in particular as content of the other component in the composition for the yield increase of the said grains, According to the objective, it can select suitably.
以下、製造例及び試験例を示して本発明を説明するが、本発明はこれらの製造例及び試験例に何ら限定されるものではない。 EXAMPLES Hereinafter, although a manufacture example and a test example are shown and this invention is demonstrated, this invention is not limited to these manufacture examples and a test example at all.
（製造例１：メタノール資化性細菌の単離）
イネの茎を試料とし、ＮＭＳ培地（Ｗｈｉｔｔｅｎｂｕｒｙ， Ｒ．， Ｋ． Ｃ． Ｐｈｉｌｌｉｐｓ， ａｎｄ Ｊ． Ｆ． Ｗｉｌｋｉｎｓｏｎ． １９７０． Ｅｎｒｉｃｈｍｅｎｔ， ｉｓｏｌａｔｉｏｎ ａｎｄ ｓｏｍｅ ｐｒｏｐｅｒｔｉｅｓ ｏｆ ｍｅｔｈａｎｅ−ｕｔｉｌｉｚｉｎｇ ｂａｃｔｅｒｉａ． Ｊ． Ｇｅｎ． Ｍｉｃｒｏｂｉｏｌ． ６１：２０５−２１８．）に０．０１％イーストエクストラクトと０．５％メタノールを添加した培地で集積培養を行った。集積培養後の培養液を同組成の平板培地に画線し、生じたコロニーを単離し、メチロバクテリウム エスピー（Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ ｓｐ．）Ｒｓｔ株を得た。
(Production Example 1: Isolation of methanol-utilizing bacteria)
Rice stems were used as samples, and NMS medium (Whitburybury, R., K. C. Phillips, and J. F. Wilkinson. 1970. Enrichment, isolation and some properties of methanol. Utils. 205-218.) Was enriched in a medium supplemented with 0.01% yeast extract and 0.5% methanol. The culture solution after enrichment culture was streaked on a plate medium having the same composition, and the resulting colonies were isolated to obtain a Methylobacterium sp. Strain Rst.
前記メチロバクテリウム エスピー（Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ ｓｐ．）Ｒｓｔ株は、独立行政法人製品評価技術基盤機構 特許微生物寄託センター（〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８）に寄託申請し、ＮＩＴＥ Ｐ−０２６２８として、２０１８年２月７日に受託された。 The Methylobacterium sp. Rst strain was filed for deposit at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818), It was commissioned on February 7, 2018 as NITE P-02628.
（製造例２：メタノール資化性細菌の細胞壁含有物の調製）
＜前培養＞
前記製造例１で得られたメチロバクテリウム エスピーＲｓｔ株を、Ｈｙｐｈｏ最少培地（Ｄｅｌａｎｅｙ Ｎ．Ｆ．， Ｋａｃｚｍａｒｅｋ Ｍ．Ｅ．， Ｗａｒｄ Ｌ．Ｍ．， Ｓｗａｎｓｏｎ Ｐ．Ｋ．， Ｌｅｅ Ｍ．Ｃ． ＆ Ｍａｒｘ Ｃ．Ｊ． Ｄｅｖｅｌｏｐｍｅｎｔ ｏｆ ａｎ ｏｐｔｉｍｉｚｅｄ ｍｅｄｉｕｍ， ｓｔｒａｉｎ ａｎｄ ｈｉｇｈ−ｔｈｒｏｕｇｈｐｕｔ ｃｕｌｔｕｒｉｎｇ ｍｅｔｈｏｄｓ ｆｏｒ Ｍｅｔｈｙｌｏｂａｃｔｅｒｉｕｍ ｅｘｔｏｒｑｕｅｎｓ． ＰＬｏＳ Ｏｎｅ ８， ｅ６２９５７ （２０１３））に０．２％コハク酸ナトリウムを添加した培地５ｍＬを用い、試験管にて、２８℃で４８時間培養した。
(Production Example 2: Preparation of cell wall-containing substance of methanol-assimilating bacteria)
<Pre-culture>
The Methylobacterium sp. Rst strain obtained in Preparation Example 1 was added to Hypho minimal medium (Delaney NF, Kaczmarek ME, Ward LM, Swanson P.K., Lee MC). & Marx C. J. Development of an optimized medium, strain and high-throughput cutting methods for Methylobacterium 6) And cultured at 28 ° C. for 48 hours.
＜本培養＞
Ｈｙｐｈｏ最少培地に０．５％メタノールを添加した培地１００ｍＬに、前記前培養した培養液をＯＤ６００が０．００２となるように加え、５００ｍＬのバッフル付き三角フラスコ（２個）にて、２８℃、１８０ｒｐｍで７２時間培養した。なお、集菌時のＯＤ６００は２．９〜３．３であった。
<Main culture>
To 100 mL of medium with 0.5% methanol added to Hypho's minimal medium, the pre-cultured culture solution was added so that the OD600 was 0.002, and in a 500 mL Erlenmeyer flask (2 pieces) with baffles, The culture was performed at 180 rpm for 72 hours. In addition, OD600 at the time of bacterial collection was 2.9 to 3.3.
＜製造例２−１：生菌体＞
前記本培養を行った後、遠心分離により培養菌体を回収した。その後、滅菌水で２回洗浄し、ペレットをＯＤ６００換算でＯＤが０．５相当になるように下記組成の展着剤にて調製した。
−展着剤−
・ ポリオキシエチレン（９）ノニルフェニルエーテル ０．２ｇ／Ｌ
・ カルボキシメチルセルロースナトリウム １ｇ／Ｌ
※ 超純水にて調製
<Production Example 2-1: Viable Cell>
After performing the main culture, the cultured cells were collected by centrifugation. Thereafter, it was washed twice with sterilized water, and the pellet was prepared with a spreading agent having the following composition so that the OD was equivalent to 0.5 in terms of OD600.
-Spreading agent-
・ Polyoxyethylene (9) nonylphenyl ether 0.2 g / L
・ Sodium carboxymethylcellulose 1g / L
* Prepared with ultrapure water
＜製造例２−２：死菌体＞
前記本培養を行った後、遠心分離により培養菌体を回収した。その後、滅菌水で２回洗浄した。洗浄後の菌体をオートクレーブで１２１℃、２０分間滅菌した。滅菌後のペレットを菌液時のＯＤ６００換算でＯＤが０．５相当になるように前記展着剤にて調製した。
<Production Example 2-2: Dead cells>
After performing the main culture, the cultured cells were collected by centrifugation. Thereafter, it was washed twice with sterilized water. The washed cells were sterilized with an autoclave at 121 ° C. for 20 minutes. The pellet after sterilization was prepared with the above spreading agent so that the OD was equivalent to 0.5 in terms of OD600 at the time of the bacterial solution.
＜製造例２−３：菌体細胞壁＞
前記本培養を行った後、遠心分離により培養菌体を回収した。その後、５０ｍＭ ＮａＣｌ水溶液で２回洗浄した。培養液量と当量（１００ｍＬ）の５０ｍＭ ＥＤＴＡと２％ ＳＤＳを含有する水溶液に懸濁した。５０ｍＬのコーニングチューブ２本に前記懸濁液を分け、３７℃の湯浴で６０分間インキュベートした。８，０００×ｇで５分間遠心し、上清を１０ｍＬずつ５０ｍＬコーニングチューブに分けた。コーニングチューブ１本に対し１ｍＬの３Ｍ酢酸ナトリウムと３０ｍＬのエタノールを加え混和した。−２０℃で一晩保存した。８，０００×ｇで５分間遠心し、ペレット状にし、７０％エタノールで１回洗浄した。ペレットを菌液時のＯＤ６００換算でＯＤが０．５相当になるように前記展着剤にて調製した。
なお、細胞壁成分の有無は、フェノール−硫酸法を用い、多糖を指標として確認した。具体的には、サンプル１００μＬに対して１００μＬの５％フェノールと５００μＬの硫酸を加え混和し、溶液の黄色への変色により、多糖の存在を確認した。
<Production Example 2-3: Cell wall of cells>
After performing the main culture, the cultured cells were collected by centrifugation. Then, it was washed twice with 50 mM NaCl aqueous solution. It was suspended in an aqueous solution containing 50 mM EDTA and 2% SDS in an amount equivalent to the culture solution (100 mL). The suspension was divided into two 50 mL Corning tubes and incubated in a 37 ° C. water bath for 60 minutes. Centrifugation was performed at 8,000 × g for 5 minutes, and 10 mL of the supernatant was divided into 50 mL Corning tubes. 1 mL of 3M sodium acetate and 30 mL of ethanol were added to one Corning tube and mixed. Stored overnight at -20 ° C. The mixture was centrifuged at 8,000 × g for 5 minutes, pelletized, and washed once with 70% ethanol. The pellet was prepared with the above spreading agent so that the OD was equivalent to 0.5 in terms of OD600 at the time of the bacterial solution.
The presence or absence of cell wall components was confirmed by using a phenol-sulfuric acid method and using polysaccharide as an index. Specifically, 100 μL of 5% phenol and 500 μL of sulfuric acid were added to and mixed with 100 μL of the sample, and the presence of the polysaccharide was confirmed by changing the color of the solution to yellow.
（試験例１）
穀物類の一例として酒米の１つである白鶴錦を用い、以下のようにして穀物類の増収を試験した。
(Test Example 1)
As an example of cereals, Hakutsuru Nishiki, one of sake rice, was used to test the increase in cereal yield as follows.
＜試料の散布＞
兵庫県多可町東安田地区の圃場にて、各試験区（下記参照）につき約１１０株の白鶴錦に対して試料を散布した。１回目の散布は８月初旬の穂肥前（出穂の約２０日前）に行い、２回目の散布は１回目の散布の１ヵ月後（出穂の約１０日後）に実施した。１回あたりの散布量は約４００ｍＬであり、噴霧器を用いて稲全体に均一に散布した。
−試験区−
・ 対照散布試験区 ・・・ 展着剤のみ
・ 生菌体散布試験区 ・・・ 製造例２−１で調製した生菌体
・ 死菌体散布試験区 ・・・ 製造例２−２で調製した死菌体
・ 菌体細胞壁散布試験区 ・・・ 製造例２−３で調製した菌体細胞壁
<Spreading sample>
Samples were sprayed on approximately 110 Hakutsuru Nishiki for each test zone (see below) in a field in the Higashi Yasuda area of Takao-cho, Hyogo Prefecture. The first spraying was carried out before Hotei in early August (about 20 days before heading), and the second spraying was carried out one month after the first spraying (about 10 days after heading). The amount of spraying per one time was about 400 mL, and sprayed uniformly over the whole rice using a sprayer.
-Test area-
・ Control spray test area ・ ・ ・ Spreading agent only ・ Viable cell spray test area ・ ・ ・ Live cells prepared in Production Example 2-1 ・ Dead cell spray test area ・ ・ ・ Prepared in Production Example 2-2 Dead cell and fungus cell wall spray test area ... Cell wall prepared in Production Example 2-3
＜各試験区の解析＞
稲刈りは通常時期に行い、各試験区の株数と面積を算出した。圃場にて３週間乾燥した後に脱穀、籾摺り、小型米選機による選別を行い、精玄米重を算出した。結果を表１に示す。
<Analysis of each test section>
Rice harvesting was performed at normal times, and the number and area of each test plot were calculated. After drying in the field for 3 weeks, threshing, mashing and sorting with a small rice sorter were performed to calculate the weight of the refined rice. The results are shown in Table 1.
表１に示されるように、各試料の面積あたりの精玄米重、株あたりの精玄米重を比較したところ、生菌体散布試験区、死菌体散布試験区、及び菌体細胞壁散布試験区は、対照試験区より増加していた。また、菌体細胞壁散布試験区で最も増加したことから、メタノール資化性細菌の菌体成分のうち細胞壁が穀物類の増収に関わっている可能性が示唆された。 As shown in Table 1, when the weight of the purified brown rice per area of each sample and the weight of the purified brown rice per strain were compared, a live cell spray test area, a dead cell spray test area, and a cell wall spray test area Increased from the control plot. In addition, the largest increase in the bacterial cell wall spraying test area suggests that the cell wall may be involved in increased yields of cereals among the bacterial components of methanol-utilizing bacteria.
ＮＩＴＥ Ｐ−０２６２８
NITE P-02628
Claims (11)
A microorganism characterized in that it is a Metylobacterium sp. Rst strain having an accession number of NITE P-02628.
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