JP6653097B2 - Microorganisms with plant protection ability - Google Patents
Microorganisms with plant protection ability Download PDFInfo
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- JP6653097B2 JP6653097B2 JP2015118395A JP2015118395A JP6653097B2 JP 6653097 B2 JP6653097 B2 JP 6653097B2 JP 2015118395 A JP2015118395 A JP 2015118395A JP 2015118395 A JP2015118395 A JP 2015118395A JP 6653097 B2 JP6653097 B2 JP 6653097B2
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、植物保護能力を有する新規微生物に関する。より具体的には、本発明は、特定の塩基配列からなる核酸を含む微生物、当該微生物を含む生物農薬、及び当該微生物を利用した植物の保護方法に関する。 The present invention relates to a novel microorganism having a plant protection ability. More specifically, the present invention relates to a microorganism containing a nucleic acid having a specific base sequence, a biological pesticide containing the microorganism, and a method for protecting a plant using the microorganism.
植物を病害から保護する効果を有する細菌はバイオコントロール細菌と呼ばれており、それらの中には微生物農薬として既に有効利用されている系統がある。微生物農薬による植物の病害防除技術は、化学農薬による防除技術（いわゆる、ケミカルコントロール）と比べて環境への負荷が低いことなど多くの利点を有している。その一方で、生物機能を利用するが故に、化学農薬と比較して効果が持続的に発揮されないことや農薬としての取り扱いが難しいことなど、生物素材ならではの問題点も多く、使用する微生物の生態や表現型に理解を深めつつ、更なる改良を加えることが望まれている。 Bacteria that have the effect of protecting plants from disease are called biocontrol bacteria, and some of them have already been effectively used as microbial pesticides. The disease control technology for plants using a microbial pesticide has many advantages, such as a lower burden on the environment, than a control technology using a chemical pesticide (so-called chemical control). On the other hand, there are many problems unique to biological materials, such as the lack of sustained effects compared to chemical pesticides and the difficulty of handling as pesticides due to the use of biological functions. It is hoped that further improvements will be made while deepening the understanding of phenotypes and phenotypes.
Ｐｓｅｕｄｏｍｏｎａｓ（シュードモナス）属細菌は環境中に広く生息するグラム陰性細菌であり、そのうちのＰｓｅｕｄｏｍｏｎａｓ ｆｌｕｏｒｅｓｃｅｎｓに属する細菌には微生物農薬として利用されている系統がいくつか含まれている。そのような細菌が用いられた微生物農薬としては、セル苗元気（多木化学、特許文献１）、ベジキーパー（登録商標）水和剤（セントラル硝子）等が挙げられる。Ｐ． ｆｌｕｏｒｅｓｃｅｎｓの一部は近年ではＰ． ｐｒｏｔｅｇｅｎｓと称されており、Ｐｆ−５株やＣＨＡ０株等の優れた効果を有する種類も存在している。しかしながら、前述したように効果の不安定性が指摘されることなどから、化学農薬の代替利用が期待されてはいるものの、実際のところは農業現場における普及は進んでいないのが現状である。 Bacteria belonging to the genus Pseudomonas are Pseudomonas fluorescens that widely inhabit the environment, and among the bacteria belonging to Pseudomonas fluorescens, there are several strains used as microbial pesticides. Microbial pesticides using such bacteria include Cell Seed Genki (Taki Kagaku, Patent Document 1), Veggiekeeper (registered trademark) wettable powder (Central Glass), and the like. P. Some fluorescens have recently been described by P.C. Protegens, and there are also types having excellent effects such as Pf-5 strain and CHA0 strain. However, as mentioned above, although the instability of the effect is pointed out, although alternative use of chemical pesticides is expected, in reality, it is not widely used in agricultural fields.
本発明は、上記の問題点等に鑑みてなされたものであり、本発明が解決しようとする課題は、優れた植物保護能力を有しており、且つ実用化において利用価値の高い新規な微生物を提供することにある。また、本発明が解決しようとする課題は、当該微生物を利用した効果的な植物の保護技術を提供することにある。 The present invention has been made in view of the above problems and the like, and an object of the present invention is to provide a novel microorganism having excellent plant protection ability and having high utility value in practical use. Is to provide. Another object of the present invention is to provide an effective plant protection technique using the microorganism.
本発明者はこれまで、バイオコントロール細菌の一つであるＰｓｅｕｄｏｍｏｎａｓ ｐｒｏｔｅｇｅｎｓ ＣＨＡ０株（旧学名Ｐｓｅｕｄｏｍｏｎａｓ ｆｌｕｏｒｅｓｃｅｎｓ ＣＨＡ０株）をモデルとして、植物保護能力に関与する因子を明らかにしてきた。かかる知見を利用して、本発明者は、日本国内の植物根圏から分離したおよそ２８００株のＰｓｅｕｄｏｍｏｎａｓ属細菌をスクリーニングし、既知の抗菌性物質合成遺伝子の特定の組み合わせから、これまでには報告されていない極めて効果の高い新規微生物を単離することに成功した。この新規に得られた微生物を利用して、本発明者は、本発明を完成するに至った。 The present inventors have clarified the factors involved in plant protection ability by using Pseudomonas protegens strain CHA0 (formerly known as Pseudomonas fluorescens CHA0 strain), which is one of the biocontrol bacteria, as a model. Utilizing such knowledge, the present inventors screened about 2,800 strains of the genus Pseudomonas isolated from the plant rhizosphere in Japan, and, based on a specific combination of known antibacterial substance synthesis genes, We have succeeded in isolating novel microorganisms that have not been highly effective. Using this newly obtained microorganism, the present inventors have completed the present invention.
本発明は、好ましくは以下に記載するような態様により行われるが、これに限定されるものではない。
［態様１］配列番号１又は２で表される塩基配列と９５％以上の同一性を有する塩基配列からなる核酸を含む、微生物。
［態様２］シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属細菌である、態様１の微生物。
［態様３］ｐｈｌ遺伝子クラスター及びｈｃｎ遺伝子クラスターを有する、態様１又は２の微生物。
［態様４］ｐｌｔ遺伝子クラスター及びｐｒｎ遺伝子クラスターを有さない、態様１〜３のいずれか１の微生物。
［態様５］ｒｚｘ類縁体遺伝子クラスターをさらに有する、態様１〜４のいずれか１の微生物。
［態様６］シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）ｓｐ． Ｏｓ１７株（受領番号ＮＩＴＥ ＡＰ−０２０５３）又はシュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）ｓｐ． Ｓｔ２９株（受領番号ＮＩＴＥ ＡＰ−０２０５４）である、態様１の微生物。
［態様７］態様１〜６のいずれか１の微生物を含む、生物農薬。
［態様８］植物保護剤である、態様７の生物農薬。
［態様９］ピシウム（Ｐｙｔｈｉｕｍ）属菌又はフザリウム（Ｆｕｓａｒｉｕｍ）属菌に関連する植物病に対する植物保護剤である、態様８の生物農薬。
［態様１０］態様１〜６のいずれか１の微生物を植物に接触させる工程を含む、植物の保護方法。
The present invention is preferably carried out in the following manner, but is not limited thereto.
[Aspect 1] A microorganism comprising a nucleic acid comprising a nucleotide sequence having 95% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 or 2.
[Aspect 2] The microorganism of Aspect 1, which is a bacterium belonging to the genus Pseudomonas.
[Aspect 3] The microorganism according to aspect 1 or 2, which has a phl gene cluster and an hcn gene cluster.
[Aspect 4] The microorganism according to any one of Aspects 1 to 3, which does not have a plt gene cluster and a prn gene cluster.
[Aspect 5] The microorganism according to any one of Aspects 1 to 4, further comprising an rzx analog gene cluster.
[Aspect 6] Pseudomonas sp. Os17 strain (Accession number NITE AP-02053) or Pseudomonas sp. The microorganism of aspect 1, which is strain St29 (Accession number NITE AP-02054).
[Aspect 7] A biological pesticide comprising the microorganism of any one of Aspects 1 to 6.
[Aspect 8] The biological pesticide according to Aspect 7, which is a plant protective agent.
[Aspect 9] The biological pesticide of Aspect 8, which is a plant protectant against a plant disease associated with a bacterium belonging to the genus Pythium or a genus Fusarium.
[Aspect 10] A method for protecting a plant, comprising a step of contacting the microorganism of any one of Aspects 1 to 6 with a plant.
本発明によって、極めて優れた植物保護能力を有する微生物を提供することができる。また、その優れた植物保護能力を利用して、従前の微生物農薬と同等、或いはそれよりも優れた微生物農薬を提供することができる。さらに、本発明の微生物を利用することによって、効果的な植物の保護方法を提供することもできる。 According to the present invention, a microorganism having extremely excellent plant protection ability can be provided. In addition, by utilizing the excellent plant protection ability, it is possible to provide a microbial pesticide which is equivalent to or superior to the conventional microbial pesticide. Furthermore, by using the microorganism of the present invention, an effective method for protecting plants can be provided.
本発明の微生物は、日本国内の植物根圏から得られたことから、日本国内での実用化が可能である。そのため、本発明の微生物を用いた生物農薬及び植物の保護方法も、日本国内での利用が十分に可能である。 Since the microorganism of the present invention was obtained from the plant rhizosphere in Japan, it can be put to practical use in Japan. Therefore, the method for protecting biological pesticides and plants using the microorganism of the present invention can be sufficiently used in Japan.
以下に、本発明について詳述するが、本発明はこれらに限定されるものではない。本明細書で特段に定義されない限り、本発明に関連して用いられる科学用語及び技術用語は、当業者によって一般に理解される意味を有するものである。 Hereinafter, the present invention will be described in detail, but the present invention is not limited thereto. Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.
（１）本発明の微生物
本発明は、新規な微生物を提供するものであり、本発明の微生物は、配列番号１又は２で表される塩基配列と９５％以上の同一性を有する塩基配列からなる核酸を含むことを特徴とする。
(1) Microorganism of the Present Invention The present invention provides a novel microorganism. The microorganism of the present invention comprises a base sequence having 95% or more identity with the base sequence represented by SEQ ID NO: 1 or 2. Characterized by containing a nucleic acid.
本明細書において核酸とは、ヌクレオチドがリン酸エステル結合で連結した高分子を意味し、ポリヌクレオチド及びオリゴヌクレオチドの用語と互換可能に使用される。核酸の構造は、１本鎖及び２本鎖のいずれであってもよく、好ましくは２本鎖である。核酸には、デオキシリボ核酸（ＤＮＡ）、リボ核酸（ＲＮＡ）及びこれらの混成物（例えば、ＤＮＡ−ＲＮＡのハイブリッド２本鎖、ＤＮＡ及びＲＮＡが１本鎖に連結されたキメラ核酸）が含まれる。本発明における核酸は、好ましくはデオキシリボ核酸（ＤＮＡ）である。核酸の構成単位には、主としてアデニン（Ａ）、グアニン（Ｇ）等のプリン塩基及びチミン（Ｔ）、シトシン（Ｃ）、ウラシル（Ｕ）等のピリミジン塩基が含まれ、これらの修飾物も含まれる。 As used herein, the term “nucleic acid” refers to a macromolecule in which nucleotides are linked by a phosphate bond, and is used interchangeably with the terms polynucleotide and oligonucleotide. The structure of the nucleic acid may be either single-stranded or double-stranded, and is preferably double-stranded. The nucleic acid includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and a mixture thereof (for example, a hybrid double-stranded DNA-RNA, a chimeric nucleic acid in which DNA and RNA are linked to a single strand). The nucleic acid in the present invention is preferably deoxyribonucleic acid (DNA). Structural units of nucleic acids mainly include purine bases such as adenine (A) and guanine (G) and pyrimidine bases such as thymine (T), cytosine (C) and uracil (U), and modified products thereof. It is.
本発明における核酸は、配列番号１又は２で表される塩基配列と９５％以上の同一性を有する塩基配列を含むことを特徴とし、その配列同一性は、好ましくは９６％以上、９７％以上、９８％以上、又は９９％以上である。本発明における核酸は、好ましくは配列番号１で表される塩基配列と９５％以上（好ましくは９６％以上、９７％以上、９８％以上、又は９９％以上）の同一性を有する塩基配列を含む。なお、配列番号１で表される塩基配列と配列番号２で表される塩基配列との間の同一性は９８．８１％（９８．５％以上）である。 The nucleic acid of the present invention is characterized by containing a base sequence having 95% or more identity with the base sequence represented by SEQ ID NO: 1 or 2, and the sequence identity is preferably 96% or more, and 97% or more. , 98% or more, or 99% or more. The nucleic acid of the present invention preferably contains a base sequence having 95% or more (preferably 96% or more, 97% or more, 98% or more, or 99% or more) identity with the base sequence represented by SEQ ID NO: 1. . The identity between the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 is 98.81% (98.5% or more).
本明細書において塩基配列の同一性とは、対象とする２つの核酸間の塩基配列の同一性をいい、当該技術分野において公知の数学的アルゴリズムを用いて作成された塩基配列の最適なアラインメントにおいて一致する塩基の割合（％）によって表される。塩基配列の同一性は、視覚的検査及び数学的計算により決定することができる。また、コンピュータープログラムを用いて同一性を決定することもできる。配列比較コンピュータープログラムとしては、当業者に周知のホモロジー検索プログラム（例えば、ＢＬＡＳＴ、ＦＡＳＴＡ）や配列整列プログラム（例えば、ＣｌｕｓｔａｌＷ））、あるいは遺伝情報処理ソフトウェア（例えば、ＧＥＮＥＴＹＸ（登録商標））などを用いることができる。本明細書における塩基配列の同一性は、具体的には、ＪＳｐｉｅｃｉｅｓのウェブサイト（http://imedea.uib-csic.es/jspecies/）で公開されている解析プログラム（ＢＬＡＳＴソフトウェアに基づいたＪＳｐｉｅｃｉｅｓプログラム（Richter, M., and Rossello-Mora, R. 2009. Proc. Natl. Acad. Sci. U.S.A. 106:19126-19131.））を用いて、デフォルトの設定条件で求めることができる。 As used herein, the nucleotide sequence identity refers to the identity of a nucleotide sequence between two nucleic acids of interest, and in an optimal alignment of a nucleotide sequence created using a mathematical algorithm known in the art. It is represented by the percentage of matching bases. Nucleotide sequence identity can be determined by visual inspection and mathematical calculation. The identity can also be determined using a computer program. As a sequence comparison computer program, a homology search program (for example, BLAST, FASTA) or a sequence alignment program (for example, ClustalW) or a genetic information processing software (for example, GENETYX (registered trademark)) known to those skilled in the art is used. be able to. Specifically, the identity of the nucleotide sequence in the present specification can be determined by the analysis program (JSpies based on BLAST software) published on the website of JSpies (http://imedea.uib-csic.es/jspecies/). Using a program (Richter, M., and Rossello-Mora, R. 2009. Proc. Natl. Acad. Sci. USA 106: 19126-19131.)), It can be obtained under default setting conditions.
本発明の微生物には、細菌、真菌（酵母、糸状菌等）等のあらゆる微生物が含まれる。本発明の微生物は、好ましくは細菌であり、より好ましくはシュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属細菌である。シュードモナス属細菌としては、シュードモナス フルオレッセンス（Ｐｓｅｕｄｏｍｏｎａｓ ｆｌｕｏｒｅｓｃｅｎｓ）、シュードモナス プロテゲンス（Ｐｓｅｕｄｏｍｏｎａｓ ｐｒｏｔｅｇｅｎｓ）、シュードモナス シリンゲ（Ｐｓｅｕｄｏｍｏｎａｓ ｓｙｒｉｎｇａｅ）、シュードモナス クロロラフィス（Ｐｓｅｕｄｏｍｏｎａｓ ｃｈｌｏｒｏｒａｐｈｉｓ）、シュードモナス シンキサンタ（Ｐｓｅｕｄｏｍｏｎａｓ ｓｙｎｘａｎｔｈａ）、又はシュードモナス ブラシカセアルム（Ｐｓｅｕｄｏｍｏｎａｓ ｂｒａｓｓｉｃａｃｅａｒｕｍ）等が挙げられる。本発明の微生物は、既存の種類に属さない新種のシュードモナス属細菌であってもよい。 The microorganism of the present invention includes all microorganisms such as bacteria, fungi (yeast, filamentous fungi, etc.). The microorganism of the present invention is preferably a bacterium, more preferably a bacterium of the genus Pseudomonas. The bacteria belonging to the genus Pseudomonas, Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas Purotegensu (Pseudomonas protegens), Pseudomonas syringae (Pseudomonas syringae), Pseudomonas chlororaphis (Pseudomonas chlororaphis), Pseudomonas Shinki Santa (Pseudomonas synxantha), or Pseudomonas brush hank alum ( Pseudomonas brassicaearum) and the like. The microorganism of the present invention may be a new species of Pseudomonas bacteria that does not belong to existing types.
本発明における核酸には、好ましくはｐｈｌ遺伝子クラスター及びｈｃｎ遺伝子クラスターが含まれる。ｐｈｌ遺伝子クラスターは、２，４−ジアセチルフロログルシノール（２，４−Ｄｉａｃｅｔｙｌｐｈｌｏｒｏｇｌｕｃｉｎｏｌ（ＤＡＰＧ））の生合成遺伝子ｐｈｌを含む遺伝子クラスターである。また、ｈｃｎ遺伝子クラスターは、シアン化水素（ｈｙｄｒｏｇｅｎ ｃｙａｎｉｄｅ（ＨＣＮ））の生合成遺伝子ｈｃｎを含む遺伝子クラスターである。当該核酸はまた、ａｐｒＡ遺伝子クラスターを含むこともできる。ａｐｒＡ遺伝子クラスターは、プロテアーゼＡｐｒＡの生合成遺伝子ａｐｒＡを含む遺伝子クラスターである。ｐｈｌ遺伝子クラスター、ｈｃｎ遺伝子クラスター及びａｐｒＡ遺伝子クラスターは、例えば、配列番号１で表される塩基配列と配列番号２で表される塩基配列とに照らし合わせると下記の通り示される。 The nucleic acids according to the present invention preferably include the phl gene cluster and the hcn gene cluster. The phl gene cluster is a gene cluster containing the biosynthetic gene phl of 2,4-diacetylphloroglucinol (DAPG). The hcn gene cluster is a gene cluster containing the biosynthetic gene hcn of hydrogen cyanide (HCN). The nucleic acid can also include the aprA gene cluster. The aprA gene cluster is a gene cluster containing the biosynthetic gene aprA of the protease AprA. The phl gene cluster, the hcn gene cluster, and the aprA gene cluster are shown as follows, for example, in light of the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2.
また、本発明における核酸には、ｐｌｔ遺伝子クラスター及びｐｒｎ遺伝子クラスターが含まれないことが好ましい。ｐｌｔ遺伝子クラスターは、ピオルテオリン（ｐｙｏｌｕｔｅｏｒｉｎ）の生合成遺伝子ｐｌｔを含む遺伝子クラスターである。また、ｐｒｎ遺伝子クラスターは、ピロールニトリン（ｐｙｒｒｏｌｎｉｔｒｉｎ）の生合成遺伝子ｐｒｎを含む遺伝子クラスターである。 Further, it is preferable that the nucleic acid in the present invention does not include the plt gene cluster and the prn gene cluster. The plt gene cluster is a gene cluster including the biosynthetic gene plt of pyroluteolin. Further, the prn gene cluster is a gene cluster including a biosynthetic gene prn of pyrrolnitrin.
また、本発明における核酸には、好ましくはｒｚｘ類縁体遺伝子クラスターがさらに含まれる。ｒｚｘ類縁体遺伝子クラスターは、リゾキシン（ｒｈｉｚｏｘｉｎ）類縁体の生合成遺伝子ｒｚｘを含む遺伝子クラスターである。ｒｚｘ類縁体遺伝子クラスターは、例えば、配列番号１で表される塩基配列に照らし合わせると下記の通り示される。
本発明の微生物は、上述の通り、好ましくはシュードモナス属細菌であり、より好ましくは、新規に単離されたシュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）ｓｐ． Ｏｓ１７株又はシュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）ｓｐ． Ｓｔ２９株であり、最も好ましくはＰｓｅｕｄｏｍｏｎａｓ ｓｐ． Ｏｓ１７株である。Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ． Ｏｓ１７株及びＰｓｅｕｄｏｍｏｎａｓ ｓｐ． Ｓｔ２９株は、独立行政法人製品評価技術基盤機構（Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅ ｏｆ Ｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ｅｖａｌｕａｔｉｏｎ（ＮＩＴＥ）） バイオテクノロジーセンター 特許微生物寄託センター（ＮＰＭＤ） 日本国千葉県木更津市かずさ鎌足２−５−８において、それぞれ受託番号ＮＩＴＥ Ｐ−０２０５３及び受託番号ＮＩＴＥ Ｐ−０２０５４として寄託されている（受託日：２０１５年５月２０日）。なお、配列番号１で表される塩基配列はＰｓｅｕｄｏｍｏｎａｓ ｓｐ． Ｏｓ１７株の全ゲノム配列に相当し、配列番号２で表される塩基配列はＰｓｅｕｄｏｍｏｎａｓ ｓｐ． Ｓｔ２９株の全ゲノム配列に相当する。 As described above, the microorganism of the present invention is preferably a bacterium of the genus Pseudomonas, and more preferably a newly isolated Pseudomonas sp. Os17 strain or Pseudomonas sp. St29, most preferably Pseudomonas sp. Os17 strain. Pseudomonas sp. Os17 strain and Pseudomonas sp. The St29 strain was obtained from the National Institute of Technology and Evaluation (NITE) Biotechnology Center Patent and Microorganisms Depositary Center (NPMD) at 2-5-8 Kazusa Kamashita, Kisarazu, Chiba, Japan It has been deposited as Accession No. NITE P-02053 and Accession No. NITE P-02054 (Accession date: May 20 , 2015). In addition, the base sequence represented by SEQ ID NO: 1 was obtained from Pseudomonas sp. The nucleotide sequence represented by SEQ ID NO: 2 corresponds to the entire genome sequence of the Os17 strain, and Pseudomonas sp. This corresponds to the entire genome sequence of St29 strain.
本発明の微生物は、液体培地又は固体培地（寒天培地）を用いて培養することができる。かかる液体培地又は固体培地は、本発明の微生物が増殖可能なものであれば特に限定されず、当業者に周知の培地材料を用いて作製することができる。そのような培地材料としては、例えば、ペプトン類（カゼインペプトン、獣肉ペプトン、心筋ペプトン、ゼラチンペプトン、大豆ペプトン等）、エキス類（肉エキス、酵母エキス等）、無機物（塩化ナトリウム、塩化カリウム、炭酸カルシウム、炭酸ナトリウム、硫酸マグネシウム等）、ビタミン類等が挙げられる。本発明の微生物の培地は、微生物の種類等に応じて適宜含有量を調整して自体公知の方法により作製することができる。また、本発明の微生物の培地には、既に調製済みの市販の液体培地又は固体培地を用いることもできる。本発明の微生物の培養に関する条件（温度、湿度、時間等）も当業者の周知技術に基づいて適宜設定することができる。 The microorganism of the present invention can be cultured using a liquid medium or a solid medium (agar medium). Such a liquid medium or solid medium is not particularly limited as long as the microorganism of the present invention can grow, and can be prepared using a medium material known to those skilled in the art. Examples of such medium materials include peptones (casein peptone, meat peptone, myocardial peptone, gelatin peptone, soy peptone, etc.), extracts (meat extract, yeast extract, etc.), and inorganic substances (sodium chloride, potassium chloride, carbonated Calcium, sodium carbonate, magnesium sulfate, etc.) and vitamins. The medium of the microorganism of the present invention can be prepared by a method known per se by appropriately adjusting the content according to the type of the microorganism and the like. Further, as the medium of the microorganism of the present invention, a commercially available liquid medium or solid medium that has already been prepared can also be used. The conditions (temperature, humidity, time, etc.) for culturing the microorganism of the present invention can also be appropriately set based on well-known techniques of those skilled in the art.
本発明の微生物の培地の具体例としては、液体培地としてＬＢ培地、ＮＹＢ培地等が挙げられ、固体培地としてＬＢ寒天培地、ＮＡ培地等が挙げられる。また、本発明の微生物は、通常１０〜４０℃の範囲、好ましくは２５〜３０℃の範囲で培養することができ、その培養時間は、通常１２時間〜２週間、好ましくは１〜２日間である。 Specific examples of the culture medium of the microorganism of the present invention include LB medium and NYB medium as a liquid medium, and LB agar medium and NA medium as a solid medium. The microorganism of the present invention can be cultured usually in the range of 10 to 40 ° C., preferably in the range of 25 to 30 ° C., and the culturing time is usually 12 hours to 2 weeks, preferably 1 to 2 days. is there.
（２）生物農薬
本発明はまた、上記（１）で説明した本発明の微生物を含む生物農薬を提供することができる。本明細書において生物農薬とは、生物を利用することにより得られる農薬を意味する。本発明の生物農薬は、本発明の微生物を利用することから微生物農薬と称することもできる。
(2) Biological pesticide The present invention can also provide a biological pesticide containing the microorganism of the present invention described in (1) above. In this specification, a biological pesticide means a pesticide obtained by utilizing an organism. The biological pesticide of the present invention can also be referred to as a microbial pesticide because it utilizes the microorganism of the present invention.
本発明の生物農薬は、本発明の微生物の植物保護作用を利用することから、植物保護剤として有用である。本明細書において植物保護とは、病原微生物、害虫及び雑草等の有害生物からの植物の保護を意味する。そのため、前記の植物保護剤は、有害生物からの植物の保護剤、又は有害生物の防除剤と換言することもできる。なお、本発明における植物保護能力については、抗菌性のみならず、植物（例えば、根圏、茎、葉等）への定着性や細菌の運動性等も含めた上でその効果の程度が決定される。そのため、たとえ抗菌性が著しく高いとしても、必ずしも植物保護能力も高くなるとは限らない。 The biological pesticide of the present invention is useful as a plant protective agent because it utilizes the plant protective action of the microorganism of the present invention. As used herein, plant protection refers to protection of plants from pests such as pathogenic microorganisms, pests and weeds. Therefore, the above-mentioned plant protective agent can also be said as an agent for protecting plants from pests or an agent for controlling pests. In the present invention, the degree of its effect is determined based on not only the antibacterial property but also the fixation to plants (eg, rhizosphere, stems, leaves, etc.) and the motility of bacteria. Is done. Therefore, even if the antibacterial property is extremely high, the plant protection ability is not always high.
本発明で対象とされる有害生物は、好ましくは病原微生物であり、より好ましくは糸状菌である。そのような糸状菌としては、例えば、ピシウム（Ｐｙｔｈｉｕｍ）属菌（ピシウム ウルティマム（Ｐｙｔｈｉｕｍ ｕｌｔｉｍｕｍ）、ピシウム アファニデルマタム（Ｐｙｔｈｉｕｍ ａｐｈａｎｉｄｅｒｍａｔｕｍ）、ピシウム メガラカンタム（Ｐｙｔｈｉｕｍ ｍｅｇａｌａｃａｎｔｈｕｍ）等）、フザリウム（Ｆｕｓａｒｉｕｍ）属菌（フザリウム オキシスポラム（Ｆｕｓａｒｉｕｍ ｏｘｙｓｐｏｒｕｍ）、フザリウム グラミネアラム（Ｆｕｓａｒｉｕｍ ｇｒａｍｉｎｅａｒｕｍ）、フザリウム ソラニ（Ｆｕｓａｒｉｕｍ ｓｏｌａｎｉ）等）、リゾクトニア（Ｒｈｉｚｏｃｔｏｎｉａ）属菌、ティエラビオプシス（Ｔｈｉｅｌａｖｉｏｐｓｉｓ）属菌等が挙げられる。本発明では、特に限定されないが、ピシウム属菌又はフザリウム属菌を対象とすることが好ましく、上記の植物保護剤は、ピシウム属菌又はフザリウム属菌に関連する植物病に対する植物保護剤とすることができる。なお、ピシウム属菌の中ではＰ． ｕｌｔｉｍｕｍを対象とすることが好ましく、フザリウム属菌の中ではＦ． ｏｘｙｓｐｏｒｕｍを対象とすることが好ましい。 The pests targeted in the present invention are preferably pathogenic microorganisms, more preferably filamentous fungi. Examples of such filamentous fungi include, for example, Pythium genus (Pythium ultimum), Pythium aphanidermatum (Pythium aphanidermatum), Pythium megalacanthum (Pythium megalacanthum), and the like. (Fusarium oxysporum), Fusarium graminearum, Fusarium solani (Fusarium solani), etc., Rhizoctonia (Rhizoctonia) genus, and the genus Thielabiopsis (Thierviopsis v. Etc.). In the present invention, although it is not particularly limited, it is preferable to target the genus Picium or the genus Fusarium, and the above-mentioned plant protectant is a plant protectant against a plant disease related to the genus Picium or the genus Fusarium. Can be. In addition, among the bacteria belonging to the genus Pysium, P. ultimum, and among Fusarium spp. It is preferable to target oxysporum.
ピシウム属菌に関連する植物病としては、例えば、イチゴ果実腐敗病、インゲンマメ苗立枯病、オーチャードグラス根腐病、オオムギ黄枯病、オクラ苗立枯病、カトレア苗黒腐病、カンラン苗黒腐病、キクピシウム立枯病、キャベツピシウム腐敗病、サツマイモ白腐病、ショウガ根茎腐敗病、スイートピー立枯病、ストック苗腐病、ダイコン腐敗病、タイサイ類ピシウム腐敗病、ダイズ苗立枯病、チューリップ根腐病、テンサイ苗立枯病、デンドロビウム類苗黒腐病、トウモロコシピシウム苗立枯病、トロロアオイ立枯病、ハクサイピシウム腐敗病、ブロッコリーピシウム腐敗病、ベゴニア根腐病、ホウレンソウ立枯病、ラッカセイ立枯病、キュウリ苗立枯病、前記以外の各種農産物の苗立枯病等が挙げられるが、これらに限定されない。 Examples of plant diseases related to the genus Picium include, for example, strawberry fruit rot, common bean seedling wilt, orchardgrass root rot, barley yellow blight, okra seedling wilt, cattleya seedling black rot, perilla seedling black Rot, kinkpisium wilt, cabbage pits rot, sweet potato white rot, ginger rhizome rot, sweet pea wilt, stock seedling rot, radish rot, picinium rot of soybean, soybean wilt, tulip Root rot, sugar beet seedling wilt, dendrobium seedling black rot, corn picycium seedling wilt, trolley mallow, Chinese cabbage rot, broccolipicium rot, begonia root rot, spinach wilt, peanut Damping-off disease, cucumber seedling-killing disease, seedling-killing disease of various agricultural products other than those described above, but are not limited thereto.
フザリウム属菌に関連する植物病としては、例えば、アカエゾマツ苗立枯病、アカエゾマツ床替苗根腐病、アカクローバ萎凋病、アカシア類苗立枯病、アサガオつる割病、アズキ萎凋病、アズキ立枯病、アスター萎凋病、アスナロ苗立枯病、アスパラガス立枯病、アマ立枯病、アルファルファ萎凋病、イチゴ萎黄病、イリス類尻腐病、インゲンマメ萎凋病、ウド萎黄病、ウメ根腐衰弱病、ウルシ苗立枯病、エンドウ萎凋病、オカヒジキ萎凋病、オクラ立枯病、カーネーション萎凋病、カシ類苗立枯病、カブ萎黄病、カラマツ球果褐変病、カラマツ苗立枯病、カラマツ床替苗根腐病、カリフラワー萎黄病、カンキツフザリウム立枯病*、カンバ類苗立枯病、カンラン腐敗病、キク葉枯病、キク萎凋病、キサラギナ萎黄病、キャベツ萎黄病、キュウリつる割病、キョウナ萎凋病、キリ苗立枯病、ギンネム苗立枯病、グアバ苗立枯病、グラジオラス乾腐病、クリ根腐病、クリムソンクローバ萎凋病、クロッカス乾腐病、ケナフ萎凋病、ケヤキ苗立枯病、ゴボウ萎凋病、ゴマ萎凋病、コマツナ萎黄病、コリアンダー株枯病、コンニャク乾腐病、ササゲ萎凋病、サツマイモつる割病、サトイモ萎凋病、サボテン腐敗病、シクラメン萎凋病、ジャガイモ萎凋病、ジャガイモ乾腐病、シュンギク萎凋病、ショウガ立枯病、ジョチュウギク立枯病、シロウリつる割病、スイートピー株枯病、スイカつる割病、スイセン乾腐病、スギ苗立枯病、スギ床替苗根腐病、ストック萎凋病、セルリー萎黄病、ソラマメ立枯病、ダイコン萎黄病、タイサイ類萎黄病、ダイズ赤かび病、ダイズ立枯病、ダグラスモミ苗立枯病、タマネギ乾腐病、チューリップ球根腐敗病、チョロギ立枯病、デルフィニウム茎腐萎凋病、デンドロビウム類腐敗病、トウガラシ萎凋病、トウガンつる割病、トウモロコシフザリウム茎腐病、トドマツ苗立枯病、トドマツ床替苗根腐病、トマト萎凋病、トマト根腐萎凋病、トマト黒すじ実腐病、トルコギキョウ立枯病、ナス半枯病、ナツシロギク萎凋病、ナラ類苗立枯病、ニガウリつる割病、ニセアカシア苗立枯病、ニラ乾腐病、ニンジン萎凋病、ニンジン黒しみ病、ニンニク乾腐病、ネギ萎凋病、ネギ根腐萎凋病、ネムノキ萎凋病、ハス腐敗病、ハスノハカズラ萎凋病、ハゼノキ苗立枯病、パセリー萎凋病、バナナパナマ病、バニラ立枯病、パパイア立枯病、ハボタン萎黄病、ヒノキ苗立枯病、ヒノキ床替苗根腐病、フェニックス類立枯病、ブナ苗立枯病、フリージア球根腐敗病、ヘチマつる割病、ベニバナ萎凋病、ベニバナインゲン萎凋病、ホウレンソウ萎凋病、ポプラ類苗立枯病、マーガレット萎凋病、マクワウリつる割病、マツ類苗立枯病、マツ類床替苗根腐病、ミツバ株枯病、ミブヨモギ立枯病、ミヤコワスレ萎黄病、メボウキ萎凋病、メロン褐色腐敗病、メロンつる割病、モモ赤かび病、モルッカネム苗立枯病、ヤマジノギク萎凋病、ヤマノイモ褐色腐敗病、ユーカリ類苗立枯病、ユウガオつる割病、ユリ類乾腐病、ラッキョウ乾腐病、ルピナス立枯病、ルリトウワタ萎凋病、レタス根腐病レッドトップフザリウム病、ワタ立枯病等が挙げられるが、これらに限定されない。 As plant diseases related to Fusarium spp., For example, Scots pine seedling wilt, red pine spruce bed root rot, red clover wilt, acacia seedling wilt, morning glory vine, adzuki wilt, adzuki wilt Disease, aster wilt, asnalo seedling wilt, asparagus wilt, alfalfa wilt, alfalfa wilt, strawberry yellow wilt, iris butt rot, common bean wilt, plum wilt, weak root rot of Japanese apricot , Sorghum seedling wilt, pea wilt, okaji wilt, okra wilt, carnation wilt, oak seedling wilt, turnip wilt, larch cotyledon, larch seedling wilt, larch bed change Seedling rot, cauliflower wilt, citrus Fusarium wilt *, birch wilt, kanran rot, chrysanthemum leaf wilt, chrysanthemum wilt, yellow wilt, cabbage wilt, cucumber Ripening wilt, Kyonna wilt, Kili seedling wilt, Ginnem seedling wilt, Guava seedling wilt, gladiolus dry rot, chestnut root rot, Crimson clover wilt, crocus dry wilt, kenaf wilt , Zelkova seedling wilt, burdock wilt, sesame wilt, komatsuna wilt, coriander strain wilt, konjac dry rot, cowpea wilt, sweet potato vine, taro wilt, cactus rot, cyclamen wilt, Potato wilt, potato dry wilt, syringa wilt, ginger wilt, jochudai wilt, white vine wilt, sweet pea wilt, watermelon wilt, narcissus wilt, cedar seedling wilt, cedar Bedbed seedling root rot, stock wilt, celery yellow wilt, broad bean wilt, radish yellow wilt, yellow rot, soybean red mold, soybean wilt, Douglas fir seedling Blight, onion dry rot, tulip bulb rot, cricket wilt, delphinium stalk wilt, dendrobium rot, capsicum wilt, poison vine wilt, corn fusarium stalk wilt, Abies sachalinensis wilt, Abies beetle bed changing root rot, tomato wilt, tomato root wilt, tomato black streak wilt, lisianthus wilt, eggplant semi-wilt, feverfew wilt, oak seedling wilt, bitter gourd wilt , Pseudomonas aeruginosa seedling wilt, Chinese chive dry rot, carrot wilt, carrot black spot, garlic dry rot, green onion wilt, green onion root wilt, nemonica wilt, lotus rot, lotus moss wilt, hazenoki seedling Damping-off, Parsley wilt, Banana-Panama, Vanilla wilt, Papaya wilt, Habotan yellow wilt, Cypress seedling wilt, Cypress flooring root rot, Phoenix Damping-off, beech seedling wilt, freesia bulb rot, loofah vine wilt, safflower wilt, safflower bean wilt, spinach wilt, poplar seedling wilt, margaret wilt, Makwauri wilt, pine Seedling blight, pine bed replacement root rot, honeysuckle strain blight, mibomimogi blight, yellow wilt, mebuki wilt, melon brown rot, melon wilt, peach red mold, molcanem seedling Damping-off, Yamaginogi wilt, Yamanoimo brown rot, Eucalyptus seedling wilt, Yugao vine, Lily rot, Rakkyo rot, Lupine wilt, Ruritowata wilt, Lettuce root rot Red Examples include, but are not limited to, top fusarium disease and cotton wilt.
本発明の生物農薬が保護する植物は、特に限定されないが、農産物であることが好ましい。そのような農産物の種類としては、例えば、野菜、穀物、果物、花、及び豆類等が挙げられるが、特にこれらに限定されない。また、その具体例としては、ウリ類（キュウリ、スイカ、カボチャ、ズッキーニ、ヒョウタン、ヘチマ、トウガン、テッポウウリ、ユウガオ、ツルレイシ（ニガウリ、ゴーヤ）、メロン等）、イモ類（ジャガイモ、サツマイモ、サトイモ、ナガイモ、ヤマノイモ等）、根菜類（カブ、ダイコン、ハツカダイコン、ワサビ、ホースラディッシュ、ゴボウ、チョロギ、ショウガ、ニンジン、ラッキョウ、レンコン、ユリ根等）、葉菜類（カラシナ、キャベツ、クレソン、ケール（ハゴロモカンラン）、コマツナ、サイシン、サンチュ、山東菜、シュンギク、シロナ、セリ、セロリ、タアサイ、ダイコンナ（スズシロ）、タカナ、チンゲンサイ、ニラ、菜の花、野沢菜、白菜、パセリ、ハルナ、フダンソウ（スイスチャード）、ホウレンソウ、ミズナ、ミブナ、ミツバ、メキャベツ、ルッコラ、レタス（チシャ）、はなっこりー、ワサビナ等）、果菜類（ナス、ペピーノ、トマト（ミニトマト、フルーツトマト等）、タマリロ、タカノツメ、トウガラシ、シシトウガラシ、ハバネロ、ピーマン（パプリカ、カラーピーマンを含む）、カボチャ、ズッキーニ、キュウリ、ツノニガウリ（キワノ）、シロウリ、ツルレイシ（ゴーヤ、ニガウリ）、トウガン、ヘチマ、ユウガオ、オクラ等）、穀物類（トウモロコシ等）、マメ類（アズキ、インゲンマメ、エンドウ、枝豆（エダマメ）、ササゲ、シカクマメ、ソラマメ、ダイズ、ナタマメ、ラッカセイ、レンズマメ、ゴマ等）、菌茸類（エノキタケ、エリンギ、キクラゲ、キヌガサタケ、シイタケ、シメジ、シロキクラゲ、タモギタケ、チチタケ、ナメコ、ナラタケ、ハタケシメジ、ヒラタケ、ブナシメジ、ブナピー、ポルチーニ、ホンシメジ、マイタケ、マッシュルーム、マツタケ、ヤマブシタケ等）等が挙げられる。 The plant protected by the biological pesticide of the present invention is not particularly limited, but is preferably an agricultural product. Examples of such types of agricultural products include, but are not limited to, vegetables, cereals, fruits, flowers, beans, and the like. Specific examples thereof include cucumber (cucumber, watermelon, pumpkin, zucchini, gourd, luffa, gangan, teppouri, yuugao, crane (bitter gourd, bitter gourd), melon, etc.), potatoes (potato, sweet potato, taro, yam) , Yam), root vegetables (turnip, radish, horseradish, wasabi, horseradish, burdock, cricket, ginger, carrot, raccoon, lotus root, lily root, etc.), leafy vegetables (kalasina, cabbage, watercress, kale (hagoromokankan)), Komatsuna, Saishin, Sanchu, Santosai, Shungiku, Shirona, Seri, Celery, Taasai, Daikonna (Suzushiro), Takana, Chingensai, Chive, Rape blossoms, Nozawana, Chinese cabbage, Parsley, Haruna, Fudansou (Swiss chard), Spinach, Mizuna , Mibuna, honeysuckle, cabbage, arugula, lettuce (chisha), hanakori, wasabina, etc., fruits and vegetables (eggplant, pepino, tomato (mini tomato, fruit tomato etc.), tamarilo, takanotsume, capsicum, shishi pepper, habanero) , Peppers (including paprika and colored peppers), pumpkins, zucchini, cucumber, tunony gourd (kiwano), shirouri, tsurureishi (goya, bitter gourd), gangan, loofah, yugao, okra, etc., grains (corn, etc.), beans (Adzuki bean, kidney beans, peas, green soybeans, cowpea, sword bean, fava beans, soybeans, beans, peanuts, peanuts, lentils, sesame, etc.) Chichitake Pholiota nameko, Armillaria, Hatakeshimeji, Pleurotus, Bunashimeji, Bunapi, porcini, hon-shimeji, maitake, mushrooms, matsutake, Hericium erinaceus, etc.) and the like.
本発明の生物農薬は、好適には種子や幼苗等の生育初期の段階の植物に適用される。また、本発明の生物農薬は、植物の根、葉、茎、枝、幹又は種子等のいずれの部位にも適用可能であるが、好ましくは根（根圏）又は種子に適用される。 The biological pesticide of the present invention is preferably applied to plants at an early growth stage such as seeds and seedlings. The biopesticide of the present invention can be applied to any part of a plant such as roots, leaves, stems, branches, trunks or seeds, but is preferably applied to roots (rhizosphere) or seeds.
本発明の生物農薬は、賦形剤、増粘剤、結合剤、安定化剤、防腐剤、ｐＨ調整剤、着色剤、着香剤等の添加剤を用いて組成物（農薬組成物）とすることができる。各種の添加剤は、生物農薬の技術分野において公知の材料を用いることができ、その配合量は、当業者の周知技術に基づいて適宜調整することができる。また、本発明の生物農薬の形態は、液体、固体、ゲル、ペースト等のいずれの形態であってもよく、使用状況等に応じて適宜設定することができる。 The biopesticide of the present invention is a composition (pesticide composition) using additives such as an excipient, a thickener, a binder, a stabilizer, a preservative, a pH adjuster, a coloring agent, and a flavoring agent. can do. As the various additives, known materials in the technical field of biological pesticides can be used, and the compounding amounts can be appropriately adjusted based on well-known techniques of those skilled in the art. The form of the biological pesticide of the present invention may be any form such as a liquid, a solid, a gel, a paste, and the like, and can be appropriately set according to a use situation and the like.
本発明の生物農薬における微生物の含有量は、例えば、製剤１００ｇ当たり１０４〜１０２０ＣＦＵ、好ましくは１０８〜１０１２ＣＦＵであるが、植物保護作用が得られる限り特に限定されない。当該含有量は、微生物の種類、微生物の性質（耐乾燥性など）、適用する植物の種類、製剤の形態等に応じて適宜設定することができる。本発明の生物農薬には、本発明の微生物をそのまま添加すればよく、その添加方法は特に限定されない。例えば、上述した方法で本発明の微生物を培養し、液体培地であれば遠心分離等で回収し、固体培地であれば形成コロニーを白金耳等で回収して、本発明の生物農薬に添加することができる。或いは、液体中に保存していた状態から自体公知の方法で凍結乾燥処理を行い、本発明の微生物を固体物として本発明の生物農薬に添加することができる。 The content of microorganisms in the biological pesticide of the present invention, for example, 10 4 to 10 20 CFU per formulation 100 g, but preferably 10 8 to 10 12 CFU, not particularly limited as long as the plant protection action is obtained. The content can be appropriately set according to the type of microorganism, the nature of the microorganism (eg, drought resistance), the type of plant to be applied, the form of the preparation, and the like. The microorganism of the present invention may be added as it is to the biological pesticide of the present invention, and the method of addition is not particularly limited. For example, the microorganism of the present invention is cultured by the method described above, and the liquid medium is collected by centrifugation or the like. be able to. Alternatively, the microorganisms of the present invention can be added to the biological pesticide of the present invention as solids by subjecting the microorganisms of the present invention to a freeze-drying treatment by a method known per se from the state stored in the liquid.
（３）植物の保護方法
本発明はまた、上記（１）で説明した本発明の微生物を利用した植物の保護方法を提供することができる。本発明の植物の保護方法は、本発明の微生物又は生物農薬を植物に接触させる工程を含むことを特徴とする。
(3) Plant Protection Method The present invention can also provide a plant protection method using the microorganism of the present invention described in (1) above. The method for protecting a plant of the present invention includes a step of bringing the microorganism or the biological pesticide of the present invention into contact with a plant.
本発明の微生物を植物に接触させる態様は、最終的に本発明の微生物と植物とが接触する限り、如何なる態様も取り得る。その一つの態様は、本発明の微生物を含む液体を目的の植物に接触させることである。当該液体は、目的の植物に対して全体的に散布してもよいし、部分的に塗布を行ってもよい。このとき、例えば粘性が高い液体を利用すれば、植物への定着性が高まり、それにより植物の保護効果が高くなり得る。特に、種子へのコーティング剤として当該液体を利用すれば、種子の植え始めから長時間にわたって植物保護効果を得ることができる。また、本発明の微生物を含む液体は、植物を栽培する土を介して目的の植物に接触させることもできる。例えば、植物が土中にある状態で、その土の上から本発明の微生物を含む液体をかけて、土中にしみ込んだ当該液体を目的の植物に接触させることができる。或いは、本発明の微生物を含む液体と植物を栽培する土とをあらかじめ混合して、その混合物となる土を植物の栽培に用いて本発明の微生物を当該植物に接触させることができる。前述したコーティング剤、及び本発明の微生物を含む液体と植物を栽培する土との混合物は、いずれも本発明の生物農薬として利用可能である。 The embodiment of bringing the microorganism of the present invention into contact with a plant may take any form as long as the microorganism of the present invention finally comes into contact with the plant. One embodiment is to contact a liquid containing the microorganism of the present invention with a target plant. The liquid may be sprayed on the target plant as a whole or may be partially applied. At this time, for example, if a highly viscous liquid is used, the fixing property to the plant may be enhanced, thereby increasing the protection effect of the plant. In particular, if the liquid is used as a coating agent for seeds, a plant protection effect can be obtained for a long time from the beginning of seed planting. In addition, the liquid containing the microorganism of the present invention can be brought into contact with a target plant through soil for cultivating the plant. For example, in a state where the plant is in the soil, a liquid containing the microorganism of the present invention can be applied over the soil, and the liquid that has permeated the soil can be brought into contact with the target plant. Alternatively, a liquid containing the microorganism of the present invention and soil for cultivating a plant can be mixed in advance, and the resulting mixture can be used for plant cultivation to bring the microorganism of the present invention into contact with the plant. The above-mentioned coating agent, and the mixture of the liquid containing the microorganism of the present invention and the soil for cultivating plants can all be used as the biological pesticide of the present invention.
本発明の微生物を植物に接触させる別の一態様は、本発明の微生物又はこれを含む組成物を固体物として利用して、かかる固体物を目的の植物に接触させることである。例えば、当該固体物は粉体であり、これを噴霧して植物に接触させることができる。粉体は、本発明の微生物を含む液体を自体公知の方法により凍結乾燥処理するなどして作製することができる。 Another aspect of bringing the microorganism of the present invention into contact with a plant is to use the microorganism of the present invention or a composition containing the same as a solid and bring the solid into contact with a target plant. For example, the solid is a powder, which can be sprayed and brought into contact with plants. The powder can be prepared by subjecting a liquid containing the microorganism of the present invention to freeze-drying treatment by a method known per se.
本発明の微生物の使用量、接触時間、接触温度等のあらゆる条件は、保護対象とする植物の種類や使用状況等に応じて任意に設定することができ、本発明の微生物の植物保護能力が発揮できる限り特に限定されない。 All conditions such as the amount of the microorganism of the present invention, the contact time, the contact temperature and the like can be arbitrarily set according to the type of plant to be protected and the use status, etc. There is no particular limitation as long as it can be demonstrated.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の技術的範囲を限定するためのものではない。当業者は本明細書の記載に基づいて容易に本発明に修飾及び変更を加えることができ、それらも本発明の技術的範囲に含まれる。 Hereinafter, the present invention will be described specifically with reference to Examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily make modifications and changes to the present invention based on the description in the present specification, and they are also included in the technical scope of the present invention.
（１）新規Pseudomonas属細菌の国内分離株からの探索
P. protegensは普遍的に生息すると言われながら、アジアにおける単離報告例がこれまでになかった。そこで本研究では、P. protegens近縁種の日本国内分離株からの探索を試みた。
(1) Search for new Pseudomonas bacteria from domestic isolates
Although P. protegens is said to be universally inhabited, no isolated cases have ever been reported in Asia. Therefore, in this study, we attempted to search for P. protegens related species from Japanese isolates.
まず、日本国内の圃場から単離されたPseudomonas属細菌を2800株準備し、DAPG（2,4-diacetylphloroglucinol）を生産する細菌を調べた。その方法は、DAPG合成遺伝子であるphlDの特異的プライマーPhl2a（5'-GAGGACGTCGAAGACCACCA-3'（配列番号３）及びPhl2b（5'-ACCGCAGCATCGTGTATGAG-3'（配列番号４））を用いてPCRを行った。phlD陽性単離株はdNBYG培地（Duffy and Defago 1999）上で培養し、DAPGの産生はTLC（Keel et al. 1992）により評価した。その結果、上記2800株の中から48株がスクリーニングされた。 First, 2,800 strains of the genus Pseudomonas isolated from fields in Japan were prepared, and bacteria producing DAPG (2,4-diacetylphloroglucinol) were examined. According to the method, PCR is performed using the specific primers Phl2a (5′-GAGGACGTCTCAGAGACCACCA-3 ′ (SEQ ID NO: 3) and Phl2b (5′-ACCGCAGCATCGTGTATGAG-3 ′ (SEQ ID NO: 4)) of the DAPG synthesis gene phlD. The phlD positive isolates were cultured on dNBYG medium (Duffy and Defago 1999), and DAPG production was evaluated by TLC (Keel et al. 1992) .As a result, 48 out of the above 2800 strains were screened. Was done.
次に、これら48株について、他のバイオコントロール因子をコードする遺伝子の有無をさらにPCRにて確認した。当該遺伝子は、prn、plt、hcnとし、PCRに用いたプライマーは下表の通りとした。なお、PRND1及びPRND2はSouza, J. T., and Raaijmakers, J. M. 2003. FEMS Microbiol. Ecol. 43:21-34.、PltBf及びPltBrはMavrodi, O. V., McSpadden Gardener, B. B., Mavrodi, D. V., Bonsall, R. F., Weller, D. M., and Thomashow, L. S. 2001. Phytopathology 91:35-43.、PM2及びPM7-26RはSvercel, M., Duffy, B., and Defago, G. 2007. J. Microbiol. Methods 70:209-213.をそれぞれ参照することができる。 Next, for these 48 strains, the presence or absence of genes encoding other biocontrol factors was further confirmed by PCR. The genes were prn, plt, and hcn, and the primers used for PCR were as shown in the table below. PRND1 and PRND2 are Souza, JT, and Raaijmakers, JM 2003. FEMS Microbiol. Ecol. 43: 21-34., PltBf and PltBr are Mavrodi, OV, McSpadden Gardener, BB, Mavrodi, DV, Bonsall, RF, Weller. , DM, and Thomashow, LS 2001.Phytopathology 91: 35-43., PM2 and PM7-26R are Svercel, M., Duffy, B., and Defago, G. 2007. J. Microbiol. Methods 70: 209-213. Can be referred to respectively.
その結果、全ての因子がポジティブとなったものは５菌株であり、全因子のうちprn及びpltのみがネガティブとなったものは７菌株（イネ由来５菌株、ジャガイモ由来１菌株、ヨモギ由来１菌株）であった。過去の研究では全ての因子がポジティブとなった菌株を既に入手しており（Cab57株）、これについては全ゲノム解析を実施している。スクリーニングすべき菌株は、本来的には全因子がポジティブである菌株が好ましいように考えられるが、新規なバイオコントロール因子を備えているのではないかという可能性を考えて、prn及びpltのみがネガティブとなった７菌株に着目した。これらについて16S rRNAの塩基配列を調べたところイネ由来の５菌株は全て同じ配列を示したため、この中から１菌株を単離してOs17株とした。また、イネ由来の５菌株の16S rRNAの塩基配列と１塩基しか異ならず、加えて重要性の高い農作物であるという観点からジャガイモ由来の１菌株を選択し、これをSt29株とした。 As a result, 5 strains showed that all factors were positive, and 7 strains showed that only prn and plt were negative among all the factors (5 strains derived from rice, 1 strain derived from potato, 1 strain derived from mugwort). )Met. Previous studies have already obtained strains in which all factors have become positive (Strain Cab57), for which whole genome analysis has been performed. Although strains to be screened are originally considered to be preferably strains in which all factors are positive, only prn and plt are considered in consideration of the possibility that they may have novel biocontrol factors. Attention was paid to the seven negative strains. When the nucleotide sequence of 16S rRNA was examined for these, all five strains derived from rice showed the same sequence, and thus one strain was isolated from these strains and designated as Os17 strain. In addition, one potato-derived strain was selected from the viewpoint that it was only one base different from the base sequence of the 16S rRNA of the five rice-derived strains and that it was a highly important agricultural product, and this was designated as St29 strain.
（２）新規単離菌株の抗菌性及び植物保護能力
（２）−１．抗菌性試験
新たに単離されたOs17株及びSt29株の抗菌作用を調べるため、Bacillus subtilis及び植物病原菌であるFusarium oxysporumに対する抗菌性試験を行った。
(2) Antibacterial activity and plant protection ability of the newly isolated strain (2) -1. Antibacterial test In order to examine the antibacterial activity of the newly isolated strains Os17 and St29, antibacterial tests against Bacillus subtilis and the plant pathogen Fusarium oxysporum were performed.
B. subtilisについては、B. subtilis M168株を使用した。各Pseudomonas属細菌（Os17株、St29株、及びP. protegens Cab57株）をGCM（glycerol-casamino acid medium）寒天培地（培地組成は下記の通り）にスポットした後、B. subtilisを培地表面に重層させ、阻止円の大きさで評価した。その結果、Os17株及びSt29株はいずれもP. protegens Cab57株よりも大きな阻止円を形成し、B. subtilisに対して高い抗菌作用を有することが示された（図１）。 For B. subtilis, B. subtilis strain M168 was used. After spotting each Pseudomonas genus bacterium (Os17 strain, St29 strain, and P. protegens Cab57 strain) on GCM (glycerol-casamino acid medium) agar medium (medium composition is as described below), B. subtilis is overlaid on the medium surface. And evaluated by the size of the stop circle. As a result, both the Os17 strain and the St29 strain formed a larger inhibition circle than the P. protegens Cab57 strain, and were shown to have high antibacterial activity against B. subtilis (FIG. 1).
植物病原菌については、F. oxysporum MAFF103054（農業生物資源ジーンバンク（http://www.gene.affrc.go.jp/index_j.php）を使用した。Os17株とP. protegens Cab57株との比較、及びSt29株とP. protegens Cab57株との比較を行うよう各Pseudomonas属細菌をOD600=1.5に調整し、その20μLをPDA（potato dextrose agar）培地（培地組成は下記の通り）に画線し、F. oxysporumを培地中央に接種し、培養後のF. oxysporumの菌体位置で抗菌性を評価した。その結果、F. oxysporumの菌体はOs17株よりもP. protegens Cab57株に近づいており、また、St29株よりもP. protegens Cab57株に近づいていた（図２）。これらの結果から、Os17株及びSt29株はいずれもP. protegens Cab57株よりもF. oxysporumに対する抗菌性が高いことが示された。 For plant pathogens, we used F. oxysporum MAFF103054 (Agricultural Biological Resources Genebank (http://www.gene.affrc.go.jp/index_j.php). Comparison between Os17 strain and P. protegens Cab57 strain, Each Pseudomonas genus bacterium was adjusted to OD 600 = 1.5 so as to compare the St29 strain with the P. protegens Cab57 strain, and 20 μL thereof was streaked on a PDA (potato dextrose agar) medium (medium composition is as described below). F. oxysporum was inoculated into the center of the medium, and the antimicrobial activity was evaluated at the position of the cells of F. oxysporum after culturing.As a result, the cells of F. oxysporum were closer to P. protegens Cab57 than to Os17 (FIG. 2) From these results, both the Os17 strain and the St29 strain have higher antibacterial activity against F. oxysporum than the P. protegens Cab57 strain. It was shown that.
（２）−２．植物保護能力検定試験
オートクレーブして滅菌水を吸水させたアワを、滅菌シャーレ１枚につき約14gずつ分けて入れ、ピシウム菌（Pythium ultimum、農業生物資源ジーンバンク（http://www.gene.affrc.go.jp/index_j.php）のMAFF番号、MAFF425494）を植菌して、27℃、暗黒下で１週間培養した。滅菌水をしみ込ませた滅菌濾紙の上に、表面滅菌したキュウリの種子（品種：新ときわ地這）を並べ、26℃、24時間、暗黒下に置いて発芽、発根させた。検定に供するPseudomonas属細菌のシングルコロニーをNYB培地（25g/L Nutrient broth (Oxoid)、5g/L Yeast extract (Oxoid)）に植菌し、振とう培養した（30℃、180 rpm、24時間）。Pseudomonas属細菌の培養液を遠心にかけて集菌し、滅菌水に懸濁した。OD600=0.1になるよう、50mL容のファルコンチューブにて滅菌水で調整した。バーミキュライト（（株）白元）に水を加えてなじませた。無処理用のバーミキュライトを取り分けておき、残りのバーミキュライトについて、1Lにつきピシウム菌感染アワを7gの割合で加え、よく撹拌した。バーミキュライトを、100mL容のフラスコに30mLずつ入れた。キュウリの種を３粒ずつ、発根したもののみバーミキュライトの上にまき、その種子の上にPseudomonas属細菌の調整済み水溶液を3mLずつ滴下した。上から無処理のバーミキュライトを5mLずつかぶせ、シリコン栓をして、明期16時間、暗期8時間、25℃にて１週間栽培した。栽培後、キュウリの生残数を数え、地上部と根に分けて重さを測定した。
(2) -2. Plant Protection Ability Test A millet, which has been autoclaved to absorb sterilized water, is divided into approximately 14 g per sterilized petri dish and placed therein, and Pythium ultimum, Genebank Agricultural Biological Resources (http: //www.gene.affrc) .go.jp / index_e.php) was inoculated with MAFF No. MAFF425494) and cultured at 27 ° C. in the dark for one week. Cucumber seeds (cultivar: Shin-Tokiwa Jizo) whose surface was sterilized were arranged on sterile filter paper impregnated with sterile water, germinated and rooted at 26 ° C. for 24 hours in the dark. A single colony of Pseudomonas bacteria to be assayed was inoculated on NYB medium (25 g / L Nutrient broth (Oxoid), 5 g / L Yeast extract (Oxoid)) and cultured with shaking (30 ° C., 180 rpm, 24 hours). . The culture solution of Pseudomonas bacteria was collected by centrifugation, and suspended in sterile water. It was adjusted with sterile water in a 50 mL Falcon tube so that OD 600 = 0.1. Water was added to vermiculite (Shiramoto Co., Ltd.) to make it adapt. Untreated vermiculite was set aside, and the remaining vermiculite was added with 7 g of Picium-infected millet per 1 L and stirred well. Vermiculite was placed in 100 mL flasks in 30 mL increments. Only three roots of cucumber seeds were sown on vermiculite, and 3 mL of an adjusted aqueous solution of Pseudomonas bacteria was dropped on the seeds. Untreated vermiculite was covered from above with 5 mL each, covered with a silicon stopper, and cultivated at 25 ° C for 16 hours in the light period, 8 hours in the dark period, and 1 week. After cultivation, the number of surviving cucumber was counted, and the weight was measured separately for the above-ground part and the root.
結果は下記の通りであり、Os17株は、キュウリの生存率、地上部の生体重、根の生体重の全てにおいて高い値を示し、優れた植物保護能力を有することが明らかとなった。また、St29株の結果も同様に高い数値であり、十分に高い植物保護能力を有することが示された。 The results are as follows. The Os17 strain showed high values in all of the survival rate of the cucumber, the living weight of the aerial part, and the living weight of the root, and it was revealed that the Os17 strain had excellent plant protection ability. In addition, the result of the St29 strain was similarly high, indicating that it had a sufficiently high plant protection ability.
（３）ゲノム解析
新たに単離されたOs17株及びSt29株の機能性及び特性を調べるため、各菌株について全ゲノムの塩基配列決定を行った。配列決定に供するため、Os17株及びSt29株からのゲノムＤＮＡの抽出を、キアゲンGenomic DNA buffer set and genomic-tipを用いて行った。これを用い、解析用の8 kb paired end ライブラリを作製し、Roche GS FLX Titanium による次世代シークエンシング解析を行った。シークエンシング解析後、アセンブル解析を実施し、アセンブル結果の最適化を行うことにより、ギャップ無しの単一コンティグ化、および環状化に成功した。
Os17株及びSt29株のゲノムデータは下記の通りであり、これまでに入手していたCab57株と合わせて下表に示す。
(3) Genome analysis In order to examine the functionality and characteristics of the newly isolated strains Os17 and St29, the nucleotide sequence of the entire genome was determined for each strain. Genomic DNA was extracted from the Os17 strain and the St29 strain using a Qiagen Genomic DNA buffer set and genomic-tip for sequencing. Using this, an 8 kb paired end library was prepared for analysis, and next-generation sequencing analysis was performed using Roche GS FLX Titanium. After the sequencing analysis, the assembly analysis was performed, and the assembly result was optimized, whereby the single contig with no gap and circularization were successfully achieved.
The genomic data of the Os17 strain and the St29 strain are as follows, and are shown in the table below together with the Cab57 strain obtained so far.
また、Os17株及びSt29株に、P. protegensの各種菌株（Cab57、CHA0、Pf-5）、P. fluorescens Pf0-1株、P. fluorescens SBW25株、及びP. fluorescens A506株を加えてゲノムの比較を行った。具体的には、Os17株を基準としてJSpeciesプログラム（Richter and Rossello-Mora 2009）を利用してその他の菌株との配列同一性を調べた。その結果は下記の通りであり、Os17株とSt29株とは極めて近似した全ゲノム配列を有しており、いずれの菌株もその他の従来のPseudomonas属細菌とは異なることがわかった。 In addition, Os17 strain and St29 strain, various strains of P. protegens (Cab57, CHA0, Pf-5), P. fluorescens Pf0-1 strain, P. fluorescens SBW25 strain, and P. fluorescens A506 strain were added to the genome. A comparison was made. Specifically, sequence identity with other strains was examined using the JSpecies program (Richter and Rossello-Mora 2009) based on the Os17 strain. The results are as follows. The Os17 strain and the St29 strain had very similar whole genome sequences, and it was found that each strain was different from other conventional Pseudomonas bacteria.
さらに本研究では、Os17株及びSt29株とその他のP. fluorescensグループに属する菌株との関係を調べるために、REALPHY（Reference sequence Alignment based Phylogeny builder）（Bertels et al. 2014）を利用して全ゲノム配列に基づく系統樹を作成した（図３）。その結果、Os17株及びSt29株は単系統群を形成し、全ゲノム配列として最も近いものはP. protegensであることがわかった。 Furthermore, in this study, in order to investigate the relationship between the Os17 strain and St29 strain and other strains belonging to the P. fluorescens group, the whole genome was analyzed using REALPHY (Reference sequence Alignment based Phylogeny builder) (Bertels et al. 2014). A phylogenetic tree based on the sequence was created (FIG. 3). As a result, it was found that the Os17 strain and the St29 strain formed a single phylogenetic group, and the closest whole genome sequence was P. protegens.
（４）Os17株、St29株及びCab57株のゲノム比較
本研究では、Os17株、St29株及びCab57株の３菌株に関して、BLASTpを利用して（カットオフ値：60%）タンパク質コード配列（CDS）の比較解析を行った（図４）。Os17株に特異的な256個のCDS、及びSt29株に特異的な347個のCDSの中に植物保護能力の差の原因となる遺伝子が含まれるものと考えられる。また、Os17株及びSt29株に特異的な519個のCDSの中に、これらの種特有の植物保護能力の高い遺伝子が含まれると考えられる。
(4) Genomic comparison of Os17 strain, St29 strain and Cab57 strain In this study, protein coding sequence (CDS) was used for three strains of Os17 strain, St29 strain and Cab57 strain by using BLASTp (cut-off value: 60%). Was analyzed (FIG. 4). It is considered that the genes responsible for the difference in plant protection ability are included in 256 CDSs specific to the Os17 strain and 347 CDSs specific to the St29 strain. In addition, it is considered that 519 CDSs specific to the Os17 strain and the St29 strain include genes having high plant protection ability specific to these species.
３菌株間のCDSの比較解析からは、２つの遺伝子クラスター（hcn及びphl）がOs17株及びSt29株に存在しており、一方で他の２つの遺伝子クラスター（plt及びprn）はこれらの菌株には存在していないことが示された。この結果は、スクリーニングに際して調べた内容と一致していた。その他の遺伝子クラスターとしては、aprA遺伝子クラスターがOs17株及びSt29株において存在していた。またOs17株では、Gac/Rsmシグナル伝達経路に関連する遺伝子が存在しており、100%の同一性でSt29株にも当該遺伝子のホモログが保存されていることがわかった。 From a comparative analysis of the CDS among the three strains, two gene clusters (hcn and phl) are present in the Os17 and St29 strains, while the other two gene clusters (plt and prn) are present in these strains. Was not present. This result was consistent with the contents examined at the time of screening. As other gene clusters, the aprA gene cluster was present in the Os17 strain and the St29 strain. In addition, it was found that a gene related to the Gac / Rsm signaling pathway was present in the Os17 strain, and that the homolog of the gene was conserved in the St29 strain with 100% identity.
上記３菌株についてはさらに、LASTZプログラム（Release 1.02.00）を用いてゲノム配列のアラインメントデータを作成し、R Dotplotのファイルフォーマット（version 3.0.1）を用いて当該データからドットプロットを行って菌株間のゲノム配列の比較解析を行った。その結果、Os17株とSt29株との比較からは、主にrzx類縁体遺伝子クラスター（約79kb）及びプロファージの領域の有無について相違していることがわかり、また、Os17株とCab57株との比較から、rzx類縁体遺伝子クラスターの有無について相違していると同時に、両者のゲノム配列の間では２カ所で大きく反転していることがわかった（図５）。これらの結果とゲノム配列の解析とから、Os17株にはrzx類縁体遺伝子クラスターが特異的に存在していることが判明した。 For the above three strains, genome sequence alignment data is further prepared using the LASTZ program (Release 1.02.00), and dot plots are made from the data using the R Dotplot file format (version 3.0.1) to obtain strains. The comparative analysis of the genomic sequence was performed. As a result, a comparison between the Os17 strain and the St29 strain revealed that the rzx analog gene cluster (approximately 79 kb) and the presence or absence of the prophage region were different, and that the Os17 strain and the Cab57 strain were different. From the comparison, it was found that the presence / absence of the rzx analog gene cluster was different, and that the two genomic sequences were greatly inverted at two places (FIG. 5). From these results and the analysis of the genome sequence, it was found that the rzx analog gene cluster was specifically present in the Os17 strain.
（５）rzx類縁体遺伝子クラスターの評価
上記の結果からOs17株におけるrzx類縁体遺伝子クラスターの重要性を調べるために、Os17株のrzxB欠損変異株を作製し、野生株との抗菌作用の比較を行った。rzxB欠損変異株は次の通り作製した。下記のプライマーRzxBUF/RzxBUR及びRzxBDF/RzxBDRを用いて、rzxB遺伝子の6790位〜7580位の790bpの領域及び9400位〜10180位の780bpの領域をそれぞれPCRにより増幅した。なおPCRにおいては、高正確性DNAポリメラーゼKOD Plus（東洋紡）を用い、テンプレートとしてOs17株のゲノムDNAを使用した。
(5) Evaluation of rzx analog gene cluster From the above results, in order to examine the importance of the rzx analog gene cluster in the Os17 strain, an rzxB-deficient mutant of the Os17 strain was prepared and its antibacterial activity was compared with that of a wild-type strain. went. The rzxB deletion mutant was prepared as follows. Using the following primers RzxBUF / RzxBUR and RzxBDF / RzxBDR, a 790 bp region from position 6790 to 7580 and a 780 bp region from position 9400 to 10180 of the rzxB gene were amplified by PCR, respectively. In the PCR, high-accuracy DNA polymerase KOD Plus (Toyobo) was used, and genomic DNA of Os17 strain was used as a template.
増幅させた上記２つの領域をアニールさせて、プライマーRzxBUF/RzxBDRを用いておよそ1.6kbのフラグメントとしてPCR増幅を行った。この増幅させた約1.6kbのフラグメントをpCR-Blunt II-TOPO（Invitrogen）内にクローニングした。インサート領域については、シークエンシング並びにBamHI及びHindIIIでの切り出しにより確認した。シークエンシングを行った後、前記フラグメントをpME3087のBamHI及びHindIII開裂部分にサブクローニングしてpME3087rzxBを作製した。このプラスミドを、E. coli HB101/pME497との三親接合によりE. coli DH5αからOs17株（Pseudomonas sp. Os17）に移動させた。テトラサイクリン感受性細胞の濃縮後に２回目の乗換えを通じてベクターの切除を行い、rzxB欠損変異株を得た。 The amplified two regions were annealed, and PCR amplification was performed as a fragment of about 1.6 kb using primers RzxBUF / RzxBDR. The amplified fragment of about 1.6 kb was cloned into pCR-Blunt II-TOPO (Invitrogen). The insert region was confirmed by sequencing and excision with BamHI and HindIII. After sequencing, the fragment was subcloned into the BamHI and HindIII cleavage sites of pME3087 to create pME3087rzxB. This plasmid was transferred from E. coli DH5α to strain Os17 (Pseudomonas sp. Os17) by triparental conjugation with E. coli HB101 / pME497. After enrichment of the tetracycline-sensitive cells, the vector was excised through a second crossover to obtain an rzxB-deficient mutant.
上記（２）−１と同様の抗菌性試験を実施した。得られたrzxB欠損変異株及び野生株のOs17株をOD600=1.5に調整し、その20μLをプレート培地の端の近くに画線した。抗菌性を評価する植物病原菌として、Pythium ultimum MAFF425494及びFusarium oxysporum MAFF103054を用いた。また、プレート培地は、P. ultimumに対する試験用としてDavis-gly培地とpotato dextrose agar（PDA）培地との１：１混合物を用い、F. oxysporumに対する試験用としてDavis-gly培地を用いた。その結果、P. ultimum及びF. oxysporumの菌体はいずれもrzxB欠損変異株の方に近づいており、rzxB欠損変異株は野生株に対して抗菌作用が低いことがわかった（図６）。この結果から、rzx類縁体遺伝子クラスターの存在が抗菌性を高めるために重要であることが示唆された。 An antibacterial test similar to the above (2) -1 was performed. The obtained rzxB-deficient mutant strain and the wild-type Os17 strain were adjusted to OD 600 = 1.5, and 20 μL thereof was streaked near the edge of the plate medium. Pythium ultimum MAFF425494 and Fusarium oxysporum MAFF103054 were used as plant pathogens for evaluating antibacterial properties. As a plate medium, a 1: 1 mixture of a Davis-gly medium and a potato dextrose agar (PDA) medium was used for testing against P. ultimum, and a Davis-gly medium was used for testing against F. oxysporum. As a result, the cells of P. ultimum and F. oxysporum were all closer to the rzxB-deficient mutant, and it was found that the rzxB-deficient mutant had a lower antibacterial activity than the wild-type strain (FIG. 6). These results suggested that the presence of the rzx analog gene cluster is important for enhancing antibacterial activity.
さらにrzx類縁体遺伝子クラスターの重要性の調査を追求するために、rzxB欠損変異株及び野生株のOs17株について上記（２）−２と同様の植物保護能力検定試験を実施した。また、本試験では同時に根圏定着能を調べるためのコロニーカウントも行った。コロニーカウントのため、Pseudomonas属細菌にテトラサイクリン耐性プラスミドpME6031を導入した。コロニーカウントの方法は次の通りである。重さを量った後の根を、0.9% NaClを15mL入れた50mL容のファルコンチューブに入れ、30秒間ボルテックスし、振とう培養器で30分間振とうした。この溶液の1000倍（10-3）、10000倍（10-4）、100000倍（10-5）希釈液を作製し、各希釈液から100μLずつ、NATc100プレート（40g/L Blood agar base (Oxoid)、5g/L Yeast extract (Oxoid)、100mg/L テトラサイクリン）に播種した。室温、暗黒下にて２日間培養した後、Pseudomonas属細菌のコロニーを数えた。 Further, in order to pursue an investigation of the importance of the rzx analog gene cluster, a plant protection ability test similar to the above (2) -2 was performed on the rzxB-deficient mutant strain and the wild strain Os17 strain. In this test, a colony count for investigating the rhizosphere settlement ability was also performed at the same time. For colony counting, a tetracycline resistance plasmid pME6031 was introduced into Pseudomonas bacteria. The method of colony counting is as follows. The weighed root was placed in a 50 mL Falcon tube containing 15 mL of 0.9% NaCl, vortexed for 30 seconds, and shaken in a shaking incubator for 30 minutes. Prepare 1000-fold (10 -3 ), 10,000-fold (10 -4 ), and 100,000-fold (10 -5 ) dilutions of this solution, and add 100 μL from each dilution to a NATc100 plate (40 g / L Blood agar base (Oxoid) ), 5 g / L Yeast extract (Oxoid), 100 mg / L tetracycline). After culturing at room temperature in the dark for 2 days, colonies of bacteria belonging to the genus Pseudomonas were counted.
結果は下記の通りであり、Os17株の野生株は根へのコロニー形成がrzxB欠損変異株と同程度でありながらも、野生株の方が、キュウリの生存率、地上部の生体重、根の生体重のいずれにおいてもrzxB欠損変異株よりも高い値を示した。この結果から、上記の抗菌性試験での確認と同様に、rzx類縁体遺伝子クラスターの存在が抗菌性を高めるために重要であることが示唆された。 The results are as follows.While the wild strain of the Os17 strain has the same level of colony formation in the root as the rzxB-deficient mutant, the wild strain has a higher cucumber survival rate, aboveground living weight, and root. In all of the fresh weights, the value was higher than that of the rzxB-deficient mutant. These results suggest that the presence of the rzx analog gene cluster is important for enhancing the antibacterial activity, as was the case with the antibacterial activity test described above.
本発明により提供される技術は、植物として農産物を効果的に有害生物から保護できる観点から農業分野において有用である。本発明により提供される微生物は、農薬分野、特に生物農薬の分野において利用することができる。 The technology provided by the present invention is useful in the agricultural field from the viewpoint of effectively protecting agricultural products as plants from pests. The microorganism provided by the present invention can be used in the field of agrochemicals, especially in the field of biological pesticides.
Claims (2)
ピシウム（Ｐｙｔｈｉｕｍ）属菌又はフザリウム（Ｆｕｓａｒｉｕｍ）属菌に関連する植物病に対する植物保護剤である、上記生物農薬。 Pseudomonas sp. Os17 strain (Accession number NITE P-02053) or Pseudomonas sp. A biological pesticide containing St29 strain (Accession No. NITE P-02054) ,
The above biological pesticide, which is a plant protectant against a plant disease associated with a bacterium of the genus Pythium or a genus of Fusarium.
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