JP2022033569A - Plant disease control agent and plant disease control method - Google Patents
Plant disease control agent and plant disease control method Download PDFInfo
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Abstract
Description
本発明は、植物病害防除剤及び植物病害防除方法に関する。 The present invention relates to a plant disease control agent and a plant disease control method.
従来から、病害虫や病害菌による被害を最小限に抑え農作物の生産性を高めるために、効果や経済性の面で優れる化学合成農薬が使われてきた。しかしながら、近年、環境や人畜への影響等の観点から、生物そのものや生物由来の物質を有効成分とする生物農薬への関心が高まっている。 Traditionally, chemically synthesized pesticides, which are excellent in terms of effectiveness and economy, have been used in order to minimize damage caused by pests and pests and increase the productivity of agricultural products. However, in recent years, there has been increasing interest in biopesticides containing organisms themselves or substances derived from organisms as active ingredients from the viewpoint of the effects on the environment and humans and animals.
生物農薬は、天敵昆虫、天敵線虫、微生物（ウイルス、細菌、糸状菌、原生動物等）、生物産生物質（フェロモン、ホルモン、酸性毒素、抽出物等）の４種類に大別される。中でも、微生物農薬は、農作物の病害を防除する微生物を利用した農薬であり、化学合成農薬に比べて環境への負担の軽減できること、さらに、化学合成農薬の耐性菌や耐性害虫の発生抑制効果が期待されている。 Biopesticides are roughly classified into four types: natural enemy insects, natural enemy nematodes, microorganisms (viruses, bacteria, filamentous fungi, protozoa, etc.), and biological substances (pheromones, hormones, acidic toxins, extracts, etc.). Among them, microbial pesticides are pesticides that use microorganisms to control diseases of agricultural crops, can reduce the burden on the environment compared to chemically synthesized pesticides, and have the effect of suppressing the occurrence of resistant bacteria and pests of chemically synthesized pesticides. It is expected.
例えば、バチルス・ズブチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）は、植物病害をもたらす病原菌を直接攻撃する力は有しないが、ある種の病原菌と拮抗するため、野菜類等の灰色かび病、うどんこ病等の防除剤として農薬登録されている。 For example, Bacillus subtilis does not have the ability to directly attack pathogens that cause plant diseases, but because it antagonizes certain pathogens, it is a control agent for gray mold and powdery mildew of vegetables and the like. It is registered as a pesticide.
また、バチルス属細菌を用いた植物病害防除に関する技術として、特許文献４及び５には、バチルス・ズブチリスの特定の株を有効成分として含有する植物病害防除剤が開示されている。
Further, as a technique for controlling plant diseases using Bacillus bacteria,
本発明は、上記事情に鑑みてなされたものであって、植物病害防除作用に優れる新規のバチルス属細菌、前記バチルス属細菌を含有する植物病害防除剤、及び前記植物病害防除剤を用いた植物病害防除方法を提供する。 The present invention has been made in view of the above circumstances, and is a novel bacillus genus bacterium having an excellent plant disease control effect, a plant disease control agent containing the bacillus genus bacterium, and a plant using the plant disease control agent. Provide a disease control method.
すなわち、本発明は、以下の態様を含む。
（１） バチルス・エスピー（Ｂａｃｉｌｌｕｓ ｓｐ．）ＴＴＣＣ２１１１株（ＮＩＴＥ Ｐ－０３２２７）及びバチルス・エスピー（Ｂａｃｉｌｌｕｓ ｓｐ．）ＴＴＣＣ２１２２株（ＮＩＴＥ Ｐ－０３２２８）からなる群より選択される１種以上の枯草菌の菌体又は培養物を有効成分として含有する、植物病害防除剤。
（２） 植物病害が、灰色かび病、緑かび病、青かび病及びうどんこ病からなる群より選択される、請求項１に記載の植物病害防除剤。
（３） 請求項１又は２に記載の植物病害防除剤を対象植物に施用することを含む、植物病害防除方法。
（４） バチルス・エスピー（Ｂａｃｉｌｌｕｓ ｓｐ．）ＴＴＣＣ２１１１株（ＮＩＴＥ Ｐ－０３２２７）。
（５） バチルス・エスピー（Ｂａｃｉｌｌｕｓ ｓｐ．）ＴＴＣＣ２１２２株（ＮＩＴＥ Ｐ－０３２２８）。
That is, the present invention includes the following aspects.
(1) One or more Bacillus subtilis selected from the group consisting of Bacillus sp. TTCC2111 strain (NITE P-03227) and Bacillus sp. TTCC2122 strain (NITE P-03228). A plant disease control agent containing bacterial cells or cultures as an active ingredient.
(2) The plant disease control agent according to
(3) A plant disease control method comprising applying the plant disease control agent according to
(4) Bacillus sp. TTCC2111 strain (NITE P-03227).
(5) Bacillus sp. TTCC2122 strain (NITE P-03228).
上記態様の植物病害防除剤及び植物病害防除方法によれば、植物病害を有効に防除することができる。 According to the plant disease control agent and the plant disease control method of the above-described embodiment, the plant disease can be effectively controlled.
＜植物病害防除剤＞
本発明の一実施形態に係る植物病害防除剤は、バチルス・エスピー（Ｂａｃｉｌｌｕｓ ｓｐ．）ＴＴＣＣ２１１１株（ＮＩＴＥ Ｐ－０３２２７）（以下、単に「ＴＴＣＣ２１１１株」と称する場合がある）及びバチルス・エスピー（Ｂａｃｉｌｌｕｓ ｓｐ．）ＴＴＣＣ２１２２株（ＮＩＴＥ Ｐ－０３２２８）以下、単に「ＴＴＣＣ２１２２株」と称する場合がある）からなる群より選択される１種以上の枯草菌の菌体又は培養物を有効成分として含有する。
<Plant disease control agent>
The plant disease control agent according to one embodiment of the present invention includes Bacillus sp. TTCC2111 strain (NITE P-03227) (hereinafter, may be simply referred to as "TTCC2111 strain") and Bacillus sp. sp.) Contains one or more Bacillus subtilis cells or cultures selected from the group consisting of TTCC2122 strain (NITE P-03228) and below, which may be simply referred to as "TTCC2122 strain") as an active ingredient.
なお、本発明において、「枯草菌の菌体又は培養物を有効成分として含有する」とは、枯草菌の菌体又は培養物を、植物病害を防除する効果を奏する成分として含有することを意味する。 In the present invention, "containing a Bacillus subtilis cell or culture as an active ingredient" means that the Bacillus subtilis cell or culture is contained as an ingredient having an effect of controlling plant diseases. do.
本実施形態の植物病害防除剤によれば、植物に病気を引き起こす幅広い病原菌の増殖や活動を効率的に抑制することができる。
本実施形態の植物病害防除剤は、植物に対する毒性や病原性がなく、植物病害に対して高い防除効果を有する。また、自然界に存在する細菌を有効成分とする微生物農薬であることから安全性が高く、化学農薬と比較して環境に対する影響が小さい。施用しても人畜への危険性や、環境汚染や農作物への残留等の問題もない。よって、消費者に安全で薬害のない農作物を提供することができる。また、化学農薬と比較して標的である病原菌の耐性菌出現率が非常に低い。
According to the plant disease control agent of the present embodiment, it is possible to efficiently suppress the growth and activity of a wide range of pathogens that cause diseases in plants.
The plant disease control agent of the present embodiment is not toxic or pathogenic to plants and has a high control effect on plant diseases. In addition, since it is a microbial pesticide containing bacteria existing in nature as an active ingredient, it is highly safe and has a smaller impact on the environment than chemical pesticides. Even if it is applied, there are no problems such as danger to humans and animals, environmental pollution, and residue on agricultural products. Therefore, it is possible to provide consumers with safe and phytotoxic crops. In addition, the appearance rate of resistant bacteria of the target pathogen is very low as compared with chemical pesticides.
後述する実施例に示すように、バチルス・エスピーＴＴＣＣ２１１１株は、灰色かび病菌（Ｂｏｔｒｙｔｉｓ ｃｉｎｅｒｅａ）に対する生育抑制作用を有する枯草菌のスクリーニングを行い、また、バチルス・エスピーＴＴＣＣ２１２２株は、カンキツ緑かび病菌（Ｐｅｎｉｃｉｌｌｉｕｍ ｄｉｇｉｔａｔｕｍ）に対する生育抑制作用を有する枯草菌のスクリーニングを行い、それぞれ灰色かび病菌に対する生育抑制作用を有する枯草菌、及び、カンキツ緑かび病菌に対する生育抑制作用を有する枯草菌として選抜された。 As shown in Examples described later, the Bacillus sp. TTCC2111 strain was screened for Bacillus subtilis having a growth-suppressing effect on Botrytis cinerea, and the Bacillus sp. TTCC2122 strain was a Botrytis cinerea strain. Bacillus subtilis having a growth-suppressing effect on Botrytis cinerea) was screened and selected as Bacillus subtilis having a growth-suppressing effect on Botrytis cinerea and Bacillus subtilis having a growth-suppressing effect on Botrytis cinerea, respectively.
バチルス・エスピーＴＴＣＣ２１１１株及びバチルス・エスピーＴＴＣＣ２１２２株（以下、総じて「本実施形態の枯草菌」と称する場合がある）は、新規に単離同定された枯草菌であり、植物病害を防除する効果を有する非常に有用な新規枯草菌である。そこで、発明者らは、これらの枯草菌を、２０２０年５月２８日付で、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（日本国千葉県木更津市かずさ鎌足２－５－８）に新規菌株として寄託した。受託番号は、バチルス・エスピーＴＴＣＣ２１１１株がＮＩＴＥ Ｐ－０３２２７、バチルス・エスピーＴＴＣＣ２１２２株がＮＩＴＥ Ｐ－０３２２８である。 The Bacillus SP TTCC2111 strain and the Bacillus SP TTCC2122 strain (hereinafter, may be collectively referred to as "Bacillus subtilis of the present embodiment") are newly isolated and identified Bacillus subtilis and have the effect of controlling plant diseases. It is a very useful novel Bacillus subtilis. Therefore, on May 28, 2020, the inventors transferred these bacilli to the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan). Deposited as a new strain. The accession numbers are NITE P-03227 for Bacillus SP TTCC2111 strains and NITE P-03228 for Bacillus SP TTCC2122 strains.
本実施形態の枯草菌の菌体は、生菌であることが好ましい。なお、ここでいう「生菌」とは、細菌が代謝、分裂、増殖等が可能な状態をいう。そのため、枯草菌における芽胞も発芽力を有する限り、生菌に含まれる。但し、本実施形態の植物病害防除剤は、枯草菌の死菌を含んでいてもよい。 The cells of Bacillus subtilis of the present embodiment are preferably viable bacteria. The term "live bacterium" as used herein means a state in which the bacterium is capable of metabolism, division, proliferation and the like. Therefore, as long as the spores of Bacillus subtilis have germination ability, they are included in the viable bacteria. However, the plant disease control agent of the present embodiment may contain dead Bacillus subtilis.
本実施形態の枯草菌の菌体は、菌末として用いることができる。菌末を調製する方法としては、特に限定されず、例えば、フリーズドライ（凍結乾燥）法、スプレードライ法、ドラムドライ法等の一般的に枯草菌の菌末を調製する方法から適宜選択することができる。生菌の状態で菌末とし得ることから、フリーズドライ法により調製することが好ましい。 The Bacillus subtilis cell of the present embodiment can be used as a bacterial powder. The method for preparing the bacterial powder is not particularly limited, and for example, a method for preparing the bacterial powder of Bacillus subtilis, such as a freeze-drying method, a spray-drying method, and a drum-drying method, may be appropriately selected. Can be done. It is preferable to prepare by the freeze-drying method because it can be used as bacterial powder in a viable state.
本実施形態の枯草菌の培養物は、枯草菌を培地中で培養して得られるものであれば特に限定されず、菌体を培地中で増殖させて得られる培養液であってもよく、培養液の上清（遠心分離処理等により培養液から菌体等の固形成分を除いたもの）であってもよく、培養液の濃縮物であってもよく、培養液に菌体破砕処理等を施したものであってもよく、培養液から菌体内成分を抽出したものであってもよい。すなわち、本実施形態の植物病害防除剤は、有効成分として、本実施形態の枯草菌の代謝物やその調製物（例えば、乾燥物、濃縮物、抽出物等）を含有していてもよい。なお、遠心分離処理、濃縮方法、菌体破砕処理、抽出方法、発酵方法等は、公知の方法から適宜選択して常法により行うことができる。 The culture of the bacillus of the present embodiment is not particularly limited as long as it is obtained by culturing the bacillus in a medium, and may be a culture solution obtained by growing the cells in the medium. The supernatant of the culture medium (the culture medium from which solid components such as cells have been removed by centrifugation or the like) may be used, or the concentrate of the culture medium may be used. It may be the one which has been subjected to the above-mentioned, or the one obtained by extracting the intracellular component from the culture solution. That is, the plant disease control agent of the present embodiment may contain a metabolite of Bacillus subtilis of the present embodiment or a preparation thereof (for example, a dried product, a concentrate, an extract, etc.) as an active ingredient. The centrifugation treatment, concentration method, cell crushing treatment, extraction method, fermentation method and the like can be appropriately selected from known methods and carried out by a conventional method.
本実施形態の枯草菌を培養する培地は、各枯草菌が生育し得る培地であれば、特に限定されるものではなく、枯草菌の培養において一般的に用いられる培地やその改変培地等から適宜選択して用いることができる。具体的には、Ｔｒｙｐｔｉｃ Ｓｏｙ Ａｇａｒ平板培地、Ｐｏｔａｔｏ Ｓｕｃｒｏｓｅ Ｂｒｏｔｈ（ＰＳＢ）培地等が挙げられる。 The medium for culturing Bacillus subtilis of the present embodiment is not particularly limited as long as it is a medium in which each Bacillus subtilis can grow, and is appropriately selected from a medium generally used for culturing Bacillus subtilis, a modified medium thereof, and the like. It can be selected and used. Specific examples thereof include Tryptic Soy Agar plate medium, Potato Sucrose Brost (PSB) medium and the like.
培養形式は、特に限定されるものではなく、培養スケール、植物病害防除剤の剤形等を考慮して適宜決定することができる。例えば、寒天平板培地に塗布して培養してもよく、液体培地中で培養してもよい。液体培地における培養形式として、静置培養、回分培養等が挙げられる。例えば、継代培養の場合には、簡便であるため、寒天平板培地上で培養することや、適当な液体培地中で静置培養、回分培養することが好ましい。 The culture form is not particularly limited, and can be appropriately determined in consideration of the culture scale, the dosage form of the plant disease control agent, and the like. For example, it may be applied to an agar plate medium and cultured, or may be cultured in a liquid medium. Examples of the culture type in the liquid medium include static culture and batch culture. For example, in the case of subculture, for convenience, it is preferable to culture on an agar plate medium, or to perform static culture or batch culture in an appropriate liquid medium.
本実施形態の枯草菌の培養条件は、特に限定されず、枯草菌を培養する場合に一般的に用いられる条件により培養することができる。例えば、培養温度は２０℃以上４０℃以下であることが好ましく、２５℃以上３７℃以下であることがより好ましい。また、培地のｐＨは３．５以上８．０以下であることが好ましく、４．０以上７．０以下であることがより好ましい。その他、本実施形態の枯草菌は、他の枯草菌と同様に好気的条件で培養することが好ましい。 The culture conditions for Bacillus subtilis of the present embodiment are not particularly limited, and Bacillus subtilis can be cultured under the conditions generally used for culturing Bacillus subtilis. For example, the culture temperature is preferably 20 ° C. or higher and 40 ° C. or lower, and more preferably 25 ° C. or higher and 37 ° C. or lower. The pH of the medium is preferably 3.5 or more and 8.0 or less, and more preferably 4.0 or more and 7.0 or less. In addition, the Bacillus subtilis of the present embodiment is preferably cultured under aerobic conditions like other Bacillus subtilis.
本実施形態の植物病害防除剤の有効成分である枯草菌の菌体又は培養物は、ＴＴＣＣ２１１１株及びＴＴＣＣ２１２２株のいずれか１種類の枯草菌のみの菌体又は培養物からなるものであってもよく、これら２種類の枯草菌の菌体又は培養物の混合物であってもよい。中でも、より広範な植物病害への有効性が期待できることから、ＴＴＣＣ２１２２株単独の菌体又は培養物を有効成分とすることが好ましい。 The Bacillus subtilis cell or culture which is the active ingredient of the plant disease control agent of the present embodiment may consist of a Bacillus subtilis cell or culture of only one of the TTCC2111 strain and the TTCC2122 strain. It may be a mixture of cells or cultures of these two types of Bacillus subtilis. Above all, since it can be expected to be effective against a wider range of plant diseases, it is preferable to use a cell or culture of the TTCC2122 strain alone as an active ingredient.
本実施形態の植物病害防除剤は、本実施形態の枯草菌の菌体又は培養物のみからなるものであってもよく、本実施形態の枯草菌の菌体又は培養物による植物病害に対する防除効果を阻害しない程度において、所望の剤形に応じて、その他の物質を更に含有する植物病害防除組成物（以下、単に「本実施形態の組成物」と称する場合がある）とすることができる。 The plant disease control agent of the present embodiment may consist only of the Bacillus subtilis cell or culture of the present embodiment, and the effect of controlling the plant disease by the Bacillus subtilis cell or culture of the present embodiment. A plant disease control composition (hereinafter, may be simply referred to as “composition of the present embodiment”) further containing other substances can be used, depending on the desired dosage form, to the extent that it does not inhibit the above.
その他の物質としては、農薬製剤上許容可能な担体、界面活性剤、分散剤、補助剤、保護剤等が挙げられる。 Examples of other substances include carriers, surfactants, dispersants, auxiliaries, protective agents and the like that are acceptable for pesticide formulations.
「農薬製剤上許容可能な担体」とは、植物病害防除剤の施用を容易にし、枯草菌の生存や植物病原菌に対する拮抗作用又は抑制作用を維持する物質及び／又は植物病害防除剤の作用速度を制御する物質であって、土壌若しくは水質等の環境、及び／又は、動物（特にヒト）に対する有害性がない若しくは低い物質をいう。 The "acceptable carrier for pesticide preparations" is a substance that facilitates the application of a plant disease control agent and maintains the survival of bacillus bacteria and the antagonism or suppression action against phytopathological bacteria and / or the rate of action of the plant disease control agent. A substance that is controlled and has no or low harmfulness to the environment such as soil or water quality and / or to animals (particularly humans).
このような担体として具体的には、例えば、タルク、ベントナイト、カオリン、クレー、珪藻土、ホワイトカーボン、バーミキュライト、消石灰、硫安、珪砂、尿素等の多孔質な固体担体；水、イソプロピルアルコール、メチルナフタレン、キシレン、シクロヘキサノン、アルキレングリコール等の液体担体等が挙げられる。 Specific examples of such carriers include porous solid carriers such as talc, bentonite, kaolin, clay, diatomaceous earth, white carbon, vermiculite, slaked lime, sulphate, silica sand, and urea; water, isopropyl alcohol, methylnaphthalene, and the like. Examples thereof include liquid carriers such as xylene, cyclohexanone, and alkylene glycol.
界面活性剤及び分散剤としては、例えば、ジナフチルメタンスルホン酸塩、アルコール硫酸エステル塩、リグニンスルホン酸塩、アルキルアリールスルホン酸塩、ポリオキシエチレングリコールエーテル、ポリオキシエチレンソルビタンモノアルキレート、ポリオキシエチレンアルキルアリールエーテル等が挙げられる。 Examples of the surfactant and dispersant include dinaphthylmethane sulfonate, alcohol sulfate ester salt, lignin sulfonate, alkylaryl sulfonate, polyoxyethylene glycol ether, polyoxyethylene sorbitan monoalchelate, and polyoxy. Examples thereof include ethylene alkylaryl ether.
補助剤としては、例えば、カルボキシメチルセルロース、ポリエチレングリコール、プロピレングリコール、アラビアゴム、キサンタンガム等が挙げられる。
保護剤としては、例えば、スキムミルク、ｐＨ緩衝剤等が挙げられる。
Examples of the auxiliary agent include carboxymethyl cellulose, polyethylene glycol, propylene glycol, gum arabic, xanthan gum and the like.
Examples of the protective agent include skim milk, a pH buffer, and the like.
本実施形態の組成物は、有効成分の枯草菌に影響しない範囲において、他の薬理作用を有する有効成分、具体的には、除草剤、殺菌剤、殺虫剤、肥料（例えば、尿素、硝酸アンモニウム、過リン酸塩）等を更に含有することもできる。 The composition of the present embodiment is an active ingredient having other pharmacological actions, specifically, a herbicide, a fungicide, an insecticide, a fertilizer (for example, urea, ammonium nitrate, etc.) as long as the active ingredient does not affect Bacillus subtilis. Perphosphate) and the like can also be further contained.
剤形としては、有効成分である枯草菌を生菌の状態で保持できれば、特に限定されず、例えば、液体状態、固体状態又はその組み合わせとすることができる。
液体状態としては、例えば、顆粒水和剤、水和剤、水溶剤、懸濁製剤、乳剤等が挙げられる。液体状態の場合における菌体を懸濁する溶剤としては、例えば、水（滅菌水、脱イオン水、超純水を含む）、生理食塩水、緩衝液（リン酸緩衝液、炭酸緩衝液を含む）、その細菌の培地等が挙げられる。
固体状態としては、例えば、粒剤、粉剤、ゲル剤等が挙げられる。
The dosage form is not particularly limited as long as the active ingredient Bacillus subtilis can be retained in a viable state, and may be, for example, in a liquid state, a solid state, or a combination thereof.
Examples of the liquid state include granule wettable powder, wettable powder, aqueous solvent, suspension preparation, emulsion and the like. Examples of the solvent that suspends the cells in the liquid state include water (including sterile water, deionized water, and ultrapure water), physiological saline, and a buffer solution (phosphate buffer solution, carbon dioxide buffer solution). ), The bacterial medium and the like.
Examples of the solid state include granules, powders, gels and the like.
本実施形態の植物病害防除剤の所定量あたりにおける枯草菌の菌体又は培養物の含有量は、各枯草菌の種類やその組み合わせ、施用対象植物の種類、剤形、及び施用方法等の諸条件によって異なる。通常は、本実施形態の植物病害防除剤を施用する際に枯草菌が植物病原菌に対する拮抗作用又は抑制作用を発揮する上で十分な量を含んでいることが好ましい。この含有量は、当該分野の技術常識の範囲において枯草菌の菌体又は培養物が施用後に施用対象植物の処理対象領域の所定の面積あたりに所望の（存在）量となるように各条件を勘案し、決定すればよい。本実施形態の植物病害防除剤における枯草菌の含有量は、例えば、菌体濃度として１０９ｃｆｕ／ｇ以上１０１１ｃｆｕ／ｇ以下程度の範囲とすることができる。或いは、本実施形態の植物病害防除剤が液体状態である場合に、枯草菌の含有量は、例えば、菌体濃度として、１０６ｃｅｌｌｓ／ｍＬ以上１０１０ｃｅｌｌｓ／ｍＬ以下程度の範囲とすることができる。この場合、必要に応じて施用時に、水、生理食塩水、緩衝液等で２倍以上１０００倍以下に希釈することもできる。 The content of Bacillus subtilis cells or cultures per predetermined amount of the plant disease control agent of the present embodiment includes various types and combinations of Bacillus subtilis, types of plants to be applied, dosage forms, application methods, and the like. It depends on the conditions. Usually, it is preferable that Bacillus subtilis contains a sufficient amount to exert an antagonistic action or an inhibitory action against a plant pathogen when the plant disease control agent of the present embodiment is applied. This content is set to a desired (presence) amount per predetermined area of the treatment target area of the application target plant after the application of Bacillus subtilis cells or culture within the range of common general technical knowledge in the field. You just have to take it into consideration and decide. The content of Bacillus subtilis in the plant disease control agent of the present embodiment can be, for example, in the range of 10 9 cfu / g or more and 10 11 cfu / g or less as the cell concentration. Alternatively, when the plant disease control agent of the present embodiment is in a liquid state, the content of Bacillus subtilis should be in the range of, for example, 10 6 cells / mL or more and 10 10 cells / mL or less as the cell concentration. Can be done. In this case, if necessary, it can be diluted 2-fold or more and 1000-fold or less with water, physiological saline, a buffer solution or the like at the time of application.
＜植物病害防除方法＞
本発明の一実施形態に係る植物病害防除方法は、上述した植物病害防除剤を対象植物に施用すること（以下、「施用工程」と称する場合がある）を含む。
<Plant disease control method>
The plant disease control method according to an embodiment of the present invention includes applying the above-mentioned plant disease control agent to a target plant (hereinafter, may be referred to as "application step").
本明細書において、「対象植物」とは、植物病害防除剤の施用対象となる植物を示す。対象植物は、病原菌の感染によって病害を発病し得る植物であれば、その種類は問わない。被子植物又は裸子植物のいずれであってもよい。被子植物としては、双子葉植物であってもよく、単子葉植物であってもよい。
単子葉植物としては、例えば、イネ科（Ｐｏａｃｅａｅ）植物等が挙げられる。
双子葉植物としては、例えば、ヒルガオ科（Ｃｏｎｖｏｌｖｕｌａｃｅａｅ）、バラ科（Ｒｏｓａｃｅａｅ）植物、セリ科（Ａｐｉａｃｅａｅ）、ナス科（Ｓｏｌａｎａｃｅａｅ）植物、ユリ科（Ｌｉｌｉａｃｅａｅ）、マメ科（Ｆａｂａｃｅａｅ）植物、ウリ科（Ｃｕｃｕｒｂｉｔａｃｅａｅ）植物、アブラナ科（Ｂｒａｓｓｉｃａｃｅａｅ）植物等が挙げられる。
As used herein, the term "target plant" refers to a plant to which a plant disease control agent is applied. The target plant may be of any kind as long as it is a plant that can develop a disease by infection with a pathogen. It may be either angiosperms or gymnosperms. The angiosperms may be dicotyledonous plants or monocotyledonous plants.
Examples of monocotyledonous plants include Poaceae plants and the like.
Examples of the dicotyledonous plants include Convolvulaceae, Rosaceae, Umbelliferae, Solanaceae, Lilyaceae, Fabaceae, and Fabaceae. Examples thereof include Curbicaceae plants and Brassicaceae plants.
具体的な対象植物としては、例えば、エンドウ（Ｐｉｓｕｍ ｓａｔｉｖｕｍ）、キュウリ（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ）、ピーマン（Ｃａｐｓｉｃｕｍ ａｎｎｕｕｍ Ｇｒｏｕｐ）、トマト（Ｓｏｌａｎｕｍ ｌｙｃｏｐｅｒｓｉｃｕｍ）、ナス（Ｓｏｌａｎｕｍ ｍｅｌｏｎｇｅｎａ）、イチゴ（Ｆｒａｇａｒｉａ ａｎａｎａｓｓａ）、ミカン（Ｃｉｔｒｕｓ ｕｎｓｈｉｕ）、ベゴニア（Ｂｅｇｏｎｉａ）、プリムラ（Ｐｒｉｍｕｌａ）等が挙げられる。 Specific target plants include, for example, pea (Pisum sativum), cucumber (Cucumis sativus), green pepper (Capsicum annuum Group), tomato (Solanum lycopersium), eggplant (Solanum melongena), strawberry (Fragana) and strawberry (Fragana). Unshiu), Begonia, Primula and the like.
［施用工程］
施用工程では、上述した植物病害防除剤を対象植物に施用する。
植物病害防除剤をそのまま直接施用してもよく、或いは、水等の溶剤に希釈して使用してもよい。植物病害防除剤の施用方法として具体的には、例えば、直接植物に散布する方法、土壌に散布する方法、植物の種子に直接塗布する方法、植物や土壌に添加する水や肥料に添加する方法等が挙げられる。植物病害防除剤の施用量及び施用回数は、対象病害、対象作物、施用方法、発生傾向、被害の程度、環境条件、使用する剤形等によって、適宜調整することができる。また、植物病害防除剤の施用時期についても、特に限定されず、病害発生前に防除作用を発揮させることを目的として施用してもよく、或いは、病害発生後に病原菌の増殖を抑制し、病害を治療することを目的として施用してもよい。
[Application process]
In the application step, the above-mentioned plant disease control agent is applied to the target plant.
The plant disease control agent may be directly applied as it is, or may be diluted with a solvent such as water before use. Specifically, as a method of applying a plant disease control agent, for example, a method of directly spraying on a plant, a method of spraying on soil, a method of directly applying on plant seeds, a method of adding to water or fertilizer added to plants or soil. And so on. The application rate and frequency of application of the plant disease control agent can be appropriately adjusted according to the target disease, the target crop, the application method, the tendency of occurrence, the degree of damage, the environmental conditions, the dosage form to be used, and the like. In addition, the timing of application of the plant disease control agent is not particularly limited, and the plant disease control agent may be applied for the purpose of exerting a control effect before the occurrence of the disease, or it may be applied for the purpose of suppressing the growth of pathogens after the occurrence of the disease to prevent the disease. It may be applied for the purpose of treatment.
本実施形態の植物病害防除剤は、広範囲の種類の細菌及び糸状菌に対して、優れた防除効果を発揮する。本実施形態の植物病害防除剤により防除可能な植物の病原菌としては、例えば、「カンキツ」の黒点病菌（Ｄｉａｐｏｒｔｈｅ ｃｉｔｒｉ）、そうか病菌（Ｅｌｓｉｎｏｅ ｆａｗｃｅｔｔｉｉ）、褐色腐敗病菌（Ｐｈｙｔｏｐｈｔｈｏｒａ ｃｉｔｒｏｐｈｔｈｏｒａ）、緑かび病菌（Ｐｅｎｉｃｉｌｌｉｕｍ ｄｉｇｉｔａｔｕｍ）、青かび病菌（Ｐｅｎｉｃｉｌｌｉｕｍ ｉｔａｌｉｃｕｍ）；
「ウリ類」のうどんこ病菌（Ｓｐｈａｅｒｏｔｈｅｃａ ｆｕｌｉｇｉｎｅａ）、つる枯病菌（Ｄｉｄｙｍｅｌｌａ ｂｒｙｏｎｉａｅ）、炭そ病菌（Ｃｏｌｌｅｔｏｔｒｉｃｈｕｍ ｌａｇｅｎａｒｉｕｍ）；
「トマト」の輪紋病菌（Ａｌｔｅｒｎａｒｉａ ｓｏｌａｎｉ）、葉かび病菌（Ｃｌａｄｏｓｐｏｒｉｕｍ ｆｕｌｖｕｍ）、うどんこ病菌（Ｏｉｄｉｕｍ ｎｅｏｌｙｃｏｐｅｒｓｉｃｉ、Ｏｉｄｉｏｐｓｉｓ ｓｐ．、Ｌｅｖｅｉｌｌｕｌａ ｔａｕｒｉｃａ）；
「ナス」の褐紋病菌（Ｐｈｏｍｏｐｓｉｓ ｖｅｘａｎｓ）、うどんこ病菌（Ｅｒｙｓｉｐｈｅ ｃｉｃｈｏｒａｃｅａｒｕｍ）；
「イチゴ」のうどんこ病菌（Ｓｐｈａｅｒｏｔｈｅｃａ ｈｕｍｕｌｉ）、炭そ病菌（Ｇｌｏｍｅｒｅｌｌａ ｃｉｎｇｕｌａｔａ）；
「ピーマン」のうどんこ病菌（Ｏｉｄｉｏｐｓｉｓ ｓｉｃｕｌａ）；
「エンドウ」のうどんこ病菌（Ｅｒｙｓｉｐｈｅ ｐｉｓｉ）；
「種々の作物」の灰色かび病菌（Ｂｏｔｒｙｔｉｓ ｃｉｎｅｒｅａ）等が挙げられ、これらに限定されない。
The plant disease control agent of the present embodiment exhibits an excellent control effect against a wide variety of bacteria and filamentous fungi. Examples of the plant pathogens that can be controlled by the plant disease control agent of the present embodiment include black spot fungus (Diaporthe citri), scab fungus (Elsinoe fawcettii), brown rot fungus (Phytophthora cytophthora), and green fungus. (Penicillium digitatum), Penicillium italicum;
Powdery mildew bacterium, Didymella bryoniae, Colletotrichum radinarium;
"Tomato", Alternaria solani, Cladosporium fluvum, Powdery mildew (Oidium neolycoperi, Oidiopsis sp., Leveillula tarica);
"Eggplant" Phomopsis vexans, powdery mildew (Erysiphe cichoracerum);
"Strawberry" powdery mildew (Sphaerotheca humuli), anthrax (Glomerella cingulata);
"Pepper" powdery mildew (Odiopsis sicula);
Powdery mildew of "pea" (Erysiphe pisi);
Examples include, but are not limited to, Botrytis cinerea of "various crops".
以下、実施例により本発明を説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.
［実施例１］
（緑かび病を防除する納豆菌の選抜）
１．納豆菌培養液の調製
納豆菌としては、タカノフーズ株式会社が保有する納豆菌７２０菌株を用いた。納豆菌培養液は、以下の方法を用いて調製した。まず、各菌株はＴｒｙｐｔｉｃａｓｅ Ｓｏｙ Ａｇａｒ（ＴＳＡ）平板培地を用いて３７℃で１６時間培養したものを白金耳でＴｒｙｐｔｉｃａｓｅ Ｓｏｙ Ｂｒｏｔｈ（ＴＳＢ）培地に懸濁し、２８℃、１０５ｒｐｍで２４時間振とう培養した。培養後、納豆菌を遠心分離（３０００ｒｃｆ、８分間、２２℃）により集菌し、上清を除去した後に滅菌水に懸濁した。この作業を３回繰り返して培地成分を除去した後に、分光光度計を用いて培養液濃度をＯＤ６００＝１となるように調整し、実験に供試した。
[Example 1]
(Selection of natto bacteria to control green mold)
1. 1. Preparation of natto bacterium culture solution As the natto bacterium, a natto bacterium 720 strain owned by Takano Foods Co., Ltd. was used. The natto bacterium culture solution was prepared using the following method. First, each strain was cultured on a Trypticase Soy Agar (TSA) plate medium at 37 ° C. for 16 hours, suspended in a Trypticase Soy Broth (TSB) medium with a platinum loop, and shaken at 28 ° C. and 105 rpm for 24 hours. .. After culturing, Bacillus natto was collected by centrifugation (3000 rcf, 8 minutes, 22 ° C.), the supernatant was removed, and then suspended in sterile water. After repeating this operation three times to remove the medium components, the culture solution concentration was adjusted to OD 600 = 1 using a spectrophotometer and used in the experiment.
２．分生子懸濁液の調製
カンキツにおける緑かび病菌Ｐｅｎｉｃｉｌｌｉｕｍ ｄｉｇｉｔａｔｕｍは、茨城大学農学部で保存しているＩＵＰｄ２株を用いた。分生子形成は、本菌をＰｏｔａｔｏ Ｓｕｃｒｏｓｅ Ａｇａｒ（ＰＳＡ）平板培地で３日間培養後、ブラックライトブルーランプを３日間照射し、その後３日間暗所に置くことで誘導した。培地上の分生子は滅菌水に懸濁した後に、滅菌ティッシュペーパーでろ過することで菌糸を除去した、分生子を遠心分離（１７００ｒｃｆ、５分間、２２℃）により回収し、上澄みを除去した後に滅菌水に再懸濁した。この作業を３回繰り返すことで分生子を洗浄した。分生子懸濁液は血球計算盤を用いて濃度を１．０×１０５ｃｏｎｉｄｉａ／ｍＬに調製し、実験に供試した。
2. 2. Preparation of conidia suspension As the Penicillium digitatum in citrus, IUPd2 strain preserved in the Faculty of Agriculture, Ibaraki University was used. Conidia formation was induced by culturing this bacterium in Potato Sucross Agar (PSA) plate medium for 3 days, irradiating it with a black light blue lamp for 3 days, and then placing it in a dark place for 3 days. The conidia on the medium were suspended in sterile water and then filtered through sterile tissue paper to remove hyphae. The conidia were collected by centrifugation (1700 rcf, 5 minutes, 22 ° C.) and the supernatant was removed. Resuspended in sterile water. The conidia were washed by repeating this operation three times. The conidia suspension was prepared at a concentration of 1.0 × 105 conidia / mL using a hemocytometer and used in the experiment.
３．対峙培養試験
ＰＳＡ平板培地にＰ． ｄｉｇｉｔａｔｕｍ分生子懸濁液を１００μＬ滴下し、コンラージ棒で塗布した後、納豆菌培養液に浸漬したペーパーディスク（径５ｍｍ）を平板培地の中央に置床した。これを２５℃の暗所で３日間培養後、ペーパーディスクの周囲に形成された阻止円の直径を測定した。試験は２反復行った。
3. 3. Confrontation culture test P.A. on PSA plate medium. 100 μL of the digitatum conidia suspension was added dropwise, applied with a spreader, and then a paper disk (
対峙培養試験の結果、供試した全ての菌株でＰ． ｄｉｇｉｔａｔｕｍの菌糸生育に対する抑制効果が見られ、中でも、ＴＴＣＣ２１２２株が他の株と比べて強い抑制効果を示した。
なお、優れた抑制効果が確かめられたＴＴＣＣ２１２２株は、独立行政法人産業技術総合研究所特許微生物寄託センター（住所：日本国千葉県木更津市かずさ鎌足２－５－８）に２０２０年５月２８日付けで、受託番号ＮＩＴＥ Ｐ－０３２２８として寄託されているものである。
As a result of the confrontation culture test, P. An inhibitory effect on the growth of hyphae of digitatum was observed, and among them, the TTCC2122 strain showed a stronger inhibitory effect as compared with other strains.
The TTCC2122 strain, whose excellent inhibitory effect was confirmed, was sent to the National Institute of Advanced Industrial Science and Technology, Patent Microorganisms Depositary Center (Address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan) on May 28, 2020. It was deposited under the accession number NITE P-03228 on the date.
［実施例２］
（灰色かび病を防除する納豆菌の選抜）
１．納豆菌培養液の調製
実施例１の「１．」と同様の方法を用いて、７２０種の納豆菌について納豆菌培養液を調製した。
[Example 2]
(Selection of natto bacteria to control gray mold)
1. 1. Preparation of natto bacterium culture solution A natto bacterium culture solution was prepared for 720 kinds of natto bacteria by the same method as in "1." of Example 1.
２．分生子懸濁液の調製
灰色かび病菌Ｂｏｔｒｙｔｉｓ ｃｉｎｅｒｅａは、茨城大学農学部で保存しているＴＶ３３５株を用いた。実施例１の「２．」と同様の方法を用いて、分生子懸濁液を調製した。
2. 2. Preparation of conidia suspension For Botrytis cinerea, a TV335 strain stored at the Faculty of Agriculture, Ibaraki University was used. A conidia suspension was prepared using the same method as in "2." of Example 1.
３．対峙培養試験
ＰＳＡ平板培地にＢ． ｃｉｎｅｒｅａ分生子懸濁液を１００μＬ滴下し、コンラージ棒で塗布した後、納豆菌培養液に浸漬したペーパーディスク（径５ｍｍ）を平板培地の中央に置床した。これを２５℃の暗所で３日間培養後、ペーパーディスクの周囲に形成された阻止円の直径を測定した。試験は２反復行った。
3. 3. Confrontation culture test B. on PSA plate medium. 100 μL of the cinerea conidia suspension was added dropwise, applied with a spreader, and then a paper disk (
対峙培養試験の結果、供試した全ての菌株でＢ． ｃｉｎｅｒｅａの菌糸生育に対する抑制効果が見られ、中でも、ＴＴＣＣ２１１１株が他の株と比べて強い抑制効果を示した。
なお、優れた抑制効果が確かめられたＴＴＣＣ２１１１株は、独立行政法人産業技術総合研究所特許微生物寄託センター（住所：日本国千葉県木更津市かずさ鎌足２－５－８）に２０２０年５月２８日付けで、受託番号ＮＩＴＥ Ｐ－０３２２７として寄託されているものである。
As a result of the confrontation culture test, all the strains tested were B. An inhibitory effect on mycelial growth of cerinerea was observed, and among them, the TTCC2111 strain showed a stronger inhibitory effect as compared with other strains.
The TTCC2111 strain, whose excellent inhibitory effect was confirmed, was sent to the National Institute of Advanced Industrial Science and Technology, Patent Microorganisms Depositary Center (Address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan) on May 28, 2020. It was deposited under the accession number NITE P-03227 on the date.
［実施例３］
（貯蔵時のみかんにおける保存性向上確認試験）
実施例１の「１．」と同様の方法を用いて、ＴＴＣＣ２１２２株の培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。参考として茨城大学で保有していた納豆菌Ｎｏ．２株についても同様に培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。また、カンキツにおける緑かび病菌Ｐ． ｄｉｇｉｔａｔｕｍ分生子懸濁液（１．０×１０５ｃｏｎｉｄｉａ／ｍＬ）についても、実施例１の「２．」と同様の方法を用いて、準備した。さらに、カンキツにおける青かび病菌Ｐ． ｉｔａｌｉｃｕｍ（茨城大学農学部で保存、ＩＵＰｉ１株）についても同様に、分生子懸濁液（１．０×１０５ｃｏｎｉｄｉａ／ｍＬ）を調製した。
[Example 3]
(Confirmation test for improving storage stability of mandarin oranges during storage)
A culture solution (mycelium concentration: 1 × 10 8 cells / mL) of the TTCC2122 strain was prepared using the same method as in “1.” of Example 1. As a reference, Bacillus natto No. held at Ibaraki University. For the two strains, a culture solution (cell concentration: 1 × 10 8 cells / mL) was prepared in the same manner. In addition, green mold fungus P. citrus in citrus. A digitatum conidia suspension (1.0 × 10 5 conidia / mL) was also prepared using the same method as in “2.” of Example 1. In addition, Penicillium fungus P. citrus in citrus. Similarly, a conidia suspension (1.0 × 105 conida / mL) was prepared for italicum (preserved at the Faculty of Agriculture, Ibaraki University, IUPi1 strain).
次いで、みかんの表面に４か所の深さ１ｍｍ、幅３ｍｍの傷を付け、ＴＴＣＣ２１２２株の培養液を１か所あたり１５μｌ滴下処理した。処理１６時間後、Ｐ． ｄｉｇｉｔａｔｕｍ及びＰ． ｉｔａｌｉｃｕｍ（茨城大学農学部で保存、ＩＵＰｉ１株）の分生子懸濁液（１．０×１０５ｃｏｎｉｄｉａ／ｍＬ）をそれぞれ傷１か所あたり１５μｌ滴下接種した。分生子の接種から４日後にみかんの表面を観察し、接種試験に供したみかんの傷に対する、かびが観察された傷の割合（百分率）を病害発生率（％）として算出した。結果を図１に示す。なお、図１において「コントロール」群は、ＴＴＣＣ２１２２株の培養液の滴下処理を行なわずに、緑かび病菌（Ｐ． ｄｉｇｉｔａｔｕｍ）又は青かび病菌（Ｐ． ｉｔａｌｉｃｕｍ）を接種したみかんである。 Next, the surface of the mandarin orange was scratched at 4 places with a depth of 1 mm and a width of 3 mm, and 15 μl of the culture solution of the TTCC2122 strain was dropped per place. After 16 hours of processing, P.I. digitatum and P.I. A conidia suspension (1.0 × 105 conidia / mL) of italicum (preserved at the Faculty of Agriculture, Ibaraki University, IUPi1 strain) was inoculated in a drop of 15 μl per wound. The surface of the mandarin orange was observed 4 days after the inoculation of the conidia, and the ratio (percentage) of the mandarin oranges in which mold was observed to the mandarin oranges used in the inoculation test was calculated as the disease incidence rate (%). The results are shown in FIG. In addition, in FIG. 1, the "control" group is a mandarin orange inoculated with P. digitatum or P. italicum without dripping the culture solution of the TTCC2122 strain.
図１に示すように、ＴＴＣＣ２１２２株の培養液による滴下処理を施した傷では、緑かび病菌（Ｐ． ｄｉｇｉｔａｔｕｍ）又は青かび病菌（Ｐ． ｉｔａｌｉｃｕｍ）のいずれを接種した場合においても病害発生率が顕著に抑制されていた。この抑制効果は、参考として使用した納豆菌Ｎｏ．２株と比較しても顕著であった。 As shown in FIG. 1, in the wounds treated with the culture solution of the TTCC2122 strain, the disease incidence rate is remarkable regardless of whether the green mold fungus (P. digitatum) or the blue mold fungus (P. italicum) is inoculated. Was suppressed. This inhibitory effect was obtained by Bacillus natto No. 1 used as a reference. It was remarkable even when compared with two strains.
［実施例４］
（納豆菌培養液のエンドウうどんこ病に対する治療効果及び防除効果確認試験）
ＴＴＣＣ２１１１株及びＴＴＣＣ２１２２株はＴｒｙｐｔｉｃａｓｅ Ｓｏｙ Ａｇａｒ（ＴＳＡ）平板培地を用いて３７℃で１６時間培養したものを白金耳でＰｏｔａｔｏ Ｓｕｃｒｏｓｅ Ｂｒｏｔｈ（ＰＳＢ）培地に懸濁した。これらを２８℃、１０５ｒｐｍで２４時間振とう培養し、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液（各菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。
[Example 4]
(Test to confirm the therapeutic effect and control effect of Bacillus natto culture solution on pea powdery mildew)
The TTCC2111 strain and the TTCC2122 strain were cultured at 37 ° C. for 16 hours using Trypticase Soy Agar (TSA) plate medium and suspended in Potato Sucrose Bros (PSB) medium with a loopful. These were shake-cultured at 28 ° C. and 105 rpm for 24 hours to prepare a culture solution of TTCC2111 strain and a culture solution of TTCC2122 strain (each cell concentration: 1 × 10 8 cells / mL).
予めうどんこ病菌を２週間前に接種したエンドウ葉から多くの分生子を形成している葉を選び、絵筆を用いて葉面上の分生子を掃い落とした。次いで、調製したＴＴＣＣ２１２２株の培養液をスプレーで噴霧した。培養液の噴霧量は、エンドウ葉１枚当たり０．３ｍＬ以上０．５ｍＬ以下程度であった。コントロールには、培養液の代わりに滅菌水を噴霧処理した。噴霧処理から２日後に、エンドウ葉を固定液に浸漬させて固定した。固定液としては、ＦＡＡ（Ｆｏｒｍａｌｉｎ／Ａｃｅｔｉｃ ａｃｉｄ／Ａｌｃｏｈｏｌ）固定液を用いた。ＦＡＡ固定液の組成は、容量比でホルマリン：酢酸：エタノール＝１：１：１である。次いで、固定後のエンドウ葉についてエンドウうどんこ病菌の菌体をメチルブルーで染色した。葉１枚当たり１０コロニーをランダムに選び、コロニー当たりに形成された分生子数を数えた。結果を図２に示す。 The leaves forming many conidia were selected from the leaves of pea that had been inoculated with powdery mildew two weeks ago, and the conidia on the leaf surface were swept off using a paintbrush. Then, the prepared culture solution of TTCC2122 strain was sprayed. The amount of the culture solution sprayed was about 0.3 mL or more and 0.5 mL or less per pea leaf. Controls were sprayed with sterile water instead of culture medium. Two days after the spraying treatment, the pea leaves were immersed in a fixing solution and fixed. As the fixative, FAA (Formaldehyde / Acetic acid / Alcohol) fixative was used. The composition of the FAA fixative is formalin: acetic acid: ethanol = 1: 1: 1 in volume ratio. Then, the pea leaves after fixation were stained with methyl blue. Ten colonies were randomly selected per leaf and the number of conidia formed per colony was counted. The results are shown in FIG.
図２に示すように、ＴＴＣＣ２１２２株の培養液の噴霧処理群では、コントロール群と比較して、形成された分生子数の減少が確認された。また、ＴＴＣＣ２１２２株の培養液を噴霧処理した葉におけるエンドウうどんこ病菌の菌糸を観察したところ、細胞死様の染色箇所が見られた（図示せず）。
以上のことから、ＴＴＣＣ２１２２株の培養液は、エンドウうどんこ病に対する治療効果を有することが示唆された。
As shown in FIG. 2, in the spray-treated group of the culture solution of the TTCC2122 strain, a decrease in the number of conidia formed was confirmed as compared with the control group. Moreover, when the hyphae of the pea udon scab fungus were observed in the leaves spray-treated with the culture solution of the TTCC2122 strain, cell death-like stained spots were observed (not shown).
From the above, it was suggested that the culture solution of the TTCC2122 strain has a therapeutic effect on powdery mildew.
次いで、１週齢のエンドウ葉表面に、調製したＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液をそれぞれスプレーで噴霧した。培養液の噴霧量は、エンドウ葉１枚当たり０．３ｍＬ以上０．５ｍＬ以下程度であった。コントロールには、培養液の代わりに滅菌水を噴霧処理した。翌日、充分にエンドウ葉表面を乾かした後、罹病エンドウ葉に形成された分生子を絵筆で処理葉全体に乗せて接種した。接種から３日後に接種葉をＦＡＡ固定液で固定した後、エンドウうどんこ病菌の菌体をメチルブルーで染色した。分生子よりも長い発芽管を形成したものを「発芽」とした。分生子数に対する発芽数の割合（百分率）を発芽率（％）として算出した。結果を図３に示す。図３において、各群について独立した試験を２回行った結果を示している。 Then, the prepared culture solution of TTCC2111 strain and the culture solution of TTCC2122 strain were sprayed on the surface of 1-week-old pea leaves, respectively. The amount of the culture solution sprayed was about 0.3 mL or more and 0.5 mL or less per pea leaf. Controls were sprayed with sterile water instead of culture medium. The next day, after the surface of the pea leaf was sufficiently dried, the conidia formed on the diseased pea leaf were placed on the entire treated leaf with a paintbrush and inoculated. Three days after inoculation, the inoculated leaves were fixed with FAA fixative, and then the cells of pea udon scab were stained with methyl blue. Those having a germination tube longer than the conidia were referred to as "germination". The ratio of the number of germination to the number of conidia (percentage) was calculated as the germination rate (%). The results are shown in FIG. FIG. 3 shows the results of two independent tests for each group.
図３に示すように、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液の噴霧処理群では、コントロール群と比較して、発芽率の減少が確認された。また、ＴＴＣＣ２１２２株の培養液の噴霧処理した葉におけるエンドウうどんこ病菌の発芽管を観察したところ、細胞死様の染色箇所が見られた（図示せず）。
以上のことから、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液は、エンドウうどんこ病に対する防除効果を有することが示唆された。
As shown in FIG. 3, in the spray-treated group of the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain, a decrease in the germination rate was confirmed as compared with the control group. Moreover, when the germination tube of the pea scab was observed in the leaves treated with the spray of the culture solution of the TTCC2122 strain, cell death-like stained spots were observed (not shown).
From the above, it was suggested that the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain have a control effect on powdery mildew of pea.
［実施例５］
（納豆菌培養液のプリムラ花弁での灰色かび病に対する発病抑制効果確認試験）
実施例１の「１．」と同様の方法を用いて、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液（各菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。次いで、プリムラ花弁の表面に、調製したＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液をそれぞれスプレーで噴霧した。各培養液の噴霧量は、プリムラ花弁１枚当たり０．３ｍＬ以上０．５ｍＬ以下程度であった。コントロールには、培養液の代わりに滅菌水を噴霧処理した。噴霧処理から１６時間後に、灰色かび病菌の分生子懸濁液（１×１０５ｃｏｎｉｄｉａ／ｍＬ）を用いて噴霧接種した。接種から２４時間後に形成されたプリムラ花弁１ｃｍ２当たりの病斑数（個）を算出した。結果を図４に示す。
[Example 5]
(Test to confirm the effect of natto bacterium culture solution on the onset of gray mold on Primula petals)
Using the same method as in "1." of Example 1, a culture solution of TTCC2111 strain and a culture solution of TTCC2122 strain (each cell concentration: 1 × 10 8 cells / mL) were prepared. Then, the prepared culture solution of TTCC2111 strain and the culture solution of TTCC2122 strain were sprayed on the surface of the Primula petals, respectively. The spray amount of each culture solution was about 0.3 mL or more and 0.5 mL or less per Primula petal. Controls were sprayed with sterile water instead of culture medium. 16 hours after the spray treatment, the conidia suspension of Botrytis cinerea (1 × 105 conida / mL) was spray-inoculated. The number of lesions (pieces) per 1 cm 2 of Primula petals formed 24 hours after inoculation was calculated. The results are shown in FIG.
図４に示すように、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液の噴霧処理群では、灰色かび病の発病抑制効果がみられた。その効果はやや弱いものであったが、これは、花弁表面が疎水性であることから、培養液が一様に広がらないことが原因であるものと推察された。灰色かび病菌による感染行動を観察したところ、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液の噴霧処理群では、発芽菌糸の伸長が抑制されていることが確認された（図示せず）。よって、培養液の組成及び処理方法を改善することで、灰色かび病の発病抑制効果を向上できるものと考えられる。 As shown in FIG. 4, in the spray-treated group of the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain, the effect of suppressing the onset of Botrytis cinerea was observed. The effect was rather weak, but it was speculated that this was due to the fact that the culture solution did not spread uniformly because the petal surface was hydrophobic. When the infection behavior by the gray mold fungus was observed, it was confirmed that the growth of germinated hyphae was suppressed in the spray-treated group of the culture solution of TTCC2111 strain and the culture solution of TTCC2122 strain (not shown). Therefore, it is considered that the effect of suppressing the onset of Botrytis cinerea can be improved by improving the composition and treatment method of the culture solution.
［実施例６］
（納豆菌培養液のキュウリうどんこ病に対する発病抑制効果確認試験）
実施例４と同様の方法を用いて、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液（各菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。次いで、２週齢のキュウリ苗の子葉に培養液をそのまま噴霧処理した。各培養液の噴霧量は、子葉全体が培養液で覆われるまで行った。コントロールには、培養液の代わりに滅菌水を噴霧処理した。噴霧処理から２４時間後に、うどんこ病菌の胞子を絵筆で処理葉全体に乗せて、接種を行った。接種後のキュウリ苗は２５℃で栽培した。接種から２週間後に各子葉に発生したコロニー数及び面積を調査し、以下に示す基準によって発病指数を判定した。コロニー面積はｉｍａｇｅＪソフトを用いて計測した。
[Example 6]
(Test to confirm the effect of natto culture solution on cucumber powdery mildew)
Using the same method as in Example 4, a culture solution of TTCC2111 strain and a culture solution of TTCC2122 strain (each cell concentration: 1 × 108 cells / mL) were prepared. Then, the cotyledons of 2-week-old cucumber seedlings were sprayed with the culture solution as they were. The amount of each culture solution sprayed was continued until the entire cotyledon was covered with the culture solution. Controls were sprayed with sterile water instead of culture medium. Twenty-four hours after the spray treatment, spores of powdery mildew were placed on the entire treated leaf with a paintbrush and inoculated. The cucumber seedlings after inoculation were cultivated at 25 ° C. The number and area of colonies generated in each
（基準）
０：発病なし
１：コロニーが１個以上２個以下認められる
２：コロニーが葉面積の１／４未満を占める
３：コロニーが葉面積の１／４以上１／２未満を占める
４：コロニーが葉面積の１／２以上を占める
(standard)
0: No disease 1: 1 or more and 2 or less colonies are observed 2: Colonies occupy less than 1/4 of the leaf area 3: Colonies occupy 1/4 or more and less than 1/2 of the leaf area 4: Colonies occupy Occupies more than half of the leaf area
次いで、判定した発病指数に基づいて、次の式を用いて発病度を算出した。結果を図５に示す。なお、各群の調査葉数（Ｎ）は１０枚である。 Then, based on the determined disease onset index, the disease onset was calculated using the following formula. The results are shown in FIG. The number of surveyed leaves (N) in each group is 10.
発病度＝［Σ｛（発病指数）×（葉数）｝／｛４×（調査葉数）｝］×１００ Disease rate = [Σ {(onset index) x (number of leaves)} / {4 x (number of surveyed leaves)}] x 100
図５に示すように、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液の噴霧処理群では、キュウリうどんこ病の発病抑制効果がみられた。特に、ＴＴＣＣ２１２２株の培養液の噴霧処理群では、キュウリうどんこ病の発病抑制効果が顕著であった。 As shown in FIG. 5, in the spray-treated group of the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain, the effect of suppressing the onset of powdery mildew of cucumber was observed. In particular, in the spray-treated group of the culture solution of the TTCC2122 strain, the effect of suppressing the onset of powdery mildew of cucumber was remarkable.
［実施例７］
（納豆菌培養液のキュウリうどんこ病に対する発病抑制効果確認試験２）
実施例４と同様の方法を用いて、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液（各菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。また、参考として、市販のバチルス属菌製剤の１０００倍希釈液（推奨希釈率、推定菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）も準備した。次いで、これらを用いて、実施例６と同様の方法で、キュウリうどんこ病に対する発病抑制効果確認試験を行った。各群の調査葉数（Ｎ）は１０枚である。結果を図６に示す。
[Example 7]
(Test to confirm the effect of natto culture solution on cucumber powdery mildew)
Using the same method as in Example 4, a culture solution of TTCC2111 strain and a culture solution of TTCC2122 strain (each cell concentration: 1 × 108 cells / mL) were prepared. In addition, as a reference, a 1000-fold diluted solution of a commercially available Bacillus bacterium preparation (recommended dilution ratio, estimated cell concentration: 1 × 108 cells / mL) was also prepared. Then, using these, a disease suppression effect confirmation test for cucumber powdery mildew was conducted by the same method as in Example 6. The number of surveyed leaves (N) in each group is 10. The results are shown in FIG.
図６に示すように、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液の噴霧処理群では、キュウリうどんこ病の発病抑制効果がみられた。一方で、市販のバチルス属菌製剤の希釈液を噴霧処理した群では、コントロール群と比べて、大きな差が見られず、キュウリうどんこ病の発病抑制効果が確認できなかった。
また、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液を噴霧処理した葉、並びに、滅菌水を噴霧処理した葉（コントロール群）をＦＡＡ固定液で固定した後、キュウリうどんこ病菌の菌体をメチルブルーで染色した。これら葉におけるキュウリうどんこ病菌の発芽管を光学顕微鏡で観察したところ、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液の噴霧処理群では、分生子の発芽が抑制されていることが確認された（図示せず）。
以上のことから、ＴＴＣＣ２１１１株の培養液及びＴＴＣＣ２１２２株の培養液は、キュウリうどんこ病に対する発病抑制効果を有することが示唆された。
As shown in FIG. 6, in the spray-treated group of the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain, the effect of suppressing the onset of powdery mildew of cucumber was observed. On the other hand, in the group spray-treated with the diluted solution of the commercially available Bacillus bacterium preparation, no significant difference was observed as compared with the control group, and the effect of suppressing the onset of cucumber powdery mildew could not be confirmed.
In addition, after fixing the leaves sprayed with the culture solution of TTCC2111 strain and the culture solution of TTCC2122 strain and the leaves spray-treated with sterile water (control group) with FAA fixative, the cells of cucumber udonko disease fungus were methylated. Stained in blue. When the germination tubes of cucumber udonko disease bacteria in these leaves were observed with an optical microscope, it was confirmed that the germination of conidia was suppressed in the spray-treated group of the culture solution of TTCC2111 strain and the culture solution of TTCC2122 strain (. Not shown).
From the above, it was suggested that the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain have an effect of suppressing the onset of cucumber powdery mildew.
［実施例８］
（納豆菌培養液及び培養上清のキュウリうどんこ病に対する発病抑制効果確認試験）
実施例４と同様の方法を用いて、得られた培養液をＴＴＣＣ２１２２株の培養液（菌体含有、菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）として、培養上清をＴＴＣＣ２１２２株の培養上清（菌体不含）として用いた。また、参考として、市販のバチルス属菌製剤の１０００倍希釈液（推奨希釈率、推定菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）も準備した。次いで、２週齢のキュウリ苗の第一本葉に培養液（菌体含有）又は培養上清（菌体不含）をそのまま噴霧処理した。各処理液の噴霧量は、葉全体が培養液で覆われるまで行った。コントロールには、培養液の代わりに滅菌水を噴霧処理した。噴霧処理から２４時間後及び１週間後に、うどんこ病菌の胞子を絵筆で処理葉全体に乗せて、接種を行った。接種後のキュウリ苗は２５℃で栽培した。接種から２週間後に各子葉に発生したコロニー数及び面積を調査し、実施例６と同様の方法を用いて、発病度を算出した。各群の調査葉数（Ｎ）は１０枚である。結果を図７に示す。
[Example 8]
(Test to confirm the effect of natto bacterium culture solution and culture supernatant on cucumber powdery mildew)
Using the same method as in Example 4, the obtained culture solution was used as the culture solution of the TTCC2122 strain (cell-containing, cell concentration: 1 × 108 cells / mL), and the culture supernatant was used on the culture of the TTCC2122 strain. It was used as Qing (free of bacterial cells). In addition, as a reference, a 1000-fold diluted solution of a commercially available Bacillus bacterium preparation (recommended dilution ratio, estimated cell concentration: 1 × 108 cells / mL) was also prepared. Then, the first true leaf of the 2-week-old cucumber seedling was spray-treated with the culture solution (containing cells) or the culture supernatant (without cells) as it was. The amount of each treatment solution sprayed was continued until the entire leaf was covered with the culture solution. Controls were sprayed with sterile water instead of culture medium. Twenty-four hours and one week after the spray treatment, powdery mildew spores were placed on the entire treated leaf with a paintbrush and inoculated. The cucumber seedlings after inoculation were cultivated at 25 ° C. The number and area of colonies generated in each
図７に示すように、ＴＴＣＣ２１２２株の培養液（菌体含有）及びＴＴＣＣ２１２２株の培養上清（菌体不含）の噴霧処理群では、いずれにおいてもキュウリうどんこ病の発病抑制効果がみられた。噴霧処理から１週間後にうどんこ病菌を接種した場合も、噴霧処理から２４時間後に接種した場合と同様に、キュウリうどんこ病の発病抑制効果がみられた。特に、噴霧処理から１週間後にうどんこ病菌を接種した場合において、ＴＴＣＣ２１２２株の培養上清（菌体不含）の噴霧処理群では、キュウリうどんこ病の発病抑制効果が顕著であった。 As shown in FIG. 7, in the spray-treated group of the culture solution of the TTCC2122 strain (containing cells) and the culture supernatant of the TTCC2122 strain (without cells), the effect of suppressing the onset of powdery mildew of cucumber was observed in both of them. rice field. When powdery mildew was inoculated one week after the spray treatment, the effect of suppressing the onset of cucumber powdery mildew was observed as in the case of inoculation 24 hours after the spray treatment. In particular, when powdery mildew was inoculated one week after the spray treatment, the effect of suppressing the onset of powdery mildew of cucumber was remarkable in the spray-treated group of the culture supernatant (without cells) of the TTCC2122 strain.
［実施例９］
（納豆菌培養液のキュウリうどんこ病に対する発病抑制効果確認試験３）
実施例４と同様の方法を用いて、ＴＴＣＣ２１２２株の培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。調製した培養液に滅菌水を加えて、５倍、１０倍及び２０倍希釈液を調製した。これらを用いて、実施例６と同様の方法で、キュウリうどんこ病に対する発病抑制効果確認試験を行った。また、防除価を次の式を用いて算出した。結果を表１に示す。
[Example 9]
(Test to confirm the effect of natto culture solution on cucumber powdery mildew)
A culture solution (cell concentration: 1 × 10 8 cells / mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterilized water was added to the prepared culture solution to prepare 5-fold, 10-fold and 20-fold diluted solutions. Using these, a disease suppression effect confirmation test for cucumber powdery mildew was carried out by the same method as in Example 6. In addition, the control value was calculated using the following formula. The results are shown in Table 1.
防除価＝｛１－（噴霧処理群での発病度の平均）／（無処理（コントロール）群での発病度の平均）｝×１００ Control value = {1- (average of the degree of disease in the spray-treated group) / (average of the degree of disease in the untreated (control) group)} × 100
表１に示すように、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液、１０倍希釈液及び２０倍希釈液の噴霧処理群において、防除価が３５．５以上であり、キュウリうどんこ病の発病抑制効果がみられた。 As shown in Table 1, in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution, the 10-fold diluted solution and the 20-fold diluted solution, the control value was 35.5 or more, and cucumber powdery mildew. The effect of suppressing the onset of the disease was observed.
［実施例１０］
（納豆菌培養液のキュウリ及びベゴニアでの灰色かび病に対する発病抑制効果確認試験）
実施例４と同様の方法を用いて、ＴＴＣＣ２１２２株の培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。調製した培養液に滅菌水を加えて、５倍、１０倍及び２０倍希釈液を調製した。また、参考として、市販のバチルス属菌製剤の１０００倍希釈液（推奨希釈率、推定菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）も準備した。次いで、これらを３週齢のキュウリ第一本葉及びベゴニアの切り葉にスプレーを用いて噴霧処理した。各液の噴霧量は、液が葉から滴るまで行った。コントロールには、培養液及び希釈液の代わりに滅菌水を噴霧処理した。噴霧処理後、２５℃の人工気象器内で維持し、キュウリは噴霧処理から２４時間後に処理葉を切り取った。これら処理葉にＰＳＡ平板培地で２日間培養したＢ． ｃｉｎｅｒｅａの菌叢プラグ（径５ｍｍ）を処理葉１枚あたり２箇所に接種した。接種葉は、イオン交換水で湿らせたペーパータオルを敷いたプラスチックボックスに入れて２２℃で維持し、接種から２日後に病斑直径を測定した。試験には各処理群について３葉を用い、同様の試験を３反復行った。結果を図８（キュウリでの病斑直径）及び図９（ベゴニアでの病斑直径）に示す。
[Example 10]
(Test to confirm the effect of Bacillus natto culture solution on the onset of Botrytis cinerea in cucumber and begonia)
A culture solution (cell concentration: 1 × 10 8 cells / mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterilized water was added to the prepared culture solution to prepare 5-fold, 10-fold and 20-fold diluted solutions. In addition, as a reference, a 1000-fold diluted solution of a commercially available Bacillus bacterium preparation (recommended dilution ratio, estimated cell concentration: 1 × 108 cells / mL) was also prepared. These were then spray-treated on the first true leaves of cucumber and the cut leaves of begonia at the age of 3 weeks. The amount of each liquid sprayed was until the liquid drips from the leaves. Controls were sprayed with sterile water instead of culture and diluent. After the spray treatment, the cucumber was maintained in an artificial meteorological instrument at 25 ° C., and the treated leaves were cut off 24 hours after the spray treatment. B. The treated leaves were cultured on a PSA plate medium for 2 days. The cinerea flora plug (
図８に示すように、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液、１０倍希釈液及び２０倍希釈液の噴霧処理群では、コントロール群と比べて、病斑直径の大きさが抑えられており、キュウリ灰色かび病に対する発病抑制効果が見られた。一方で、市販のバチルス属菌製剤の希釈液を噴霧処理した群では、コントロール群と比べて、病斑直径の大きさに差が見られず、キュウリ灰色かび病に対する発病抑制効果が確認できなかった。また、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液及び１０倍希釈液の噴霧処理群において、キュウリ灰色かび病に対する発病抑制効果が特に顕著であった。 As shown in FIG. 8, in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution, the 10-fold diluted solution and the 20-fold diluted solution, the size of the lesion diameter was suppressed as compared with the control group. It has been shown to have an effect of suppressing the onset of cucumber Botrytis cinerea. On the other hand, in the group spray-treated with the diluted solution of the commercially available Bacillus genus, there was no difference in the size of the lesion diameter as compared with the control group, and the effect of suppressing the onset of cucumber Botrytis cinerea could not be confirmed. rice field. In addition, in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution and the 10-fold diluted solution, the effect of suppressing the onset of cucumber Botrytis cinerea was particularly remarkable.
図９に示すように、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液、１０倍希釈液及び２０倍希釈液の噴霧処理群では、コントロール群と比べて、病斑直径の大きさが抑えられており、ベゴニア灰色かび病に対する発病抑制効果が見られた。一方で、市販のバチルス属菌製剤の希釈液を噴霧処理した群では、コントロール群と比べて、病斑直径の大きさに差が見られず、ベゴニア灰色かび病に対する発病抑制効果が確認できなかった。また、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液、１０倍希釈液及び２０倍希釈液の噴霧処理群いずれにおいても、ベゴニア灰色かび病に対する発病抑制効果が顕著であった。 As shown in FIG. 9, in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution, the 10-fold diluted solution and the 20-fold diluted solution, the size of the lesion diameter was suppressed as compared with the control group. It has been shown to have an effect of suppressing the onset of begonia gray mold. On the other hand, in the group spray-treated with the diluted solution of the commercially available Bacillus genus, there was no difference in the size of the lesion diameter as compared with the control group, and the effect of suppressing the onset of Begonia gray mold could not be confirmed. rice field. In addition, the effect of suppressing the onset of begonia Botrytis was remarkable in all of the spray-treated groups of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution, the 10-fold diluted solution, and the 20-fold diluted solution.
［実施例１１］
（納豆菌培養液のイチゴうどんこ病に対する発病抑制効果確認試験）
実施例４と同様の方法を用いて、ＴＴＣＣ２１２２株の培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。調製した培養液に滅菌水を加えて、５倍及び１０倍希釈液を調製した。これらをイチゴ（品種：ローズベリーレッド）の小葉裏面にスプレーを用いて噴霧処理した。小葉はイチゴ苗の展葉後間もない（１週間から２週間程度）複葉から切り取って実験に供試した。各液の噴霧量は、小葉裏面を処理液が覆うまで行った。コントロールには、培養液及び希釈液の代わりに滅菌水を噴霧処理した。噴霧処理から２４時間後に、イチゴうどんこ病罹病葉の病斑から分生胞子を絵筆で払い落とし、処理葉全体に接種を行った。接種後のイチゴ葉は湿らせた濾紙を敷いたプラスチックトレイ内に並べて密閉し、２０℃、蛍光灯照明下（１日当たり１２時間の照明）に維持した。接種から２週間後に葉に発生したコロニー数及び面積を実体顕微鏡下で調査し、実施例６と同様の方法で、発病度を算出し、実施例９と同様の方法で、防除価を算出した。結果を表２に示す。
[Example 11]
(Test to confirm the effect of natto culture solution on strawberry powdery mildew)
A culture solution (cell concentration: 1 × 10 8 cells / mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterilized water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were spray-treated on the back surface of the leaflets of strawberry (variety: rose berry red) using a spray. The leaflets were cut from the compound leaves shortly after the strawberry seedlings were expanded (about 1 to 2 weeks) and used in the experiment. The amount of each liquid sprayed was continued until the back surface of the leaflets was covered with the treatment liquid. Controls were sprayed with sterile water instead of culture and diluent. Twenty-four hours after the spray treatment, conidia were wiped off from the lesions of the strawberry powdery mildew-affected leaves with a paintbrush, and the entire treated leaves were inoculated. After inoculation, the strawberry leaves were lined up in a plastic tray lined with moistened filter paper and sealed, and maintained at 20 ° C. under fluorescent lighting (12 hours per day). The number and area of colonies generated on the
表２に示すように、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液及び１０倍希釈液の噴霧処理群において、防除価が５４．２以上であり、十分なイチゴうどんこ病の発病抑制効果がみられた。 As shown in Table 2, in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution and the 10-fold diluted solution, the control value was 54.2 or more, and the onset of strawberry powdery mildew was sufficiently suppressed. The effect was seen.
また、各処理群の葉の裏面を実体顕微鏡で観察したところ、５倍希釈液の噴霧処理群及び１０倍希釈液の噴霧処理群の葉面には毛茸の下にうどんこ病菌のコロニーが観察できるが、それらは、滅菌水の噴霧処理群（コントロール群）で見られるコロニーよりも菌糸密度が低いことが明らかとなった（図示せず）。 In addition, when the back surface of the leaves of each treated group was observed with a stereomicroscope, colonies of Udonko disease fungi were found under the hair mushrooms on the leaf surfaces of the 5-fold diluted solution spray-treated group and the 10-fold diluted solution spray-treated group. Observably, they were found to have a lower hyphal density than the colonies found in the sterile water spray treatment group (control group) (not shown).
［実施例１２］
（納豆菌培養液のピーマンうどんこ病に対する発病抑制効果確認試験）
実施例４と同様の方法を用いて、ＴＴＣＣ２１２２株の培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。調製した培養液に滅菌水を加えて、５倍及び１０倍希釈液を調製した。これらをピーマンうどんこ病菌に感受性であるシシトウ葉の裏面にスプレーを用いて噴霧処理した。シシトウの葉は播種後８週齢の苗から切り取って実験に供試した。各液の噴霧量は、葉面を処理液が覆うまで行った。コントロールには、培養液及び希釈液の代わりに滅菌水を噴霧処理した。噴霧処理から２４時間後に、ピーマンうどんこ病罹病葉の病斑から分生胞子を絵筆でかき取り、処理葉全体になすりつけて接種を行った。接種後のシシトウ葉は湿らせた濾紙を敷いたプラスチックトレイ内に並べて密閉し、２１℃、蛍光灯照明下（１日当たり１２時間の照明）に維持した。接種から３週間後に葉に発生したコロニー数及び面積を実体顕微鏡下で調査し、実施例６と同様の方法で、発病度を算出し、実施例９と同様の方法で、防除価を算出した。結果を表３に示す。
[Example 12]
(Test to confirm the effect of Bacillus natto culture solution on the onset of powdery mildew)
A culture solution (cell concentration: 1 × 10 8 cells / mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterilized water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were spray-treated on the back surface of Shishito leaves, which are susceptible to powdery mildew, using a spray. The leaves of Shishito were cut from seedlings 8 weeks old after sowing and used in the experiment. The amount of each liquid sprayed was continued until the leaf surface was covered with the treatment liquid. Controls were sprayed with sterile water instead of culture and diluent. Twenty-four hours after the spray treatment, conidia were scraped from the lesions of the leaves affected by powdery mildew of bell pepper with a paintbrush and rubbed on the entire treated leaves for inoculation. After inoculation, the leaves of Shishito were placed side by side in a plastic tray lined with moistened filter paper and sealed, and maintained at 21 ° C. under fluorescent lighting (12 hours of lighting per day). The number and area of colonies generated on the
表３に示すように、ＴＴＣＣ２１２２株の培養液（原液）及び５倍希釈液の噴霧処理群において、防除価が３８．６以上であり、ピーマンうどんこ病の発病抑制効果がみられた。一方で、１０倍希釈液の噴霧処理群では、ピーマンうどんこ病の発病抑制効果がみられなかった。 As shown in Table 3, in the spray-treated group of the culture solution (stock solution) and the 5-fold diluted solution of the TTCC2122 strain, the control value was 38.6 or more, and the effect of suppressing the onset of powdery mildew was observed. On the other hand, in the spray-treated group of the 10-fold diluted solution, the effect of suppressing the onset of powdery mildew was not observed.
また、各処理群の葉の裏面を実体顕微鏡で観察したところ、各処理群に形成されたコロニーに顕著な違いは見られなかった（図示せず）。これは、他のうどんこ病菌が表面寄生性であるのに対し、本菌が内部寄生性であるためと推察された。 Moreover, when the back surface of the leaves of each treatment group was observed with a stereomicroscope, no remarkable difference was observed in the colonies formed in each treatment group (not shown). It was speculated that this is because this fungus is endoparasitic while other powdery mildew fungi are superficially parasitic.
［実施例１３］
（納豆菌培養液のトマトうどんこ病に対する発病抑制効果確認試験）
実施例４と同様の方法を用いて、ＴＴＣＣ２１２２株の培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。調製した培養液に滅菌水を加えて、５倍及び１０倍希釈液を調製した。これらを３週齢のトマト（品種：ポンテローザ）の本葉にスプレーを用いて噴霧処理した。各液の噴霧量は、葉面を処理液が覆うまで行った。コントロールには、培養液及び希釈液の代わりに滅菌水を噴霧処理した。噴霧処理から２４時間後に、トマトうどんこ病罹病葉の病斑から分生胞子を絵筆でかき取り、処理葉全体になすりつけて接種を行った。接種後のトマト苗は２５℃で栽培した。接種から１０日後に葉に発生したコロニー数及び面積を実体顕微鏡下で調査し、実施例６と同様の方法で、発病度を算出し、実施例９と同様の方法で、防除価を算出した。結果を表４に示す。
[Example 13]
(Test to confirm the effect of natto culture solution on tomato powdery mildew)
A culture solution (cell concentration: 1 × 10 8 cells / mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterilized water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were spray-treated on the true leaves of 3-week-old tomatoes (variety: Ponterosa) using a spray. The amount of each liquid sprayed was continued until the leaf surface was covered with the treatment liquid. Controls were sprayed with sterile water instead of culture and diluent. Twenty-four hours after the spray treatment, conidia were scraped from the lesions of the tomato powdery mildew-affected leaves with a paintbrush and rubbed on the entire treated leaves for inoculation. The tomato seedlings after inoculation were cultivated at 25 ° C. The number and area of colonies generated on the
表４に示すように、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液及び１０倍希釈液の噴霧処理群において、防除価が３６．４以上であり、トマトうどんこ病の発病抑制効果がみられた。 As shown in Table 4, in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution and the 10-fold diluted solution, the control value was 36.4 or more, and the effect of suppressing the onset of tomato powdery mildew was obtained. It was seen.
また、各処理群の接種葉の表面におけるトマトうどんこ病菌の分生子及び菌糸の形態を目視で観察したところ、ＴＴＣＣ２１２２株の培養液（原液）の噴霧処理群ではコントロール群と比べて顕著な発芽抑制効果が見られた（図示せず）。また、５倍希釈液の噴霧処理群では、コントロール群に比べてコロニーを形成する菌糸の密度が薄くなっていた（図示せず）。１０倍希釈液の噴霧処理群ではコントロール群との差異は見られなかった（図示せず）。 In addition, when the morphology of conidia and hyphae of tomato powdery mildew on the surface of the inoculated leaves of each treatment group was visually observed, the germination was remarkable in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain as compared with the control group. An inhibitory effect was seen (not shown). Further, in the 5-fold diluted solution spray-treated group, the density of hyphae forming colonies was lower than that in the control group (not shown). No difference was observed in the 10-fold diluted solution spray-treated group from the control group (not shown).
［実施例１４］
（納豆菌培養液のナスうどんこ病に対する発病抑制効果確認試験）
実施例４と同様の方法を用いて、ＴＴＣＣ２１２２株の培養液（菌体濃度：１×１０８ｃｅｌｌｓ／ｍＬ）を調製した。調製した培養液に滅菌水を加えて、５倍及び１０倍希釈液を調製した。これらを３週齢のナス（品種：千両二号）の本葉にスプレーを用いて噴霧処理した。各液の噴霧量は、葉面を処理液が覆うまで行った。コントロールには、培養液及び希釈液の代わりに滅菌水を噴霧処理した。噴霧処理から２４時間後にナスうどんこ病罹病葉の病斑から分生胞子を絵筆でかき取り、処理葉全体になすりつけて接種を行った。接種後のナス苗は２５℃で栽培した。接種から１０日後に葉に発生したコロニー数及び面積を調査し、実施例６と同様の方法で、発病度を算出し、実施例９と同様の方法で、防除価を算出した。結果を表５に示す。
[Example 14]
(Test to confirm the effect of natto culture solution on eggplant powdery mildew)
A culture solution (cell concentration: 1 × 10 8 cells / mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterilized water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were spray-treated on the true leaves of 3-week-old eggplants (variety: Senryo No. 2) using a spray. The amount of each liquid sprayed was continued until the leaf surface was covered with the treatment liquid. Controls were sprayed with sterile water instead of culture and diluent. Twenty-four hours after the spray treatment, conidia were scraped from the lesions of the leaves affected by powdery mildew of eggplant with a paintbrush and rubbed on the entire treated leaves for inoculation. After inoculation, eggplant seedlings were cultivated at 25 ° C. The number and area of colonies generated on the
表５に示すように、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液及び１０倍希釈液の噴霧処理群において、防除価が２５．４以上であり、ナスうどんこ病の発病抑制効果がみられた。 As shown in Table 5, in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution and the 10-fold diluted solution, the control value was 25.4 or more, and the effect of suppressing the onset of powdery mildew of eggplant was obtained. It was seen.
また、各処理群の接種葉の表面におけるナスうどんこ病菌の分生子及び菌糸の形態を目視で観察したところ、ナスうどんこ病菌は他のうどんこ病菌に比べて感染率が低く、コロニーも他に比べて進展しないものであった。また、ＴＴＣＣ２１２２株の培養液（原液）、５倍希釈液及び１０倍希釈液の噴霧処理群において、ナスうどんこ病の発病抑制効果は見られたものの、これまで検定した他の植物種のうどんこ病菌に対する発病抑制効果に比べて低く見えた。これは、上述したように、ナスうどんこ病菌は他のうどんこ病菌に比べて感染率が低いことから、コントロール群との差異が見にくかったためであると推察された。 In addition, when the morphology of conidia and hyphae of Eggplant powdery mildew on the surface of the inoculated leaves of each treatment group was visually observed, the infection rate of Eggplant powdery mildew was lower than that of other powdery mildews, and the colonies were also other. It did not progress as compared to. In addition, although the effect of suppressing the onset of powdery mildew of eggplant was observed in the spray-treated group of the culture solution (stock solution) of the TTCC2122 strain, the 5-fold diluted solution and the 10-fold diluted solution, udon noodles of other plant species tested so far. It seemed to be lower than the disease-suppressing effect on this fungus. It was presumed that this was because, as described above, the powdery mildew bacterium of Eggplant had a lower infection rate than the other powdery mildew bacterium, and therefore it was difficult to see the difference from the control group.
以上のことから、ＴＴＣＣ２１１１株及びＴＴＣＣ２１２２株は、各種植物病害に対する防除効果を有することが明らかとなった。 From the above, it was clarified that the TTCC2111 strain and the TTCC2122 strain have a control effect against various plant diseases.
本実施形態の植物病害防除剤及び植物病害防除方法によれば、植物病害を有効に防除することができる。 According to the plant disease control agent and the plant disease control method of the present embodiment, plant diseases can be effectively controlled.
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