JP2015181423A - Strain belonging to bacillus, microbiological agent, and plant cultivation method - Google Patents
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- Cultivation Of Plants (AREA)
Abstract
Description
本発明は、抗生物質を体外へ産生・分泌することによる植物病害防除及び植物に病害抵抗性を誘導し免疫機能を強化することを特徴とする微生物に関する。更には、バチルス・アミロリクエファシエンスに属する微生物が生産する物質を有効成分として含有する菌株、植物病害の防除剤、及びそれを利用した植物病害の防除方法に関する。
The present invention relates to a microorganism characterized by controlling plant diseases by producing and secreting antibiotics outside the body and inducing disease resistance in plants to enhance immune functions. Furthermore, the present invention relates to a strain containing a substance produced by a microorganism belonging to Bacillus amyloliquefaciens as an active ingredient, a plant disease control agent, and a plant disease control method using the same.
我が国の農薬市場は低迷が続いている。対照的に、世界の農薬市場は人口爆発による食糧需要の拡大により、右肩上がりである。よって、国内の農薬メーカーにとって、海外でも拡販できる農薬開発が必須である。そのためには、「食の安全・安心」「環境への負荷軽減」といった付加価値の強い農薬原体の開発が必要になってきた。特に「食の安全・安心」は、質の高い国民生活を実現するために達成するべき喫緊の課題である。「安心・安全な食を守るため、ご自身で取り組んでいることはありますか」というアンケートに対し、一般消費者の79％が「農薬不使用や減農薬の野菜・果物を選んでいる」を選択していることからも、「食の安全・安心」に対する農薬が果たす役割は非常に大きい。
このような状況に置かれた農薬市場において、「微生物農薬」が脚光を浴び始めている。微生物農薬は、自然界に生存する微生物を利用して、農作物の病気を抑える戦略を取るため、農作物、人間、そして環境に対する負荷が少なく、食の安全・安心に大きく貢献し、「安全で高品質な食料・食品生産」という社会基盤を実現する農業技術になることが期待されている。
微生物農薬が分類される生物農薬の我が国の市場規模は、30億円程度と推察されており、現時点では農薬市場（約3,300億円）の１％に満たない（日経バイオ年鑑2010）。ただし、有機および減農薬栽培を試行する生産者の増加や、残留農薬に関する昨今の消費者意識の向上および残留農薬規制が強化された改正食品衛生法の施行などの影響により、「脱化学農薬」の流れに乗って、2015年には市場規模が100億円に達する予測もある。
加えて、2009年EU（欧州連合）が農薬の使用制限および認可ルールを厳しくする規則並びに農薬の持続的可能な使用のための目標および要件を定める枠組み指令（2009/128/EC）を採択したことによって、現在、EUでは脱化学農薬の動きが盛んとなり、微生物農薬を含む生物農薬の市場が活性化されている。
このように、国内外において微生物農薬の開発、使用が急速に熟成中である。
The Japanese agrochemical market continues to be sluggish. In contrast, the global pesticide market is on the rise as food demand grows due to population explosions. Therefore, it is essential for agrochemical manufacturers in Japan to develop agrochemicals that can expand sales overseas. To that end, it has become necessary to develop a high-value-added pesticide active ingredient such as “food safety and security” and “reducing environmental burdens”. In particular, “food safety and security” is an urgent issue to be achieved in order to realize a high-quality national life. In response to the questionnaire, “Are you doing anything on your own to protect safe and secure foods?”, 79% of general consumers said they chose “pesticide-free and reduced pesticide vegetables and fruits” From the selection, the role of pesticides for “food safety and security” is very large.
In the agrochemical market under such circumstances, “microbial pesticides” are beginning to attract attention. Microbial pesticides use microorganisms that live in nature and take strategies to reduce crop diseases, so they have a low impact on crops, people, and the environment, and contribute greatly to food safety and security. It is expected to become an agricultural technology that realizes the social foundation of “food and food production”.
Japan's market scale of bio-pesticides classified as microbial pesticides is estimated to be about 3 billion yen, which is currently less than 1% of the agrochemical market (about 330 billion yen) (Nikkei Bio Yearbook 2010). However, due to the effects of an increase in the number of producers trying to grow organic and reduced pesticides, the recent improvement in consumer awareness of pesticide residues, and the enforcement of the revised Food Sanitation Law with stricter regulations on pesticide residues, Based on this trend, the market size is forecast to reach 10 billion yen in 2015.
In addition, the 2009 EU (European Union) adopted a Framework Directive (2009/128 / EC) that establishes rules that tighten the restrictions and authorization rules for the use of pesticides and targets and requirements for the sustainable use of pesticides As a result, the movement of dechemical pesticides has become active in the EU and the market for biological pesticides including microbial pesticides has been activated.
In this way, the development and use of microbial pesticides are rapidly maturing both at home and abroad.
本発明は、植物病害の防除及び植物免疫機能の強化作用の効果を有する微生物を自然界から分離して提供することである。また、前記の微生物を有効菌として含有する微生物製剤として使用できる植物病害防除剤及びそれを利用した植物病害の防除方法を提供することにある。
An object of the present invention is to provide a microorganism having an effect of controlling plant diseases and enhancing the function of plant immunity separated from the natural world. Another object of the present invention is to provide a plant disease control agent that can be used as a microorganism preparation containing the above microorganisms as effective bacteria, and a plant disease control method using the same.
上記課題を解決するために、本発明の請求項１による菌株または菌株の培養物は、植物病害防除作用及び植物免疫機能の強化作用の効果を示すバチルス・アミロリクエファシエンスS13-3株（NITE P-1121）であることを特徴とするものである。
また、請求項２による微生物製剤は、請求項１に記載の菌株または菌株の培養物を有効成分として含有することを特徴とするものである。
また、請求項３による栽培方法は、請求項２に記載の微生物製剤で植物を処理することを特徴とするものである。
In order to solve the above-mentioned problems, a strain or a culture of a strain according to claim 1 of the present invention is a Bacillus amyloliquefaciens S13-3 strain (NITE) exhibiting the effects of controlling plant diseases and enhancing the functions of plant immune functions. P-1121).
A microorganism preparation according to claim 2 contains the strain or culture of the strain according to claim 1 as an active ingredient.
A cultivation method according to claim 3 is characterized in that a plant is treated with the microorganism preparation according to claim 2.
本発明のバチルス・アミロリクエファシエンスS13-3株、バチルス・アミロリクエファシエンスS13-3株の培養物は、抗生物質生産能（直接的に病原菌に作用し、病気を防ぐ）による植物病害防除作用と植物免疫誘導能（植物のもつ抵抗性を誘導し、間接的に病気を防ぐ）による植物免疫機能強化作用という多機能性を有している。
また、本発明のバチルス・アミロリクエファシエンスS13-3株を有効成分として含有する微生物製剤、または微生物製剤を使用して植物を処理する栽培方法として実用化することによって化学農薬の削減による食の安全・安心の実現に貢献することができる。
The culture of Bacillus amyloliquefaciens strain S13-3 and Bacillus amyloliquefaciens strain S13-3 of the present invention controls plant diseases by the ability to produce antibiotics (acts directly on pathogens and prevents diseases) It has multi-functionality of plant immunity enhancement by action and ability to induce plant immunity (inducing resistance of plants and indirectly preventing diseases).
In addition, a microbial preparation containing the Bacillus amyloliquefaciens S13-3 strain of the present invention as an active ingredient, or a cultivation method for treating a plant using the microbial preparation for practical use as a food by reducing chemical pesticides. It can contribute to the realization of safety and security.
以下、本発明を詳細に説明する。
本発明の植物病害防除剤は、バチルス・アミロリクエファシエンスに属し、植物病原菌の生育を抑制する物質を生産する微生物を培養し、その培養産物を採取し、必要に応じて製剤化することにより製造できる。バチルス・アミロリクエファシエンスに属する微生物としては、植物病原菌の生育を抑制する物質を生産し、且つ宿主植物へ病害抵抗性を誘導することによって植物免疫機能を強化するものであれば特に限定されないが、バチルス・アミロリクエファシエンスS13-3株を使用するのが好ましい。
Hereinafter, the present invention will be described in detail.
The plant disease control agent of the present invention belongs to Bacillus amyloliquefaciens, cultivates a microorganism that produces a substance that suppresses the growth of plant pathogenic bacteria, collects the culture product, and formulates it as necessary. Can be manufactured. The microorganism belonging to Bacillus amyloliquefaciens is not particularly limited as long as it produces a substance that suppresses the growth of phytopathogenic fungi and enhances the plant immune function by inducing disease resistance to the host plant. Bacillus amyloliquefaciens strain S13-3 is preferably used.
〔実施例１〕 in vitroでの植物病原菌とバチルス・アミロリクエファシエンスS13-3株の対峙培養試験
バチルス・アミロリクエファシエンスS13-3株は、公益財団法人 新技術開発財団が所有する植物研究園から分離した細菌約20,000株の中から、寒天培地上の対峙培養によりブドウやイチゴに炭疽病を引き起こす病原菌（コレトトリカム・グロエスポリオイダス）に対して最も強い生育抑制を示した菌株であり（図１Ａ）、16S rDNA塩基配列からバチルス・アミロリクエファシエンス(Bacillus amyloliquefaciens)として同定された分離株である。
病原菌との対峙培養を実施したところ、バチルス・アミロリクエファシエンスS13-3株により生育抑制を示した病原菌の菌糸先端は、病原菌の種類に関わらず、膨潤し、破裂した（図１Ｂ）。この菌糸生育抑制はバチルス・アミロリクエファシエンスS13-3株が生産・分泌する抗生物質イツリンA(iturinA)によるものであると同定した。
バチルス・アミロリクエファシエンスS13-3株培養液中のイツリンA濃度は、培養6日後には0.064 mg/mLであり、病原菌の菌糸成長を抑制するのに必要なイツリンA最低濃度の0.01 mg/mL を超える生産量であった。また、バチルス・アミロリクエファシエンスS13-3株はトマトなどに青枯れ病を引き起こす土壌病害細菌（ラルストニア・ソラナセラム）に対しても、生育抑制効果を示した（図１Ｃ）。
[Example 1] In vitro culture test of phytopathogenic fungi and Bacillus amyloliquefaciens S13-3 strain
Bacillus amyloliquefaciens strain S13-3 is an anthracite cultivated from the plant research institute owned by the New Technology Development Foundation. It is a strain that showed the strongest growth suppression against the causative fungus (Colletotricum gloesporoidas) (Fig. 1A), an isolate identified as Bacillus amyloliquefaciens from the 16S rDNA base sequence. is there.
When opposing culture with the pathogen was carried out, the hyphal tip of the pathogen that showed growth inhibition by the Bacillus amyloliquefaciens S13-3 strain swelled and ruptured regardless of the type of the pathogen (FIG. 1B). This hyphal growth inhibition was identified to be caused by the antibiotic Iturin A (iturinA) produced and secreted by the Bacillus amyloliquefaciens S13-3 strain.
The iturin A concentration in the culture solution of Bacillus amyloliquefaciens strain S13-3 is 0.064 mg / mL after 6 days of culture, and 0.01 mg / mL, the lowest concentration of iturin A necessary to suppress mycelial growth of pathogenic bacteria. The production volume exceeded mL. Moreover, Bacillus amyloliquefaciens S13-3 strain also showed a growth inhibitory effect against a soil disease bacterium (Ralstonia solanacerum) that causes bacterial wilt disease in tomato and the like (FIG. 1C).
〔実施例２〕 ブドウ圃場散布試験
圃場散布試験において、バチルス・アミロリクエファシエンスS13-3株は自然発生するブドウ晩腐病（コレトトリカム・グロエスポリオイダスによる）の発生を抑制した。2009年度に行ったブドウ房および葉面への散布試験では、ブドウ晩腐病発症率は、それぞれ、コントロール（無処理）40.9%、SCD（Soybean-Casein Digest）液体培地91.1%、バチルス・アミロリクエファシエンスS13-3 株培養液0%であった（図２Ａ）。コントロールとSCD液体培地を比較すると、SCD液体培地自体はブドウ晩腐病の発症率の抑制に繋がらないことが明らかであり、逆に栄養分となる培地を散布したことによりブドウ晩腐病菌の感染を増長した。1晩培養したバチルス・アミロリクエファシエンスS13-3株の培養液を散布したブドウでは晩腐病の発症が確認されなかったことから、バチルス・アミロリクエファシエンスS13-3 株の培養液はブドウ晩腐病に対する高い防除効果があることが示された。2010年度に行った同散布試験のブドウ晩腐病発症率は、それぞれ、コントロール（無処理）70.2%、SCD液体培地95.4%、バチルス・アミロリクエファシエンスS13-3株 培養液30.7%であった（図２Ｂ）。
一方、コレトトリカム・グロエスポリオイダスによるイチゴ炭疽病に対しても、バチルス・アミロリクエファシエンスS13-3株は防除効果を示した（図３）。本散布試験では市販微生物農薬「タフパール」を対照区として使用したが、イチゴ炭疽病に対するタフパールとバチルス・アミロリクエファシエンスS13-3株の防除効果は等しいことが示された（図３）。バチルス・アミロリクエファシエンスS13-3株の適用範囲の拡大を目的にバチルス・アミロリクエファシエンスS13-3 株の抗菌スペクトルを検討した結果、供試した植物病原菌25種の内、24種に対し拮抗作用を示した(表１)。
[Example 2] Grape field spray test In the field spray test, Bacillus amyloliquefaciens S13-3 strain suppressed the occurrence of naturally occurring grape late rot (due to Choletotricum gloespolioidus). In 2009, the incidence of late grape rot was 40.9% for control (no treatment), 91.1% for SCD (Soybean-Casein Digest) liquid medium, and Bacillus amylolique. The culture solution of Facience S13-3 strain was 0% (FIG. 2A). Comparing the control and SCD liquid medium, it is clear that the SCD liquid medium itself does not lead to the suppression of the incidence of grape late rot, and conversely, the infection of the grape late rot fungus by spraying the nutrient medium. Increased. Since the onset of late rot was not confirmed in grapes sprayed with overnight culture of Bacillus amyloliquefaciens strain S13-3, the culture solution of Bacillus amyloliquefaciens strain S13-3 It was shown to have a high control effect against late rot. The incidence of late grape rot in the same spraying test conducted in 2010 was 70.2% for control (no treatment), 95.4% for SCD liquid medium, and 30.7% for Bacillus amyloliquefaciens strain S13-3. (FIG. 2B).
On the other hand, Bacillus amyloliquefaciens S13-3 strain also showed a control effect against strawberry anthracnose caused by Coretotricum gloesporoidas (FIG. 3). In this spraying test, the commercially available microbial pesticide “Tuff Pearl” was used as a control, but it was shown that the control effects of Tuff Pearl and Bacillus amyloliquefaciens S13-3 against strawberry anthracnose were equal (FIG. 3). As a result of investigating the antibacterial spectrum of Bacillus amyloliquefaciens S13-3 for the purpose of expanding the scope of application of Bacillus amyloliquefaciens S13-3, 24 out of 25 phytopathogenic fungi tested Antagonism was shown (Table 1).
病原菌との拮抗試験（阻止帯の平均距離、及び拮抗度）
〔実施例３〕 青枯れ病菌とバチルス・アミロリクエファシエンスS13-3株のトマトポット苗試験
本病害は土壌病原細菌によるものであることから、本試験ではポット苗の土壌へバチルス・アミロリクエファシエンスS13-3株の処理試験を実施した。
バチルス・アミロリクエファシエンスS13-3株をTSB(Tryptic soy broth)培地100mLに37℃で一晩培養した培養液5mL (1.8-5.6×108cfu/mL)をラルストニア・ソラナセラムによるトマト青枯れ病菌5mL(1.0×107 cfu/mL)とともに、本葉6-7葉展開のトマト苗(9cmポット苗)の土壌に接種した。その後、植物培養器にて明期16時間、25℃、暗期8時間、18℃にて培養を行い、バチルス・アミロリクエファシエンスS13-3株の防除効果を検討した結果、バチルス・アミロリクエファシエンスS13-3株処理区では、無処理区で認められた「土際部の褐変」、「苗の萎凋」が全く認められなかった（図４）。従って、トマト青枯れ病に対してバチルス・アミロリクエファシエンスS13-3株は防除効果があると判断された。
[Example 3] Tomato pot seedling test of bacterial wilt and Bacillus amyloliquefaciens strain S13-3 Since this disease is caused by soil pathogenic bacteria, in this test Bacillus amyloliquefacii A processing test of ENS S13-3 strain was performed.
Bacillus amyloliquefaciens S13-3 strain cultured in TSB (Tryptic soy broth) medium at 37 ° C overnight at 37 ° C. 5 mL (1.8-5.6 × 10 8 cfu / mL) of tomato bacterial wilt caused by Ralstonia solanacerum Along with 5 mL (1.0 × 10 7 cfu / mL), the soil of 6-7 leaves of tomato seedlings (9 cm pot seedlings) with real leaves was inoculated. Thereafter, the cells were cultured in a plant incubator for 16 hours in the light period, 25 ° C., 8 hours in the dark period, and 18 ° C., and the control effect of the Bacillus amyloliquefaciens S13-3 strain was examined. As a result, Bacillus amylolique In the treatment group of Facience S13-3 strain, “browning of the shore part” and “wilt of seedlings” observed in the untreated group were not observed at all (FIG. 4). Therefore, it was judged that Bacillus amyloliquefaciens S13-3 strain has a controlling effect against tomato bacterial wilt.
〔実施例４〕 うどんこ病菌とバチルス・アミロリクエファシエンスS13-3株の土壌接種処理によるトマトポット苗試験
本病害は、トマトの温室栽培において、深刻な病気の1つであり、生きたトマトにしか感染しない絶対寄生菌によるものである。バチルス・アミロリクエファシエンスS13-3株をトマト根に接種することによって、トマトが植物免疫を獲得し、うどんこ病を抑制するかどうかを確認するため、本試験ではポット苗の土壌へバチルス・アミロリクエファシエンスS13-3株の処理試験を実施した。
上記実施例３と同様の条件にて培養したバチルス・アミロリクエファシエンスS13-3株培養液とトマト葉から採取したうどんこ病菌の胞子を滅菌水に懸濁して約104 spores/mLに調整した胞子懸濁液を準備した。上記バチルス・アミロリクエファシエンスS13-3株培養液5mLを土壌接種処理し、うどんこ病菌胞子懸濁液5mLをトマト葉に散布処理して、直ちに植物培養器で明期16時間、25℃、暗期8時間、18℃にて培養を行った。その結果、地上部のトマト葉にはうどんこ病の病徴がほとんど認められなかった。従って、バチルス・アミロリクエファシエンスS13-3株の土壌処理はうどんこ病に対する植物免疫を誘導したと判断した（図５）。バチルス・アミロリクエファシエンスS13-3株の土壌処理により、土壌とは離れた地上部の葉で、病原性関連タンパク質（PRプロテイン）であるキチナーゼおよびβ-1，3-グルカナーゼの遺伝子発現および酵素活性が顕著に増加した（図６）。これはバチルス・アミロリクエファシエンスS13-3株がトマトに植物免疫（全身獲得抵抗性）を誘導したことを強く示唆した。この植物免疫により、トマトうどんこ病を効率よく防除できると判断された（図5）。
[Example 4] Tomato pot seedling test by soil inoculation treatment with powdery mildew fungus and Bacillus amyloliquefaciens strain S13-3 This disease is one of serious diseases in greenhouse cultivation of tomato, and live tomato It is due to an absolute parasitic bacterium that only infects worms. To inoculate Bacillus amyloliquefaciens strain S13-3 on tomato roots, to confirm whether tomatoes acquire plant immunity and suppress powdery mildew, in this study, Bacillus A treatment test of amyloliquefaciens strain S13-3 was performed.
Bacillus amyloliquefaciens strain S13-3 cultured under the same conditions as in Example 3 and powdery mildew spores collected from tomato leaves were suspended in sterile water and adjusted to about 10 4 spores / mL. A prepared spore suspension was prepared. The above-mentioned Bacillus amyloliquefaciens S13-3 strain culture solution 5mL is inoculated with soil, powdery mildew fungus spore suspension 5mL is sprayed on tomato leaves, immediately in the plant incubator for 16 hours, 25 ° C, Culture was performed at 18 ° C. for 8 hours in the dark period. As a result, almost no symptoms of powdery mildew were observed on the tomato leaves above the ground. Therefore, it was determined that soil treatment of Bacillus amyloliquefaciens S13-3 strain induced plant immunity against powdery mildew (FIG. 5). Soil treatment of Bacillus amyloliquefaciens strain S13-3, and the gene expression and enzymes of pathogenicity related proteins (PR proteins) chitinase and β-1,3-glucanase on the leaves above the soil The activity increased significantly (FIG. 6). This strongly suggested that Bacillus amyloliquefaciens strain S13-3 induced plant immunity (systemic acquired resistance) in tomato. It was determined that this plant immunity could effectively control tomato powdery mildew (Fig. 5).
〔実施例５〕 バチルス・アミロリクエファシエンスS13-3株の葉面散布処理によるトマトポット苗試験
続いて、バチルス・アミロリクエファシエンスS13-3株の葉面処理によっても、処理葉において、植物免疫が誘導されることを確認した（図７）。植物免疫誘導物質（植物免疫エリシター）はバチルス・アミロリクエファシエンスS13-3株により細胞外へ分泌される物質であった（図８）。
[Example 5] Tomato pot seedling test by foliar spraying treatment of Bacillus amyloliquefaciens S13-3 strain Subsequently, by foliar treatment of Bacillus amyloliquefaciens S13-3 strain, It was confirmed that immunity was induced (FIG. 7). The plant immunity inducer (plant immunity elicitor) was a substance secreted extracellularly by the Bacillus amyloliquefaciens S13-3 strain (FIG. 8).
上記実施例より、バチルス・アミロリクエファシエンスS13-3株は、農薬登録基準をクリアーできるレベルで、ブドウ、イチゴ、トマトの病害に適用できる。
加えて、抗生物質生産能並びに植物免疫誘導能という多機能性をバチルス・アミロリクエファシエンスS13-3株が有するため、植物病害防除効果および植物免疫機能強化の向上につながる使用法例として以下に示す。
土壌処理による直接的な土壌病害防除、すなはち、育苗時に1回、移植後2週間毎に1回処理を実施する。続いて、移植後、1週間毎に1回葉面散布処理による直接的な地上部病害防除および植物免疫誘導による間接的な地上部病害防除により、効果的に上記病害を抑制することができる（図９）。
From the above examples, the Bacillus amyloliquefaciens S13-3 strain can be applied to diseases of grapes, strawberries, and tomatoes at a level that can satisfy the pesticide registration standard.
In addition, since the Bacillus amyloliquefaciens S13-3 strain has the multifunctionality of antibiotic production ability and plant immunity induction ability, the following usage examples lead to improvement of plant disease control effect and enhancement of plant immune function .
Direct soil disease control by soil treatment, that is, treatment once at the time of raising seedlings and once every 2 weeks after transplanting. Subsequently, after transplantation, the above-mentioned diseases can be effectively suppressed by direct control of the above-ground diseases by foliar spray treatment once a week and indirect control of the above-ground diseases by induction of plant immunity ( FIG. 9).
Claims (3)
A cultivation method for treating a plant with the microorganism preparation according to claim 2.
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| JP7084414B2 (en) | 2017-03-27 | 2022-06-14 | テンフォールド テクノロジーズ リミティッド ライアビリティ カンパニー | Methods and Agricultural Compositions for Preventing or Controlling Plant Diseases |
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| JP7122765B2 (en) | 2017-06-21 | 2022-08-22 | フンダシオン ウニヴェルシタリア サン パブロ | Inhibitory Potential of Raspberry and Strawberry Extracts on Secondary Metabolic Stimulants Bacillus Amyloliquefaciens QV15 and Metabolic Syndrome Associated Enzymes for Secondary Metabolism of Phenolic Compounds |
| JP2020535108A (en) * | 2017-09-28 | 2020-12-03 | ローカス アグリカルチャー アイピー カンパニー エルエルシー | Treatment of plant mosaic virus and bacterial infections |
| JP7390191B2 (en) | 2017-09-28 | 2023-12-01 | ローカス アグリカルチャー アイピー カンパニー エルエルシー | Treatment of mosaic virus and bacterial infections in plants |
| JP2020002063A (en) * | 2018-06-28 | 2020-01-09 | 国立研究開発法人農業・食品産業技術総合研究機構 | Plant disease inhibitory composition for agricultural and horticultural use, and method for culturing agricultural and horticultural plants |
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