JPH02209803A - Prevention of plant disease with bacterium - Google Patents
Prevention of plant disease with bacteriumInfo
- Publication number
- JPH02209803A JPH02209803A JP1029686A JP2968689A JPH02209803A JP H02209803 A JPH02209803 A JP H02209803A JP 1029686 A JP1029686 A JP 1029686A JP 2968689 A JP2968689 A JP 2968689A JP H02209803 A JPH02209803 A JP H02209803A
- Authority
- JP
- Japan
- Prior art keywords
- bacteria
- strain
- bacillus subtilis
- culture
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
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- 208000015181 infectious disease Diseases 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 235000021332 kidney beans Nutrition 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
【発明の詳細な説明】
皮呈上段剋■立国
本発明は、微生物により植物の病害を抑制し、健全な野
菜を栽培する方法およびそれに用いるバチルス属に属す
る新規な微生物に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of suppressing plant diseases using microorganisms and cultivating healthy vegetables, and a novel microorganism belonging to the genus Bacillus used therein.
狐米（２）韮歪
従来、農園芸用作物の病害を防除するため多（の合成殺
菌剤が用いられているが、環境への影響や薬剤耐性をも
った病原菌の出現などの問題があり、生物農薬が注目さ
れている。すなわち、病害菌に対し拮抗作用を有する微
生物を植物体に直接または畑作上に、添加処理して作物
の病害菌による感染、畑作土壌内での病害菌の生育ある
いは病害作用を抑制する生物防除の方法が開発されてい
る。Fox rice (2) Dwarf strain Traditionally, synthetic fungicides have been used to control diseases in agricultural and horticultural crops, but there are problems such as the impact on the environment and the emergence of drug-resistant pathogenic bacteria. , biopesticides are attracting attention.In other words, microorganisms that have an antagonistic effect on disease-causing bacteria are added directly to plants or on field crops to prevent infection of crops by disease-causing bacteria and the growth of disease-causing bacteria in the soil of field crops. Alternatively, biological control methods have been developed to suppress the effects of the disease.
一方、微生物の生産する抗生物質が種々の植物病害に効
果のあることが見出されてきている０例えば、バチルス
属に属するある種の菌株は園芸作物の灰色かび病、菌核
病、うどんこ病、灰星病に対し顕著な防除効果を有する
抗かび抗生物質であるプルマイシン（４−Ｎ−Ｄ−アラ
ニル−２，４−ジアミノ−２，４−ジデオキシアラビノ
ーズ）を生産することが知られており （特開昭５４−
１５７８９８号）、またバチルス属に属する他のある種
の菌株は灰色かび病、炭そ病、いもち病、紋枯病に対し
て高い防除効果を有するペプチド系抗生物質イツリンＡ
を生産することが知られている（特開昭６１−２８００
９５号、Ｔｅｔｒａ−ｈｅｄｒｏｎ Ｌｅｔｔｅｒｓ
Ｔｈ３０．３０６５〜３０６８．１９８２） 。On the other hand, it has been discovered that antibiotics produced by microorganisms are effective against various plant diseases. Purmycin (4-N-D-alanyl-2,4-diamino-2,4-dideoxyarabinose) is known to produce purmycin (4-N-D-alanyl-2,4-diamino-2,4-dideoxyarabinose), an antifungal antibiotic that has a remarkable control effect on P. (Unexamined Japanese Patent Publication No. 1973-
157898), and some other strains belonging to the genus Bacillus contain iturin A, a peptide antibiotic that has a high control effect against botrytis, anthracnose, blast, and sheath blight.
It is known to produce (JP-A-61-2800
No. 95, Tetra-hedron Letters
Th30.3065-3068.1982).
しかし、これらプルマイシンやイツリンＡの効果は微生
物より分離して施用した場合の効果であって、微生物自
体を作物に施用し、灰色かび病やうどんこ病を防除する
有効な微生物は知られていない。However, the effects of purmycin and iturin A are only when applied separately from the microorganism, and there are no known microorganisms that are effective in controlling gray mold and powdery mildew by applying the microorganisms themselves to crops. .
■が”° しようとする課
上述のような微生物から生産される抗生物質を微生物か
ら単離することなく、抗生物質を生産する微生物を直接
作物に適用し得れば極めて好都合であり、そのような微
生物農薬の開発が望まれている。``°'' It would be extremely convenient if microorganisms that produce antibiotics could be directly applied to crops without isolating them from the microorganisms mentioned above. The development of microbial pesticides is desired.
本発明は、プルマイシンを生産する能力を有する微生物
を自然界より広く検索し、灰色かび病菌、うどんこ病菌
、べと病菌、赤錆病菌等に原因する病害を抑制し得る微
生物及び該微生物を利用して主として野菜類の細菌によ
る植物病害を防除する方法を提供することを課題とする
。The present invention searches widely for microorganisms in nature that have the ability to produce purmycin, and utilizes microorganisms and microorganisms that can suppress diseases caused by gray mold, powdery mildew, downy mildew, rust fungi, etc. The object of the present invention is to provide a method for controlling plant diseases mainly caused by bacteria in vegetables.
量 を ンするための
本発明は、自然界より分離したバチルス属に属し、プル
マイシンを生産する能力を有する新規なバチルス・ズブ
チルスｓ、ｐ、 Ｋ Ｂ−１１１１（Ｂａｃｉｌｌｕｓ
ｓｕｂｔｉｌｉｓ ｓ、ｐ、 Ｋ Ｂ−１１１１）株（
徽工研菌寄第１７３８号）およびバチルス・ズブチルス
Ｓ、ρ、 Ｋ Ｂ−１１２２（Ｂａｃｌｌｌｕｓｓｕｂ
ｔｔｌｌｓ ｓ、ｐ、 Ｋ Ｂ−１１２２）株（８１Ｌ
工研菌寄第１７３９号）を利用するものである。The present invention is directed to the use of novel Bacillus subtilis s, p, KB-1111, which belongs to the genus Bacillus and is isolated from nature and has the ability to produce purmycin.
subtilis s, p, KB-1111) strain (
Bacillus subtilis S, ρ, K B-1122 (Bacillus subtilis S, ρ, K B-1122)
ttlls s, p, KB-1122) strain (81L
This method utilizes Koken Bibori No. 1739).
バチルス・ズブチルスｓ、ｐ、 Ｋ Ｂ−１１１１菌株
（以下単にＫ Ｂ−１１１１菌と記す）およびバチルス
・ズブチルスｓ、ｐ、Ｋ Ｂ−１１２２菌株く以下単に
ＫＢ−１１２２菌と記す）は後述するような培養条件で
培養するときプルマイシン等の抗生物質を生産し、この
生菌体を野菜に噴霧し生育させるとき灰色かび病、うど
んこ病、べと病、赤錆病等に優れた効果を示す。Bacillus subtilis s, p, KB-1111 strains (hereinafter simply referred to as KB-1111 bacteria) and Bacillus subtilis s, p, KB-1122 strains (hereinafter simply referred to as KB-1122 bacteria) are as described below. When cultivated under suitable culture conditions, it produces antibiotics such as purmycin, and when the live bacteria are sprayed onto vegetables and allowed to grow, they exhibit excellent effects against botrytis, powdery mildew, downy mildew, rust, etc.
本発明者等が新潟県寺泊の土壌から分離した菌株Ｋ Ｂ
−１１１１菌は次のような菌学的性質を有する。Bacterial strain KB isolated by the present inventors from soil in Teradomari, Niigata Prefecture
-1111 bacteria has the following mycological properties.
観察事項 Ｋ Ｂ−１１１１閉ａ）形態
（１）形および大きさ
（２）多形性
（３）運動性とべん毛
（４）胞子の有無
胞子の形
胞子の形成部位
（５）ダラム染色性
（６）抗酸性
ｂ）生育状況
（１）肉汁寒天平板培養
桿菌（両端丸みあり）
単一
有り
有り
卵円形
中心
陽性
無し
コロニーは灰白色か
クリーム色、光沢少
々あり、円形で大き
さは直径２〜４＋ａｍ、集
落***形は中門、周
縁形は波状、
粘性あり
培地表面に広がって
増殖し、色は灰白色
で光沢少々あり、粘
性あり、拡散性色素
はなし
１〜２日目で培地表面
に菌膜をつくり全体
に覆う。混濁なく、
菌体は灰白色
拡散性で層状の天日
色コロニ
肉汁ゼラチン穿刺培養 ２０℃、３０℃で培養すと液化
が始まる。そ
の聖地は層状。Observations K B-1111 closed a) Morphology (1) Shape and size (2) Pleomorphism (3) Motility and flagella (4) Presence or absence of spores Spore shape Site of spore formation (5) Durham staining ( 6) Anti-acidity b) Growth status (1) Broth agar plate culture Bacillus (rounded at both ends) Single present, oval center not positive Colonies are grayish-white or cream-colored, slightly glossy, round, and 2-4+ am in diameter The shape of the colony is a central gate, the shape of the periphery is wavy, and it is viscous. It spreads and proliferates on the surface of the medium. The color is grayish white with a little gloss, it is viscous, and there is no diffusible pigment. It forms a bacterial film on the surface of the medium in 1 to 2 days. Cover the entire structure. There is no turbidity, and the bacterial cells are grayish-white and diffusive, layered, sun-coloured colonies, and gelatin puncture culture. When cultured at 20°C or 30°C, liquefaction begins. The sacred place is layered.
テストの方法
硝酸塩肉汁 有り
駒形らの方法 無し
ばれいしょ切片
（３）肉汁液体培地
（２）肉汁寒天斜面
Ｃ）生理学的性質
１）硝酸塩の還元
２）脱窒反応
３）ＭＲ陰性
４）ＶＰ 陽性５
）インドールの生成 無し６）硫
化水素の生成 ＴＳＩ寒天 無し酢酸鉛試験
紙を用いる方法 肉汁 無し運動性検査用培地
無し
７）クエン酸の利用 Ｋｏｓｅｒ ｃｉｔｒａｔｅ ｍ
ｅｄｉｕ−有りＣｈｒｉｓｔｅｎｓｅｎ ａｇａｒ
有り８）デンプンの分解
陽性９）色素の生成 じゃがいも切片 無し
１０）無機窒素源の利用試験
硫酸アンモニウム 有り
硝酸ナトリウム 無し
グルタミン酸ソーダ 有り
カザミノ酸ｖ−１ｒｅｅ 有り
１１）ウレアーゼ ＣｈｒｉｓＬｅｎｓｅｎ尿素培地
有り１２）オキシダーゼ 陽
性１３）カタラーゼ 陽性１
４）カゼインの分解 カゼイン２％寒天 有りリドマ
スミルク ペプトン化と色素還元有りＬＶ寒天
有りブドウ糖肉汁での嫌気性発育
無し生育の範囲（肉汁培地）
４５℃における発育 有り
６５℃における発育 無し
ｐＨ５〜９における発育 有り
７％ＮａＣｌにおける発育有り
リゾチーム感受性０．００１％ブドウ糖肉汁有り糖から
酸の生成 グルコース ◎シュクロース
◎
マンノース ◎
グリセリン ◎
ソルビット ◎
フラクトース ◎
マンニット ◎
キシロース △
アラビノース ○
デンプン Δ
ラクトース ○
麦芽糖 ◎
イノジット Ｏ
トレハロース ○
ガラクトース ×
ラフィノース Δ〜０
２１）アジ化ナトリウム０．０２％生育ブイヨン無し２
２）千ロジンの分解 チロシン寒天 無し一方、
本発明者等が茨城県新治郡の土壌から分離した菌株ＫＢ
−１１２２菌は次のような国学的性質を有する。Test method With nitrate meat juice Method of Komagata et al. Without Potato sections (3) Meat juice liquid medium (2) Meat juice agar slope C) Physiological properties 1) Nitrate reduction 2) Denitrification reaction 3) MR negative 4) VP positive 5
) Production of indole None 6) Production of hydrogen sulfide TSI agar None Method using lead acetate test paper Meat juice None Motility test medium
None 7) Use of citric acid
ediu-with Christensen agar
Yes 8) Decomposition of starch
Positive 9) Pigment formation Potato slices None 10) Inorganic nitrogen source utilization test Ammonium sulfate Yes Sodium nitrate No Sodium glutamate Yes Casamino acid v-1ree Yes 11) Urease Chris Lensen urea medium Yes 12) Oxidase positive 13) Catalase Positive 1
4) Decomposition of casein Lidomus milk with casein 2% agar LV agar with peptonization and pigment reduction
Yes Anaerobic growth in glucose broth None Growth range (broth medium) Growth at 45°C Yes Growth at 65°C None Growth at pH 5-9 Yes Growth in 7% NaCl Lysozyme sensitivity 0.001% glucose in broth Sugar to acid Production of glucose ◎Sucrose
◎ Mannose ◎ Glycerin ◎ Sorbitol ◎ Fructose ◎ Mannitol ◎ Xylose △ Arabinose ○ Starch Δ Lactose ○ Maltose ◎ Inosit O Trehalose ○ Galactose × Raffinose Δ~0 21) Sodium azide 0.02% without growth broth 2
2) Decomposition of 1,000 rosin Tyrosine agar None On the other hand,
Bacterial strain KB isolated by the present inventors from soil in Niiharu District, Ibaraki Prefecture
-1122 bacterium has the following national characteristics.
観察事項 ＫＢ−１１２２凹（５）ダラム
染色性
（６）抗酸性
ｂ）生育状況
（１）肉汁寒天平板培養
肉汁寒天斜面
ａ）形態
（１）形および大きさ
（２）多形性
（３）運動性とべん毛
（４）胞子の有無
胞子の形
胞子の形成部位
桿菌（両端丸みあり）
単一
存り
有り
卵円形
中心
肉汁液体培地
ばれいしょ切片
肉汁ゼラチン穿刺培養
陽性
無し
コロニーは灰白色か
クリーム色、光沢な
く不正円形で大きさ
は直径２〜６ｍｔａ、***
形で周縁は波状
培地表面に広がって
増殖し、色は灰白色
かクリーム色
拡散性色素はなし
１〜２日目で培地表面
に菌膜をつくり全体
に覆う、混濁なく、
国体は灰白色
拡散性で皺杖の天日
色コロニ
２０℃、３０℃で培養す
と液化が始まる。そ
の形は層状。Observation items KB-1122 concave (5) Durham stainability (6) acid fast b) Growth status (1) Juice agar plate culture Juice agar slope a) Morphology (1) Shape and size (2) Pleomorphism (3) Motility and flagella (4) Presence of spores Spore shape Site of spore formation Bacillus (rounded at both ends) Single present Oval center Broth juice Liquid medium Potato section Flesh gelatin Puncture culture No positive Colony is grayish white or cream colored, glossy It is irregularly round, 2 to 6 mta in diameter, has a raised shape and a wavy edge, and grows on the surface of the medium.The color is grayish-white or cream, and there is no diffusible pigment.After 1 to 2 days, a bacterial film is formed on the surface of the medium, and the entire surface of the medium grows. The Kokutai is grayish-white and diffusive, and the sun-coloured colonies of wrinkled canes begin to liquefy when cultured at 20°C or 30°C. Its shape is layered.
Ｃ）生理学的性質 テストの方法
１）硝酸塩の還元 硝酸塩肉汁 有り２）
脱窒反応 駒形らの方法 無し３）ＭＲ
陰性
４）ＶＰ 陽性５
）インドールの生成 無し６）硫
化水素の生成 ＴＳＴ寒天 無し酢酸鉛試験
紙を用いる方法 肉汁 有り運動性検査用培地
無し
７）クエン酸の利用 Ｋｏｓｅｒ ｃｉＬｒａｔｅ ＋
ａｅｄｉｕｓ有りＣｈｒｔｓｔｅｎｓｅｎ ａｇａｒ
有り８）デンプンの分解
陽性９）色素の生成 じゃがいも切片 無
し１０）無機窒素源の利用試験
硫酸アンモニウム 有り
硝酸ナトリウム 有り
グルタミン酸ソーダ 有り
カザミノ酸ｖ−ｔｒｅｅ 有り
ウレアーゼ Ｃｈｒｉｓｔｅｎｓｅｎ尿素培地 有り
オキシダーゼ 陽性カタラーゼ
陽性カゼインの分解 カゼ
イン２％寒天 有りリドマスミルクペプトン化と色素
還元有りＬＶ寒天 有りブ
ドウ糖肉汁での嫌気性発育 無し生育の範囲（
肉汁培地）
４５℃における発育 有り
６５℃における発育 無し
ｐＨ５〜９における発育 有り
７％ＮａＣ１における発育有り
リゾチーム感受性０．００１％ブドウ糖肉汁有り糖から
酸の生成 グルコース ◎シュクロース
◎
マンノース ◎
グリセリン ◎
ソルビット ◎
フラクトース ◎
マンニット ○
キシロース ○
アラビノース ○
デンプン ○
ラクトース ○
麦芽糖 ○
イノジット Ｏ
トレハロース ○
ガラクトース Δ
ラフィノース ×
２１）アジ化ナトリウム０．０２％生育ブイヨン無し２
２）チロシンの分解 チロシン寒天 無し以上の
菌学的性質から、バジヱイズマニュアル（Ｂｅｒ’ｇｅ
ｙ’ｓ ｍａｎｕａｌ ｏｆ ｓｙｓｔｅｍａｔｉｃ
ｂａｃｔｅｒｉｏｌｏｇｙ）を参照として同定を行った
結果、２菌株共にバチルス・ズブチルス（Ｂａｃｉｌｌ
ｕｓ ５ｕｂｔｉｌｕｓ）に属する菌種と同定された。C) Physiological properties Test method 1) Nitrate reduction With nitrate broth 2)
Denitrification reaction Komagata et al. method None 3) MR
Negative 4) VP Positive 5
) Production of indole None 6) Production of hydrogen sulfide TST agar None Method using lead acetate test paper Meat juice Present Motility test medium
None 7) Use of citric acid Koser ciLrate +
Chrtstensen agar with aedius
Yes 8) Decomposition of starch
Positive 9) Pigment formation Potato slices None 10) Inorganic nitrogen source utilization test Ammonium sulfate Yes Sodium nitrate Yes Sodium glutamate Yes Casamino acid v-tree Yes Urease Christensen urea medium Yes Oxidase Positive Catalase Positive Casein degradation Casein 2% agar Yes Lidomus With milk peptonization and pigment reduction LV agar With anaerobic growth in glucose broth Without growth range (
Growth at 45°C Yes Growth at 65°C No Growth at pH 5-9 Yes Growth at 7% NaCl Lysozyme sensitivity 0.001% glucose Meat juice Production of acid from sugar Glucose ◎Sucrose
◎ Mannose ◎ Glycerin ◎ Sorbitol ◎ Fructose ◎ Mannitol ○ Xylose ○ Arabinose ○ Starch ○ Lactose ○ Maltose ○ Inosit O Trehalose ○ Galactose Δ Raffinose × 21) Sodium azide 0.02% No growth broth 2
2) Decomposition of tyrosine Tyrosine agar None Due to the above mycological properties, the Berg'is Manual (Ber'ge
y's manual of systematic
As a result of identification using Bacillus subtilis as a reference, both strains were identified as Bacillus subtilis.
It was identified as a bacterial species belonging to U.S. 5ubtilus).
培養条件
上記菌株の培養は、発酵学の分野で公知の常法に従って
行うことができる。培地としては、この菌株が資化可能
な炭素源及び窒素源を適当量含有し、必要に応じて無機
塩、微量発育促進物質、消泡剤等を添加したものが使用
される。具体的には、炭素源としては、グルコース、フ
ラクトース、マルトース、ガラクトース、リボース、サ
ッカロス、澱粉、糖蜜、廃糖蜜等の糖類、グリセロール
、マンニトール等のアルコール類、ピルビン酸、酢酸、
クエン酸等の脂肪酸類、グリシン、グルタミン酸、グル
タミン、アラニン、アスパラギン等のアミノ酸類等一般
的な炭素源より使用する微生物の資化性を考慮して、一
種又は二種以上を適宜選択し使用すればよい。Culture Conditions The above bacterial strain can be cultured according to conventional methods known in the field of fermentation science. The medium used is one containing appropriate amounts of carbon sources and nitrogen sources that can be assimilated by the strain, and to which inorganic salts, trace growth-promoting substances, antifoaming agents, etc. are added as necessary. Specifically, carbon sources include sugars such as glucose, fructose, maltose, galactose, ribose, saccharose, starch, molasses, blackstrap molasses, alcohols such as glycerol and mannitol, pyruvic acid, acetic acid,
One or more types should be selected and used as appropriate from common carbon sources such as fatty acids such as citric acid, amino acids such as glycine, glutamic acid, glutamine, alanine, and asparagine, taking into consideration the assimilation ability of the microorganisms used. Bye.
窒素源としては、肉エキス、ペプトン、酵母エキス、乾
燥酵母、大豆加水分解物、大豆粉、ミルクカゼイン、カ
ザミノ酸、各種アミノ酸、コーンスチーブリカー等動物
、植物、微生物の加水分解物等の有機窒素化合物、アム
モニア、硝酸アンモニウム、硫酸アンモニウム、塩化ア
ンモニウム等のアンモニウム塩、硝酸ナトリウム等の硝
酸塩、尿素等、無機窒素化合物より使用微生物の資化性
を考慮し、一種又は二種以上を適宜選択して使用すれば
よい。Nitrogen sources include meat extract, peptone, yeast extract, dried yeast, soybean hydrolyzate, soy flour, milk casein, casamino acids, various amino acids, corn stew liquor, and other organic nitrogen sources such as animal, plant, and microbial hydrolysates. Compounds, ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, nitrates such as sodium nitrate, urea, etc., and inorganic nitrogen compounds such as urea should be considered, and one or more of them should be selected and used as appropriate, taking into consideration the assimilation ability of the microorganisms used. Bye.
さらに、無機塩として微量のマグネシウム、マンガン、
鉄、カルシウム、カリウム、等のリン酸塩、塩酸塩、硫
酸塩、酢酸塩等の一種又は二種以上を適宜添加し、必要
に応じて植物油、界面活性剤などの消泡剤を添加しても
よい。In addition, trace amounts of magnesium, manganese,
One or more types of phosphates, hydrochlorides, sulfates, acetates, etc. of iron, calcium, potassium, etc. are added as appropriate, and if necessary, antifoaming agents such as vegetable oil and surfactants are added. Good too.
培養は、前記培地成分を含有する液体培地中で振とう培
養、通気攪拌培養、静置培養、連続培養等の通常の培養
法より使用微生物に適した培養法を選択して行う。Cultivation is carried out in a liquid medium containing the above-mentioned medium components by selecting a culture method suitable for the microorganism used from among ordinary culture methods such as shaking culture, aerated agitation culture, static culture, and continuous culture.
培養条件は、培地の種類、培養法により適宜選択すれば
よく、本菌株が増殖し、プルマイシンを生産できる条件
、あるいは本菌株が増殖しプルマイシンを生産する能力
を維持できる条件であれば特に制限はない。通常は培養
開始のｐＨを６〜８に調整し、２５〜３５℃の温度条件
で培養することが好ましい、培養日数は通常２〜７日が
適当である。The culture conditions may be selected as appropriate depending on the type of medium and culture method, and there are no particular restrictions as long as the conditions allow this strain to grow and produce purmycin, or the conditions can maintain the ability of this strain to grow and produce purmycin. do not have. Usually, it is preferable to adjust the pH at the start of culture to 6 to 8 and culture at a temperature of 25 to 35°C, and the appropriate number of culture days is usually 2 to 7 days.
以上のように本菌株を培養した後、得られた培養物その
もの、培養物から遠心分離、凝集分離等の通常の方法に
よって集菌した生菌体、または生菌体を凍結乾燥、アセ
トン乾燥等の方法によって乾燥した乾燥菌体、あるいは
これらを被覆材により被覆処理したものを作物に適用す
ることによりべと病、赤錆病、うどんこ病、灰色かび病
等の防除を期することができる。After culturing this bacterial strain as described above, the obtained culture itself, viable bacteria collected from the culture by conventional methods such as centrifugation and aggregation, or viable bacteria such as freeze-drying, acetone drying, etc. By applying dried bacterial cells dried by the above method or those coated with a coating material to crops, it is possible to control downy mildew, rust, powdery mildew, gray mold, etc.
尚、上記の菌体の被覆材としては、植物種子の被覆に用
いられる被覆材、例えばメチルセルロース、アラ−ビア
ゴム、アルギン酸塩、ポリウレタンプレポリマー、ポリ
ビニルアセテートホモポリマー等の天然もしくは合成高
分子、過酸化カルシウム、炭酸カルシウム、焼石膏、ガ
ラス綿などを用いることができる。The above-mentioned coating materials for the bacterial cells include coating materials used for coating plant seeds, such as natural or synthetic polymers such as methylcellulose, gum arabic, alginates, polyurethane prepolymers, polyvinyl acetate homopolymers, and peroxide. Calcium, calcium carbonate, calcined gypsum, glass cotton, etc. can be used.
本発明に係るＫ Ｂ−１１１１菌及びＫ Ｂ −１１２
２菌がプルマイシンを生産することは次のことから知る
ことができる。KB-1111 bacteria and KB-112 according to the present invention
It can be known from the following that the two bacteria produce purmycin.
すなわち、Ｋ Ｂ−１１１１菌およびＫ Ｂ−１１２２
菌のそれぞれの培養液から菌体を除いた上滑をｐＨ３，
０に調整し、陽イオン交換樹脂アンバーライトＩＲＣ−
５０（オルガノ社製）で処理し、その吸着成分を０．５
Ｎ−アンモニウムで溶出し、中和後アンバーライトＣＧ
−５０（オルガノ社製）で吸着処理し、その吸着成分を
０．５Ｎアムモニア水で溶出し中和後、Ｃトセファデッ
クス（ファルマシア社製）で吸着処理し、その吸着成分
を０．６モル／ｌの食塩水で溶出し、さらにセファデッ
クスＬｌｌ−６０（ファルマシア社製）で処理し、吸着
成分を水で溶出して濃縮するとき、プルマイシンを白色
結晶として得ることができる。That is, KB-1111 bacteria and KB-1122
The supernatant from which the bacterial cells were removed from each bacterial culture solution was adjusted to pH 3,
0 and cation exchange resin Amberlite IRC-
50 (manufactured by Organo), and the adsorbed components were reduced to 0.5
Elute with N-ammonium and after neutralization Amberlite CG
-50 (manufactured by Organo), the adsorbed component was eluted with 0.5N ammonia water and neutralized, and then adsorbed with C Tocephadex (manufactured by Pharmacia), and the adsorbed component was 0.6 mol. Purmycin can be obtained as white crystals by elution with /l of saline, further treatment with Sephadex Lll-60 (manufactured by Pharmacia), and elution and concentration of adsorbed components with water.
以下実施例により本発明を具体的に説明する。The present invention will be specifically explained below using Examples.
実施例１ （生菌体製造例）
肉エキス３ｉ、ペプトン１０ｇ１塩化ナトリウム５ｇ、
を精製水１０００−に溶かし、ＰＨを７．０〜７．３に
調整しオートクレーブ滅菌後ＫＢ−１１ＬＬ菌およびＫ
Ｂ１１２２菌を各々別々に接種し、３０℃、１日前培養
する。この前培養液を１〜５％下記本培養培地に接種す
る６本培養は、３０℃、５日間振盪培養する。培養後、
菌体を集菌し凍結乾燥する。Example 1 (Example of producing viable bacterial cells) Meat extract 3i, peptone 10g 1 sodium chloride 5g,
Dissolve KB-11LL bacteria and K
B1122 bacteria were inoculated separately and cultured at 30°C for 1 day. Six main cultures are inoculated with 1 to 5% of this preculture solution into the following main culture medium, and cultured with shaking at 30°C for 5 days. After culturing,
Collect the bacterial cells and freeze-dry them.
本培養培地組成
酵母エキス０．５ｇ、燐酸水素−カリウム２ｇ、燐酸水
素二カリウム７ｇ、硫酸マグネシウム・７水塩０．５ｇ
、硫酸第一鉄・７水塩２ｍｇ ％ファーマメデア２０ｇ
、ラクトース２０ｇ、に水道水１００ＯＮ１を加えｐＨ
７，２に調整し、１２０℃、２０分オートクレーブで滅
菌する。Main culture medium composition Yeast extract 0.5g, potassium hydrogen phosphate 2g, dipotassium hydrogenphosphate 7g, magnesium sulfate heptahydrate 0.5g
, ferrous sulfate heptahydrate 2mg % Pharmamedea 20g
, add 100ON1 of tap water to 20g of lactose and adjust the pH.
7.2 and sterilize in an autoclave at 120°C for 20 minutes.
実施例２
実施例１によって得た凍結乾燥菌体を、インゲンを用い
た灰色かび病防除試験を下記の方法にしたがって行った
。Example 2 The freeze-dried cells obtained in Example 1 were subjected to a botrytis mold control test using kidney beans according to the following method.
インゲン子葉灰色かび病検定方法
所定濃度に調製しておい薬液（Ｋ Ｂ−１１１１または
Ｋ Ｂ−１１２２乾燥菌体１Ｏ−１５ｄ／インゲン１個
体）をインゲン子葉（発芽後約１０日）に均一に散布す
る。薬液を風乾後にポテト・サッカロース寒天培地上に
生育させておいた灰色かび閏（Ｂｏｔｒｙｔｉｓｃｉｎ
ｅｒｅａ）のコロニー先端部をコルクポーラ−にて打ち
抜き（アガーピース）１葉当り２個、計４個を子葉上に
接種する。その後インゲンポットを２０℃、高温にたも
ち、３〜４日後に発病程度を調査する。Green bean cotyledon gray mold disease assay method: Spray the chemical solution (10-15 d of dried K B-1111 or KB-1122 dry bacterial cells/1 green bean) uniformly on the common bean cotyledons (approximately 10 days after germination). do. Botrytiscin was grown on a potato sucrose agar medium after the chemical solution was air-dried.
The tips of the colonies of A. rea) are punched out using a cork polarizer (agar pieces) and inoculated onto the cotyledons with 2 colonies per leaf, a total of 4 colonies. Thereafter, the green bean pot is kept at a high temperature of 20°C, and the degree of disease onset is investigated after 3 to 4 days.
発病程度は、インゲン葉上に形成される病斑面積の、無
処理区のインゲン葉上に形成される病斑面積に対する百
分率で表わす。The degree of disease onset is expressed as a percentage of the area of lesions formed on green bean leaves to the area of lesions formed on green bean leaves of untreated plots.
表１
結果は表１に示す、また、市販の防除剤を用いた結果も
比較として示した。Table 1 The results are shown in Table 1, and the results using a commercially available insecticide are also shown for comparison.
なお、以下の表２〜４にも市販の防除剤を用いた結果を
比較として併せて示した。Note that Tables 2 to 4 below also show results using commercially available insecticides for comparison.
実施例３ キュウリベと病防除効果試験径１０ｃ＋＋＋
の素焼体を用いて栽培した第２本葉時のキュウリ葉（品
種；相撲半白、１本播き／鉢、３鉢／処理区使用）にＫ
Ｂ−１１１１，ＫＢ−１１２２培養菌体を０．７％（乾
燥菌体重量％）に水で希釈懸濁し、ｌ鉢当り５ｍ１散布
した。散布葉を風乾後、り病葉から採取したキュウリペ
と病菌胞子の懸濁液を噴霧接種し、２０〜２２℃高湿度
条件下に２４時間保ち、その後は温室内に放置した。接
種後５〜７日目にキュウリベと病の病斑面積率を調査し
、下記式により防除価を算出した。Example 3 Cucumber and disease control effect test diameter 10c+++
K on cucumber leaves at the second true leaf stage (variety: Sumo Hanshiro, 1 sown/pot, 3 pots/treated area used) grown using a clay pot.
B-1111 and KB-1122 cultured cells were diluted and suspended in water to a concentration of 0.7% (dry cell weight %) and sprayed at 5 ml per 1 pot. After the sprayed leaves were air-dried, they were spray-inoculated with a suspension of cucumber and disease fungus spores collected from the diseased leaves, kept at 20-22°C under high humidity conditions for 24 hours, and then left in a greenhouse. Five to seven days after inoculation, the lesion area ratio of cucumber and disease was investigated, and the control value was calculated using the following formula.
結果は表２に示す。The results are shown in Table 2.
実施例４ コムギうどんこ病防除効果試験径１０ｃｍの
素焼体を用いて栽培した第２葉期の幼苗コムギ（品種；
農林６４号、１６本／鉢、３鉢／処理区使用）にＫ Ｂ
４１１１、ＫＢ−１１２２墳養菌体を０．７％（乾燥菌
体重量％）に水で希釈懸濁し、１鉢当り５ｄ散布した。Example 4 Wheat powdery mildew control effect test Second leaf stage seedling wheat (variety;
Agriculture and Forestry No. 64, 16 plants/pot, 3 pots/treatment area used) with K B
4111 and KB-1122 cultured microbial cells were diluted and suspended in water to 0.7% (dry microbial weight %) and sprayed at 5 d per pot.
散布葉を風乾後、り病葉から採取したこむぎうどんこ病
菌胞子の懸濁液を噴霧接種し、２０〜２４℃高湿度条件
下に２４時間保ち、その後は温室内に放置した。接種後
９〜１１日目にコムギうどんこ病の病斑面積率を調査し
、下記式により防除価を算出した。After the sprayed leaves were air-dried, they were inoculated by spraying with a suspension of powdery mildew spores collected from the leprosy leaves, kept under high humidity conditions of 20-24°C for 24 hours, and then left in a greenhouse. The lesion area ratio of wheat powdery mildew was investigated 9 to 11 days after inoculation, and the control value was calculated using the following formula.
実施例５ キュウリうどんこ病防除効果試験径１０ｃｍ
の素焼体を用いて栽培した第２本葉時のキュウリ（品種
；相撲半白、１本／鉢、３鉢／処理区使用）にＫＢ−１
１１１、ＫＢ−１１２２培養菌体を０．７％（乾燥菌体
重量％）に水で希釈懸濁し、１鉢当り５−散布した。散
布葉を風乾後、り病葉より筆で胞子をふりかけて接種し
、ガラス温室内で発病させた。接種後９〜１１日目にキ
ュウリうどんこ病の病斑面積率を調査し、下記式により
防除価を算出した。Example 5 Cucumber powdery mildew control effect test Diameter 10cm
KB-1 was applied to cucumbers at the second true leaf stage (variety: Sumo Hanshiro, 1 plant/pot, 3 pots/treated area) grown using a clay pot.
111 and KB-1122 cultured cells were diluted and suspended in water to a concentration of 0.7% (dry cell weight %) and sprayed at 5 times per pot. After the sprayed leaves were air-dried, spores were sprinkled on the diseased leaves with a brush to inoculate them, and the disease was caused to develop in a glass greenhouse. The lesion area ratio of cucumber powdery mildew was investigated 9 to 11 days after inoculation, and the control value was calculated using the following formula.
結果は表２に示す。The results are shown in Table 2.
実施例６ コムギ赤さび病防除効果試験径１０ｃｍの素
焼体を用いて栽培した第２本葉時の幼苗コムギ（品種；
農林６４号、１６本／鉢）にＫＢ−１１１１，ＫＢ−１
１２２培養菌体を０．７％（乾燥菌体重量％）に水で希
釈懸濁し、５Ｎ１／鉢の割合で散布した。散布葉を風乾
後、り病葉から採取した結果は表２に示す。Example 6 Wheat rust control effect test Young wheat seedlings at the second true leaf stage (variety;
Norin No. 64, 16 plants/pot), KB-1111, KB-1
122 cultured cells were diluted and suspended in water to 0.7% (dry cell weight %) and sprayed at a ratio of 5N1/pot. After air-drying the sprayed leaves, the results are shown in Table 2. The results are shown in Table 2.
コムギ赤さび病菌胞子の懸濁液を噴霧接種し、２０〜２
３℃高湿度条件下に２４時間保った。その後ガラス温室
内に放置し、接種から７〜１０日後にコムギ赤さび病の
病斑面積率を調査し、下記式により防除価を算出した。A suspension of wheat rust fungus spores was spray inoculated, and 20 to 2
It was kept under high humidity conditions at 3°C for 24 hours. Thereafter, the plants were left in a glass greenhouse, and 7 to 10 days after inoculation, the lesion area ratio of wheat rust was investigated, and the control value was calculated using the following formula.
結果は表２に示す。The results are shown in Table 2.
実施例７ レタス灰色かび病防除効果試験径１０ｃｍの
素焼法を用いて播種後、温室（１５〜２５℃）で約５週
間栽培したレタス（品種：キングクラウン）の葉にＫＢ
−１１１１，ＫＢ−１１２２培養菌体を所定濃度に水で
希釈懸濁し、１鉢当り５１１１散布した。Example 7 Lettuce gray mold control effect test KB was applied to leaves of lettuce (variety: King Crown) grown in a greenhouse (15-25°C) for about 5 weeks after sowing using the bisque firing method with a diameter of 10 cm.
-1111 and KB-1122 cultured cells were diluted and suspended in water to a predetermined concentration, and 5111 cells were sprayed per pot.
散布葉を風乾後、予め灰色かび病菌をふすま培地（ふす
ま５ｇ、籾からＩｇ、水５ｄ）で２０℃、８日間培養し
、つくった菌糸の塊を１６メツシユの篩にかけて、レタ
スの上から直接に接種し、２０〜２２℃高湿度条件下に
保った。接種後５日目にレタスの灰色かび病の病斑面積
率を調査し、下記式により防除価を算出した。After air-drying the sprayed leaves, the gray mold fungus was cultured in advance at 20°C for 8 days in a bran medium (5 g of bran, Ig from the rice, and 5 d of water), and the resulting mycelial mass was passed through a 16-mesh sieve and injected directly onto the lettuce. and maintained at 20-22°C under high humidity conditions. Five days after inoculation, the lesion area ratio of gray mold on lettuce was investigated, and the control value was calculated using the following formula.
結果は表３に示す。The results are shown in Table 3.
表
実施例８ イチゴの灰色かび病防除効果試験イチゴパッ
クを用いて、いちご（品種：女峰）の実を並べ、ＫＢ−
１１１１，ＫＢ−１１２２培養菌体を０．７％（乾燥菌
体重量％）に水で希釈した懸濁液を、１パンク５−の割
合で散布した。風乾後、予め砂糖加用馬鈴薯煎汁寒天培
地を用いて２０℃で１４日間培養した灰色かび病菌の胞
子を１パックク当リ１／４シャーレ相当量付着させ、２
０〜２２℃高湿度条件下に保った。接種後４日目にイチ
ゴの灰色かび病の病斑面積率を調査し、下記式により防
除価を算出した。Table Example 8 Strawberry gray mold control effect test Using a strawberry pack, strawberries (variety: Onho) were lined up and KB-
A suspension of cultured cells of 1111 and KB-1122 diluted with water to 0.7% (dry cell weight %) was sprayed at a rate of 1 puncture and 5 centimeters. After air-drying, spores of Botrytis spp., which had been cultured in advance at 20°C for 14 days using a sugar-added potato decoction agar medium, were attached to each pack in an amount equivalent to 1/4 petri dish.
It was maintained at 0-22°C under high humidity conditions. On the fourth day after inoculation, the lesion area ratio of gray mold on strawberries was investigated, and the control value was calculated using the following formula.
事件の表示 手続補正書 平成１年７月２１日 平成１年特許願第２９６８６号 発明の名称 細菌による植物病害防除方法 結果は表４に示す。Display of incidents Procedural amendment July 21, 1999 1999 Patent Application No. 29686 name of invention Method for controlling plant diseases caused by bacteria The results are shown in Table 4.
補正をする者 事件との関係person who makes corrections Relationship with the incident
Claims (3)
Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ｓ．ｐ．ＫＢ−
１１１１）株（微工研条寄第１７３８号）及び／又はバ
チルス・ズブチルスｓ．ｐ．ＫＢ−１１２２（Ｂａｃｉ
ｌｌｕｓ ｓｕｂｔｉｌｉｓ ｓ．ｐ．ＫＢ−１１２２
）株（微工研条寄第１７３９号）を野菜類に噴霧・生育
させることを特徴とする細菌に対する植物病害防除方法
。(1) Bacillus subtilis s. p. KB-1111 (
Bacillus subtilis s. p. KB-
1111) strain (Feikoken Article No. 1738) and/or Bacillus subtilis s. p. KB-1122 (Baci
llus subtilis s. p. KB-1122
) strain (Feikoken Joyori No. 1739) is sprayed on vegetables and allowed to grow, a method for controlling plant diseases against bacteria.
ブチルスｓ．ｐ．ＫＢ−１１１１（Ｂａｃｉｌｉｕｓ
ｓｕｂｔｉｌｉｓｓ．ｐ．ＫＢ−１１１１）株（微工研
条寄第１７３８号）。(2) Bacillus subtilis s. has bacterial plant disease control properties. p. KB-1111 (Bacillus
subtiliss. p. KB-1111) strain (Feikoken Joyori No. 1738).
ブチルスｓ．ｐ．ＫＢ−１１２２（Ｂａｃｉｌｌｕｓ
ｓｕｂｔｉｌｉｓｓ．ｐ．ＫＢ−１１２２）株（微工研
条寄第１７３９号）。(3) Bacillus subtilis s. has bacterial plant disease control properties. p. KB-1122 (Bacillus
subtiliss. p. KB-1122) strain (Feikoken Joyori No. 1739).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1029686A JP2673718B2 (en) | 1989-02-10 | 1989-02-10 | Bacterial plant disease control method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1029686A JP2673718B2 (en) | 1989-02-10 | 1989-02-10 | Bacterial plant disease control method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02209803A true JPH02209803A (en) | 1990-08-21 |
| JP2673718B2 JP2673718B2 (en) | 1997-11-05 |
Family
ID=12282993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1029686A Expired - Fee Related JP2673718B2 (en) | 1989-02-10 | 1989-02-10 | Bacterial plant disease control method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2673718B2 (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0591869A (en) * | 1991-10-02 | 1993-04-16 | Yuukishitsu Hiryo Seibutsu Kassei Riyou Gijutsu Kenkyu Kumiai | Plant pathogenic fungus-inhibiting microorganism and its utilization |
| WO1998021964A1 (en) * | 1996-11-18 | 1998-05-28 | Agritope, Inc. | Biological control of plant fungal infections |
| US6004774A (en) * | 1996-11-18 | 1999-12-21 | Agritope, Inc. | Biological control of plant fungal infections |
| WO2000049875A1 (en) * | 1999-02-23 | 2000-08-31 | Kureha Chemical Ind Co Ltd | Plant disease control method |
| KR100407074B1 (en) * | 2000-08-18 | 2003-11-28 | 대한민국 | Antimicrobial Peptide-Producing Microorganism from Korean Fermented Fishes, Jeotkal in Korean |
| WO2005059112A1 (en) * | 2003-12-17 | 2005-06-30 | Kt & G Co., Ltd | The novel bacillus amyloliquefaciens ktgb0202 and control method of plant pathogenic funzi using that |
| WO2005082149A1 (en) * | 2004-02-27 | 2005-09-09 | Itsuki Co., Ltd. | Method of controlling plant disease damage by using bacillus and controlling agent |
| CN102329758A (en) * | 2011-10-12 | 2012-01-25 | 淮阴师范学院 | Biological prevention bacterial strain BS1 (bacillus cereus) for preventing and controlling greenhouse cucumber downy mildew and application thereof |
| WO2022254622A1 (en) | 2021-06-02 | 2022-12-08 | 株式会社エス・ディー・エス バイオテック | Agricultural/horticultural insecticide composition and agricultural/horticultural insect pest control method |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3498097B1 (en) | 2016-08-09 | 2021-04-14 | SDS Biotech K. K. | Agricultural and horticultural fungicide composition and plant disease controlling method |
-
1989
- 1989-02-10 JP JP1029686A patent/JP2673718B2/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0591869A (en) * | 1991-10-02 | 1993-04-16 | Yuukishitsu Hiryo Seibutsu Kassei Riyou Gijutsu Kenkyu Kumiai | Plant pathogenic fungus-inhibiting microorganism and its utilization |
| WO1998021964A1 (en) * | 1996-11-18 | 1998-05-28 | Agritope, Inc. | Biological control of plant fungal infections |
| US6004774A (en) * | 1996-11-18 | 1999-12-21 | Agritope, Inc. | Biological control of plant fungal infections |
| WO2000049875A1 (en) * | 1999-02-23 | 2000-08-31 | Kureha Chemical Ind Co Ltd | Plant disease control method |
| KR100407074B1 (en) * | 2000-08-18 | 2003-11-28 | 대한민국 | Antimicrobial Peptide-Producing Microorganism from Korean Fermented Fishes, Jeotkal in Korean |
| WO2005059112A1 (en) * | 2003-12-17 | 2005-06-30 | Kt & G Co., Ltd | The novel bacillus amyloliquefaciens ktgb0202 and control method of plant pathogenic funzi using that |
| WO2005082149A1 (en) * | 2004-02-27 | 2005-09-09 | Itsuki Co., Ltd. | Method of controlling plant disease damage by using bacillus and controlling agent |
| CN102329758A (en) * | 2011-10-12 | 2012-01-25 | 淮阴师范学院 | Biological prevention bacterial strain BS1 (bacillus cereus) for preventing and controlling greenhouse cucumber downy mildew and application thereof |
| WO2022254622A1 (en) | 2021-06-02 | 2022-12-08 | 株式会社エス・ディー・エス バイオテック | Agricultural/horticultural insecticide composition and agricultural/horticultural insect pest control method |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2673718B2 (en) | 1997-11-05 |
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