JP4695070B2 - Plant disease control composition and microorganism - Google Patents
Plant disease control composition and microorganism Download PDFInfo
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- JP4695070B2 JP4695070B2 JP2006510272A JP2006510272A JP4695070B2 JP 4695070 B2 JP4695070 B2 JP 4695070B2 JP 2006510272 A JP2006510272 A JP 2006510272A JP 2006510272 A JP2006510272 A JP 2006510272A JP 4695070 B2 JP4695070 B2 JP 4695070B2
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- strain
- bacillus
- disease control
- plant disease
- disease
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Description
本発明は、各種農園芸作物の灰色かび病や菌核病、うどんこ病、炭疽病、輪斑病、青かび病、緑かび病、軟腐病、かいよう病等の植物病害に対して植物病害が発生する温度領域の低温域での防除能も有するバチルス属（Bacillus）に属する新規な微生物、該新規微生物を有する植物病害防除剤組成物、該植物病害防除剤組成物を用いた植物病害の防除方法に関する。 The present invention has a plant disease against various plant diseases such as gray mold, mycorrhizal disease, powdery mildew, anthrax, ring mold, blue mold, green mold, soft rot, and mildew of various agricultural and horticultural crops. A novel microorganism belonging to the genus Bacillus that also has the ability to control in the low temperature range of the generated temperature range, a plant disease control composition comprising the novel microorganism, and control of plant diseases using the plant disease control composition Regarding the method.
従来から、ベンズイミダゾール系殺菌剤及びチオファネート系殺菌剤が農園芸作物に寄生する種々の病原菌に対して優れた防除効果を示し、１９７０年頃より農園芸用殺菌剤として広く一般に使用され、作物増産に大きく寄与してきた。 Conventionally, benzimidazole fungicides and thiophanate fungicides have shown excellent control effects against various pathogens that parasitize agricultural and horticultural crops, and have been widely used as agricultural and horticultural fungicides since about 1970 to increase crop production. It has contributed greatly.
一方、従来の合成殺菌剤による防除に代わるべき、あるいは併用すべき手段として、環境汚染が極めて少なく、生態系に調和し、かつ防除効果の優れたものとして生物農薬が知られている。農園芸作物の病害防除、特に灰色かび病に用いられてきた微生物として、トリコデルマ属、グリオクラディウム属、バチルス属に属する微生物が挙げられ、これまでにこれらの微生物を含有する農園芸用殺菌剤組成物も数多く研究開発されてきた。 On the other hand, biological pesticides are known as means that should be used in place of or in combination with conventional synthetic fungicides and that have very little environmental pollution, are in harmony with the ecosystem, and have excellent control effects. Microorganisms belonging to the genus Trichoderma, Gliocladium, and Bacillus are examples of microorganisms that have been used for disease control of agricultural and horticultural crops, especially gray mold disease, and sterilization for agricultural and horticultural use containing these microorganisms so far Many agent compositions have been researched and developed.
例えば、バチルス属に属する細菌については、特開昭６３−２７３４７０号公報では、バチルス・ズブチリス（Bacillus subtilis）JB3株菌（NCIB１２３７５）、バチルス・ズブチリスJB３．６株菌（NCIBB１２３７６）、バチルス・ズブチルスR1株菌（NCIB１２６１６）、あるいはこれらの変異株等から得られる抗菌物質が植物の病気、動物及びヒトの微生物汚染等を抑制し、更に一般的な微生物汚染を抑制するとし、上記各菌株の培養物を用いて各種農園芸作物の病害を防除する試みがなされている（例えば特許文献１参照）。また、上記バチルス・ズブチリスJB３菌株（NCIB１２３７５）、バチルス・ズブチリスJB３．６株菌（NCIBB１２３７６）、バチルス・ズブチルスR1株菌（NCIB１２６１６）、あるいはこれらの変異株等から得られる抗菌物質を単離し、これを各種農園芸作物の病害防除に用いるという試みがなされている（例えば特許文献２参照）。その他、クワの炭疽病や白紋羽病の防除微生物として、バチルス・アミロリクエファシエンスが知られている（例えば特許文献３、非特許文献１、非特許文献２参照）。また、バチルス・ズブチリス（Bacillus subtilis）QST-713株の芽胞製剤であるインプレッション水和剤が２００３年5月に農薬登録を取得し、上市されている（例えば非特許文献３参照）。
しかし、このようなバチルス属に属する細菌を応用した農園芸作物の病害防除方法では、植物病原菌の生育適温と病害防除細菌の生育適温とのずれが大きく、何れも各種農園芸作物病害が発生する低温域での防除が必ずしも十分であるとは言えなかった。例えば、各種農園芸作物の灰色かび病は１５℃前後の低温での発病が極めて旺盛であり、２０℃から４５℃に生育適温を持つバチルス属に属する細菌では、特に気温が低く推移した場合、決して十分な防除効果が得られるとは限らなかった（例えば非特許文献３参照）。 However, in the method for controlling diseases of agricultural and horticultural crops using bacteria belonging to the genus Bacillus, there is a large difference between the optimal temperature for growth of phytopathogenic bacteria and the optimal temperature for growth of disease-controlling bacteria. Control at low temperatures was not always sufficient. For example, the gray mold disease of various agricultural and horticultural crops is very active at a low temperature of around 15 ° C., and in bacteria belonging to the genus Bacillus having a suitable temperature for growth from 20 ° C. to 45 ° C., particularly when the temperature is low, A sufficient control effect was not always obtained (see, for example, Non-Patent Document 3).
本発明は、かかる実情に鑑みてなされたものであり、本発明の課題は、各種農園芸作物等の植物の病害、特に灰色かび病等の対象植物病害に対して、１０℃から１５℃といった低温域においても一定して優れた防除能を有するバチルス属（Bacillus）に属する微生物、該微生物を含有する植物病害防除剤組成物や、これを用いて植物及び／又は土壌を処理する植物病害の防除方法、該微生物に主として由来しそのごく一部の特性を変更させた菌株、該微生物の選抜方法を提供することにある。 This invention is made | formed in view of this situation, and the subject of this invention is 10 to 15 degreeC with respect to object plant diseases, such as plant diseases, such as various agricultural and horticultural crops, especially gray mold disease. Microorganisms belonging to the genus Bacillus having constant and excellent control ability even in a low temperature range, plant disease control compositions containing the microorganisms, and plant diseases that treat plants and / or soil using the same It is an object of the present invention to provide a control method, a strain mainly derived from the microorganism and having only a part of its characteristics changed, and a method for selecting the microorganism.
本発明者らは、上記の課題を解決するため、日本曹達株式会社榛原フィールドリサーチセンターの各圃場から採取した茶葉から、植物病害防除能を有する候補微生物を単離した。各圃場から採取した茶葉の葉面を少量の滅菌水内で洗浄し、その洗浄水を標準寒天培地（日水製薬社製）上に塗抹して、生育してきた微生物のコロニーを採取し、更に単一のコロニーとなるように分離・取得した。これらの微生物の中から、常温（20℃）で灰色かび病（Botrytis cinerea）に対して防除効果を有する菌株を選抜した。選抜された菌株について低温（10℃）で生育可能な菌株を更に選抜し、再度低温（14℃）で灰色かび病に対して防除効果を有する２菌株の微生物を見出した。これら２菌株は、コロニーの形態からバチルス属（Bacillus）の細菌と予想され、その形態や菌学的性質は以下の通りであった。 In order to solve the above-mentioned problems, the present inventors isolated candidate microorganisms having plant disease control ability from tea leaves collected from each field of Nippon Soda Co., Ltd. Sugawara Field Research Center. Wash the leaves of tea leaves collected from each field in a small amount of sterilized water, smear the washed water on a standard agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), collect colonies of the growing microorganisms, and Separated and acquired so as to form a single colony. From these microorganisms, a strain having a controlling effect against gray mold (Botrytis cinerea) at room temperature (20 ° C.) was selected. From the selected strains, strains capable of growing at low temperature (10 ° C.) were further selected, and two strains of microorganisms having a control effect against gray mold at low temperature (14 ° C.) were found again. These two strains were predicted as bacteria of the genus Bacillus from the morphology of the colonies, and their morphology and mycological properties were as follows.
グラム陽性の桿菌でカタラーゼ反応陽性で、好気的に酸を生成し、バチルス型の芽胞形成が認められた。 The gram-positive bacilli were catalase-positive, aerobically produced acid, and Bacillus-type spore formation was observed.
１６ＳｒＤＮＡの約１１００bp塩基配列を基に系統解析を行い相同性検索を行った結果、４−５−１−１株 Ｎｏ．３０は、バチルス・ズブチリス（Bacillus subtilis），バチルス・アミロリクエファシエンス（Bacillus amyloliquefaciens），バチルス・ヴェレセンシス（Bacillus velesensis）と相同性一致率９９．９０６％、バチルス・リチェニホルミス（Bacillus licheniformis）と相同性一致率９９．７１９％であった。また、アピマニュアルキット（bio Merieux Industry）のAPI 50 CHを用い検討したところ、４−５−１−１株 Ｎｏ．３０は、バチルス・ズブチリス（Bacillus subtilis）との相同性は４４．７％、バチルス・リチェニホルミス（Bacillus licheniformis）との相同性は３５．１％であった。更に、嫌気的生育，プロピオン酸塩の利用の有無，55℃での生育可否について検討したところ何れも陰性であった。更に炭素化合物の利用性について調べたところ、４−５−１−１株 Ｎｏ．３０は、Glycerol、L-Arabinose、Ribose、D-Xylose、D-Glucose、D-Fructose、D-Mannose、Inositol、Mannitol、Sorbitol、α-Methyl-D-glucoside、Amygdaline、Arbutin、Esculin、Salicin、Cellobiose、Maltose、Lactose、Melibiose、Sucrose、Trehalose、D-Raffinose、Starch、Glycogen、β-Gentiobioseは、利用でき、Erythritol、D-Arabinose、L-Xylose、Adonitol、β-Methyl-D-xyloside、Galactose、L-Sorbose、Rhamnose、Dulcitol、α-Methyl-D-mannoside、N-Acethyl-glucosamine、Inuline、Melezitose、Xylitol、D-Turanose、D-Lyxose、D-Tagatose、D-Fucose、L-Fucose、D-Arabitol、L-Arabitol Gluconate、2-ceto-gluconate、5-ceto-gluconateは、利用できなかった。 As a result of phylogenetic analysis based on the approximately 1100 bp base sequence of 16S rDNA and homology search, strain 4-5-1-1 no. 30 is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velesensis, 99.906% homology match, Bacillus licheniformis and Bacillus licheniformis The rate was 99.719%. Further, when API 50 CH of the API manual kit (bio Merieux Industry) was used, the 4-5-1-1 strain No. 30 was 44.7% homology with Bacillus subtilis and 35.1% homology with Bacillus licheniformis. Furthermore, anaerobic growth, the presence or absence of use of propionate, and the possibility of growth at 55 ° C were all negative. Furthermore, when the availability of the carbon compound was examined, the 4-5-1-1 strain No. 30 is Glycerol, L-Arabinose, Ribose, D-Xylose, D-Glucose, D-Fructose, D-Mannose, Inositol, Mannitol, Sorbitol, α-Methyl-D-glucoside, Amygdaline, Arbutin, Esculin, Salicin, Cellobiose , Maltose, Lactose, Melibiose, Sucrose, Trehalose, D-Raffinose, Starch, Glycogen, β-Gentiobiose are available, Erythritol, D-Arabinose, L-Xylose, Adonitol, β-Methyl-D-xyloside, Galactose, L -Sorbose, Rhamnose, Dulcitol, α-Methyl-D-mannoside, N-Acethyl-glucosamine, Inuline, Melezitose, Xylitol, D-Turanose, D-Lyxose, D-Tagatose, D-Fucose, L-Fucose, D-Arabitol , L-Arabitol Gluconate, 2-ceto-gluconate and 5-ceto-gluconate were not available.
一方、４−５−２１ ０３０６株について、１６ＳｒＤＮＡの約７００ｂｐ塩基配列を基に系統解析を行い相同性検索を行った結果、バチルス・ズブチリスと相同性一致率９９．０％であった。また、アピマニュアルキット（bio Merieux Industry）のAPI 50 CHを用い検討したところ、４−５−２１ ０３０６株は、バチルス・ズブチリス（Bacillus subtilis）との相同性は８７．４％であった。更に炭素化合物の利用性について調べたところ、４−５−２１ ０３０６株は、Glycerol、L-Arabinose、Ribose、D-Glucose、D-Fructose、D-Mannose、Inositol、Mannitol、Sorbitol、α-Methyl-D-glucoside、Amygdaline、Arbutin、Esculin、Salicin、Cellobiose、Maltose、Lactose、Sucrose、Trehalose、D-Raffinose、Starch、Glycogenは、利用でき、Erythritol、D-Arabinose、D-Xylose、L-Xylose、Adonitol、β-Methyl-D-xyloside、Galactose、L-Sorbose、Rhamnose、Dulcitol、α-Methyl-D-mannoside、N-Acethyl-glucosamine、Melibiose、Inuline、Melezitose、Xylitol、β-Gentiobiose、D-Turanose、D-Lyxose、D-Tagatose、D-Fucose、L-Fucose、D-Arabitol、L-Arabitol Gluconate、2-ceto-gluconate、5-ceto-gluconateは、利用できなかった。 On the other hand, the strain 4-5-21 0306 was subjected to phylogenetic analysis and homology search based on the approximately 700 bp nucleotide sequence of 16S rDNA. As a result, the homology coincidence with Bacillus subtilis was 99.0%. Moreover, when examined using API 50 CH of an API manual kit (bio Merieux Industry), 4-5-21 0306 strain was 87.4% of homology with Bacillus subtilis (Bacillus subtilis). Further examination of the availability of carbon compounds revealed that the strain 4-5-21 0306 was Glycerol, L-Arabinose, Ribose, D-Glucose, D-Fructose, D-Mannose, Inositol, Mannitol, Sorbitol, α-Methyl- D-glucoside, Amygdaline, Arbutin, Esculin, Salicin, Cellobiose, Maltose, Lactose, Sucrose, Trehalose, D-Raffinose, Starch, Glycogen are available, Erythritol, D-Arabinose, D-Xylose, L-Xylose, Adonitol, β-Methyl-D-xyloside, Galactose, L-Sorbose, Rhamnose, Dulcitol, α-Methyl-D-mannoside, N-Acethyl-glucosamine, Melibiose, Inuline, Melezitose, Xylitol, β-Gentiobiose, D-Turanose, D- Lyxose, D-Tagatose, D-Fucose, L-Fucose, D-Arabitol, L-Arabitol Gluconate, 2-ceto-gluconate and 5-ceto-gluconate were not available.
以上の結果を総合すると４−５−１−１株 Ｎｏ．３０は、はバチルス・ズブチリス（Bacillus subtilis）と判断された。また、４−５−２１ ０３０６株は、バチルス・ズブチリス（Bacillus subtilis）の近縁種であるバチルス エスピー（Bacillus sp.）と判断された。これらの菌株は、独立行政法人産業技術総合研究所特許生物寄託センター(日本国茨城県つくば市東１丁目１番地１中央第６）に２００４年２月１０日にそれぞれ寄託番号ＦＥＲＭ ＢＰ−１０２４４、寄託番号ＦＥＲＭ ＢＰ−１０２４５として寄託されている。そして、上記細菌は、灰色かび病の他、菌核病やうどんこ病、炭疽病、輪斑病，青かび病，緑かび病、軟腐病、かいよう病等広範囲の病害に対し優れた防除活性を有する。 Summing up the above results, the strain 4-5-1-1 30 was determined to be Bacillus subtilis. Moreover, 4-5-21 0306 strain was judged to be Bacillus sp., A related species of Bacillus subtilis. These strains were deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st, 1st, 1st, 1st, Tsukuba City, Ibaraki, Japan) on February 10, 2004, with deposit numbers FERM BP-10244, respectively. Deposited under the number FERM BP-10245. In addition to gray mold disease, the above-mentioned bacteria have excellent control activity against a wide range of diseases such as mycorrhizal disease, powdery mildew, anthrax, ring spot disease, blue mold disease, green mold disease, soft rot disease, and mildew disease. Have.
すなわち、本発明は第１に、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０を含有することを特徴とする植物病害防除剤組成物であり、第２にバチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株を含有することを特徴とする植物病害防除組成物であり、第３にバチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０又はバチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株を含有する植物病害防除剤組成物を用いて、植物及び／又は土壌を処理することを特徴とする植物病害の防除方法であり、第４にバチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０であり、第５にバチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株であり、第６に次のa〜ｄの連続した各工程からなる、１０〜１５℃で病害防除能を有するバチルス属（Bacillus sp.）に属する微生物の選抜方法である。
a.菌株分離源から菌体を採取する工程
b.常温（１８〜２５℃）で灰色かび病（Botrytis cinerea）に対して防除効果を有する菌株を選抜する工程
c.低温（８〜１２℃）で生育可能な菌株を選抜する工程
d.低温（１３〜１５℃）で灰色かび病（Botrytis cinerea）に対して防除効果を有する菌株を再選抜する工程
That is, first, the present invention relates to Bacillus subtilis 4-5-1-1 strain No. 30. A plant disease control composition characterized by containing 30 , and secondly a plant disease control composition characterized by containing Bacillus sp. 4-5-21 0306 strain Third, Bacillus subtilis 4-5-1-1 strain No. 30 or a plant disease control method comprising using a plant disease control agent composition containing Bacillus sp. 4-5-21 0306 strain , Fourth , Bacillus subtilis 4-5-1-1 strain No. 30, 5th is Bacillus sp. 4-5-21 0306 strain, and 6th is composed of the following consecutive steps a to d, and has a disease control ability at 10 to 15 ° C. This is a method for selecting microorganisms belonging to the genus Bacillus sp.
a. Collecting cells from strain isolation source
b. A step of selecting a strain having an effect of controlling against gray mold (Botrytis cinerea) at room temperature (18-25 ° C.)
c. Process for selecting strains that can grow at low temperature (8-12 ° C)
d. Reselection of a strain having a controlling effect against gray mold (Botrytis cinerea) at a low temperature (13 to 15 ° C.)
本発明の植物病害防除剤組成物は、市販のバチルス属製剤が十分な効果を示さない低温条件下で高い病害防除能を有する微生物を含有することにより市販のバチルス属製剤が十分な効果を示さない低温条件下で高い病害防除能を示すと共に、長期にわたり病害防除能を保持することができ、また、一度に多種類の病害を防除することができる。かかる植物病害防除剤組成物を用いた本発明の植物病害の防除方法によれば、植物病害防除剤組成物を各種農園芸作物等の植物や、土壌に散布する等の方法により、植物の病害、特に灰色かび病に対して効果的に防除することができる。また本発明の選抜方法によれば、市販のバチルス属製剤が十分な効果を示さない低温条件下で高い病害防除能を有する微生物を効率よく選抜できる。 The plant disease control agent composition of the present invention has a sufficient effect of a commercially available Bacillus genus preparation by containing a microorganism having a high disease control ability under a low temperature condition in which the commercially available Bacillus genus preparation does not exhibit a sufficient effect. In addition to exhibiting high disease control ability under low temperature conditions, the disease control ability can be maintained over a long period of time, and many kinds of diseases can be controlled at once. According to the plant disease control method of the present invention using such a plant disease control agent composition, the plant disease control method is applied to plants such as various agricultural and horticultural crops or to the soil by a method such as spraying the plant disease control composition. In particular, it can be effectively controlled against gray mold. Moreover, according to the selection method of the present invention, a microorganism having a high disease control ability can be efficiently selected under low temperature conditions in which a commercially available Bacillus genus preparation does not exhibit a sufficient effect.
本発明の植物病害防除剤組成物としては、茶葉から分離することができ、灰色かび病（Botrytis cinerea）に対して防除効果を有し、且つ全ての病害発生温度域、特に低温域で一定した優れた防除能を有するバチルス属（Bacillus sp.）に属する微生物、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０（ＦＥＲＭ ＢＰ−１０２４４）、バチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株（ＦＥＲＭ ＢＰ−１０２４５）から選ばれる少なくとも１種を含有することを特徴とする植物病害防除剤組成物であれば特に制限されることはないが、上記バチルス属（Bacillus sp.）に属する微生物が、菌核病菌（S.sclerotiorum）炭疽病菌（C.lagenarium）、輪斑病菌（P.longiseta）、緑かび病菌（P.digitatum）、青かび病菌（P.italicum）、軟腐病（E.carotovora）、かいよう病（X.campestris）等に対して防除効果を有するものが好ましい。 The plant disease control agent composition of the present invention can be isolated from tea leaves, has a control effect against gray mold (Botrytis cinerea), and is constant in all disease generation temperature ranges, particularly low temperature ranges. A microorganism belonging to the genus Bacillus (Bacillus sp.) Having excellent control ability, Bacillus subtilis 4-5-1-1 strain No. 30 (FERM BP-10244), Bacillus sp (Bacillus sp.) 4-5-21 0306 strain (FERM BP-10245) at least 1 sort (s) containing, The plant disease control agent composition characterized by the above-mentioned The microorganism belonging to the genus Bacillus (Bacillus sp.) Is not particularly limited, but may be selected from the group consisting of S. sclerotiorum, C. lagenarium, P. longiseta, and green mold. Those having a control effect against P. digitatum, P. italicum, soft rot (E. carotovora), scab (X. campestris) and the like are preferable.
本発明の植物病害防除剤組成物は、灰色かび病や菌核病、うどんこ病、炭疽病、輪斑病、青かび病、緑かび病、軟腐病、かいよう病等に対して特に有効に用いることができる。 The plant disease control agent composition of the present invention is particularly effective for gray mold disease, mycorrhizal disease, powdery mildew, anthrax disease, ring spot disease, blue mold disease, green mold disease, soft rot, mold disease, and the like. be able to.
本発明の植物病害防除剤組成物の製造には、バチルス属（Bacillus sp.）に属する微生物を固体培養あるいは液体培養等の公知の手段で増殖させた菌体を用いることができる。かかる微生物の増殖方法としては、菌体が増殖する方法であれば、特に培地の種類や培養条件等を問わず、いずれの方法でもよいが、例えば、固体培養の場合、標準寒天培地や普通寒天培地、ポテトデキストロース寒天培地等における２５〜３５℃での静置培養を、液体培養の場合、上記寒天培地から寒天を除いた各種液体培地等における２５〜３５℃での振盪・攪拌培養等を挙げることができる。本発明の菌体とは栄養細胞または胞子等バチルス属の微生物が通常とりうるあらゆる形態を示す。また、菌体の使用形態としては、菌体自体のほか、その懸濁液ないし培養液又はこれらの濃縮物、ペースト状物、乾燥物、希釈物等のいずれの形態であっても適用することができる。 In the production of the plant disease control agent composition of the present invention, cells obtained by growing microorganisms belonging to the genus Bacillus by a known means such as solid culture or liquid culture can be used. As a method for growing such microorganisms, any method may be used as long as the cells grow, regardless of the type of medium, culture conditions, etc. For example, in the case of solid culture, a standard agar medium or ordinary agar is used. In the case of liquid culture, stationary culture in a medium, potato dextrose agar medium, etc. at 25 to 35 ° C., shaking / stirring culture at 25 to 35 ° C. in various liquid mediums obtained by removing agar from the agar medium be able to. The microbial cell of the present invention refers to all forms normally taken by Bacillus microorganisms such as vegetative cells or spores. In addition to the cells themselves, the cells may be used in any form such as suspensions or culture solutions or concentrates, pastes, dried products, diluted products, etc. Can do.
本発明の病害防除剤組成物におけるバチルス属（Bacillus sp.）に属する菌の濃度は、特に制限されるものではないが、１０００〜２０００倍に希釈した際に、菌体濃度に換算して、１×１０11 〜１×１０2 ｃｆｕ／ｍｌ、好ましくは１×１０9〜１×１０4 ｃｆｕ／ｍｌの範囲を好適に例示することができる。また、本発明の病害防除剤組成物には通常使用される担体、界面活性剤、分散剤、補助剤等を配合させることができ、その形態としては通常の農薬のとり得る形態、例えば、粉剤、水和剤、乳剤、フロアブル剤、粒剤等の形態を採用することができる。The concentration of the bacterium belonging to the genus Bacillus (Bacillus sp.) In the disease control agent composition of the present invention is not particularly limited, but when diluted 1000 to 2000 times, it is converted into the cell concentration, A range of 1 × 10 11 to 1 × 10 2 cfu / ml, preferably 1 × 10 9 to 1 × 10 4 cfu / ml can be suitably exemplified. The disease control agent composition of the present invention can be mixed with commonly used carriers, surfactants, dispersants, adjuvants, and the like, and the form can be taken by ordinary agricultural chemicals, for example, powders. , Wettable powders, emulsions, flowables, granules and the like can be employed.
本発明の農薬製剤には、例えば、炭酸カルシウムや塩化カリウム、硫酸ナトリウム、硫酸カルシウム、硫酸アンモニウム等の無機塩類、クエン酸、リンゴ酸、ステアリン酸等の有機酸及びそれらの塩、乳糖、ショ糖等の糖類等、アルミナ粉、シリカゲル、ゼオライト、ヒドロキシアパタイト、リン酸ジルコニウム、リン酸チタン、酸化チタン、酸化亜鉛、ハイドロタルサイト、カオリナイト、モンモリロナイト、タルク、クレー、珪藻土、ベントナイト、ホワイトカーボン、カオリン、バーミキュライト等の固体担体を挙げることができる。また、必要に応じて、酸化防止剤、着色剤、滑剤、紫外線吸収剤、耐電防止剤などを添加することができる。更に、補助剤として、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、アラビアゴム、澱粉等を添加することができる。 Examples of the agricultural chemical preparation of the present invention include inorganic salts such as calcium carbonate, potassium chloride, sodium sulfate, calcium sulfate and ammonium sulfate, organic acids such as citric acid, malic acid and stearic acid, and salts thereof, lactose, sucrose and the like. Saccharides, alumina powder, silica gel, zeolite, hydroxyapatite, zirconium phosphate, titanium phosphate, titanium oxide, zinc oxide, hydrotalcite, kaolinite, montmorillonite, talc, clay, diatomaceous earth, bentonite, white carbon, kaolin, A solid carrier such as vermiculite can be mentioned. Moreover, an antioxidant, a coloring agent, a lubricant, an ultraviolet absorber, an antistatic agent, etc. can be added as needed. Furthermore, as an auxiliary agent, for example, carboxymethylcellulose, polyethylene glycol, gum arabic, starch and the like can be added.
界面活性剤および分散剤としては、通常の農薬用製剤に使用できるものであれば特に限定されるものではなく、具体的には、非イオン性界面活性剤としてはソルビタン脂肪酸エステル（Ｃ12〜18），ＰＯＥソルビタン脂肪酸エステル（Ｃ12〜18），ショ糖脂肪酸エステルなどの糖エステル型で界面活性剤、ＰＯＥ脂肪酸エステル（Ｃ12〜18），ＰＯＥ樹脂酸エステル，ＰＯＥ脂肪酸ジエステル（Ｃ12〜18）などの脂肪酸エステル型界面活性剤、ＰＯＥアルキルエーテル（Ｃ12〜18）等のアルコール型界面活性剤、ＰＯＥアルキル（Ｃ8〜12）フェニルエーテル，ＰＯＥジアルキル（Ｃ8〜12）フェニルエーテル，ＰＯＥアルキル（Ｃ8〜12）フェニルエーテルホルマリン縮合物などのアルキルフェノール型界面活性剤、ポリオキシエチレン・ポリオキシプロピレンブロックポリマー，アルキル（Ｃ12〜18）ポリオキシエチレン・ポリオキシプロピレンブロックポリマーエーテルなどのポリオキシエチレン・ポリオキシプロピレンブロックポリマー型界面活性剤、ＰＯＥアルキルアミン（Ｃ12〜18），ＰＯＥ脂肪酸アミド（Ｃ12〜18）などのアルキルアミン型界面活性剤、ＰＯＥ脂肪酸ビスフェニルエーテルなどのビスフェノール型界面活性剤、ＰＯＡベンジルフェニル（またはフェニルフェニル）エーテル，ＰＯＡスチリルフェニル（またはフェニルフェニル）エーテルなどの多芳香環型界面活性剤、ＰＯＥエーテルおよびエステル型シリコンおよびフッ素系界面活性剤などのシリコン系、フッ素系界面活性剤、ＰＯＥヒマシ油，ＰＯＥ硬化ヒマシ油などの植物油型界面活性剤、アニオン性界面活性剤としてはアルキルサルフェート（Ｃ12〜18，Ｎａ，ＮＨ4，アルカノールアミン），ＰＯＥアルキルエーテルサルフェート（Ｃ12〜18，Ｎａ，ＮＨ4，アルカノールアミン），ＰＯＥアルキルフェニルエーテルサルフェート（Ｃ12〜18，ＮＨ4，アルカノールアミン，Ｃa），ＰＯＥベンジル（またはスチリル）フエニル（またはフェニルフェニル）エーテルサルフェート（Ｎａ，ＮＨ4，アルカノールアミン），ポリオキシエチレン，ポリオキシプロピレンブロックポリマーサルフェート（Ｎａ，ＮＨ4，アルカノールアミン）などのサルフェート型界面活性剤、パラフィン（アルカン）スルホネート（Ｃ12〜22，Ｎａ，Ｃａ，アルカノールアミン），ＡＯＳ（Ｃ14〜16，Ｎａ，アルカノールアミン），ジアルキルスルホサクシネート（Ｃ8〜12，Ｎａ，Ｃａ，Ｍg），アルキルベンゼンスルホネート（Ｃ12，Ｎａ，Ｃａ，Ｍｇ，ＮＨ4 ，アルキルアミン，アルカノール，アミン，シクロヘキシルアミン），モノまたはジアルキル（Ｃ3〜6）ナフタレンスルホネート（Ｎａ，ＮＨ4 ，アルカノールアミン，Ｃａ，Ｍｇ），ナフタレンスルホネート・ホルマリン縮合物（Ｎａ，ＮＨ4），アルキル（Ｃ8〜12）ジフェニルエーテルジスルホネート（Ｎａ，ＮＨ4），リグニンスルホネート（Ｎａ，Ｃa），ＰＯＥアルキル（Ｃ8〜12）フエニルエーテルスルホネート（Ｎａ），ＰＯＥアルキル（Ｃ12〜18）エーテルスルホコハク酸ハーフエステル（Ｎａ）などのスルホネート型界面活性剤、カルボン酸型脂肪酸塩（Ｃ12〜18，Ｎａ，Ｋ，ＮＨ4，アルカノールアミン），Ｎ-メチル-脂肪酸サルコシネート（Ｃ12〜18，Ｎａ），樹脂酸塩（Ｎａ，Ｋ）などＰＯＥアルキル（Ｃ12〜18）エーテルホスフェート（Ｎａ，アルカノールアミン）、ＰＯＥモノまたはジアルキル（Ｃ8〜12）フェニルエーテルホスフェート（Ｎａ，アルカノールアミン），ＰＯＥベンジル（またはスチリル）化フェニル（またはフェニルフェニル）エーテルホスフェート（Ｎａ，アルカノールアミン），ポリオキシエチレン・ポリオキシプロピレンブロックポリマー（Ｎａ，アルカノールアミン），ホスファチジルコリン・ホスファチジルエタノールイミン（レシチン），アルキル（Ｃ8〜12）ホスフェートなどのホスフェート型界面活性剤、カチオン性界面活性剤としてはアルキルトリメチルアンモニウムクロライド（Ｃ12〜18），メチル・ポリオキシエチレン・アルキルアンモニウムクロライド（Ｃ12〜18），アルキル・Ｎ-メチルピリジウムブロマイド（Ｃ12〜18），モノまたはジアルキル（Ｃ12〜18）メチル化アンモニウムクロライド，アルキル（Ｃ12〜18）ペンタメチルプロピレンジアミンジクロライドなどのアンモニウム型界面活性剤、アルキルジメチルベンザルコニウムクロライド（Ｃ12〜18），ベンゼトニウムクロライド（オクチルフェノキシエトキシエチルジメチルベンジルアンモニウムクロライド）などのベンザルコニウム型界面活性剤、両性界面活性剤としてはジアルキル（Ｃ8〜12）ジアミノエチルベタイン、アルキル（Ｃ12〜18）ジメチルベンジルベタイン等のベタイン型界面活性剤、ジアルキル（Ｃ8〜12）ジアミノエチルグリシン、アルキル（Ｃ12〜18）ジメチルベンジルグリシンなどのグリシン型界面活性剤等を例示することができる。これらは、１種単独で、または２種以上を混合して用いることができる。The surfactant and the dispersant are not particularly limited as long as they can be used in ordinary agricultural chemical preparations. Specifically, as the nonionic surfactant, sorbitan fatty acid ester (C 12-18) is used. ), POE sorbitan fatty acid ester (C 12-18 ), sugar ester type such as sucrose fatty acid ester, surfactant, POE fatty acid ester (C 12-18 ), POE resin acid ester, POE fatty acid diester (C 12-18 ) Fatty acid ester type surfactants, alcohol type surfactants such as POE alkyl ether (C 12-18 ), POE alkyl (C 8-12 ) phenyl ether, POE dialkyl (C 8-12 ) phenyl ether, POE alkylphenol type surfactants such as alkyl (C 8 to 12) phenyl ether formalin condensates, polyoxyethylene Po Polyoxypropylene-block polymers, alkyl (C 12 to 18) polyoxyethylene-polyoxypropylene block polymer surfactants such as polyoxyethylene-polyoxypropylene block polymer ether, POE alkylamine (C 12 to 18), POE fatty Alkylamine type surfactants such as amide (C 12-18 ), bisphenol type surfactants such as POE fatty acid bisphenyl ether, POA benzylphenyl (or phenylphenyl) ether, POA styrylphenyl (or phenylphenyl) ether, etc. Polyaromatic surfactants, silicon-based surfactants such as POE ether and ester-type silicones and fluorosurfactants, fluorosurfactants, vegetable oil-type surfactants such as POE castor oil and POE hydrogenated castor oil, Alkyl sulfates as the emission surfactant (C 12~18, Na, NH 4 , alkanolamine), POE alkyl ether sulfates (C 12~18, Na, NH 4 , alkanolamine), POE alkylphenyl ether sulfate (C 12 to 18, NH 4, alkanolamine, Ca), POE benzyl (or styryl) phenyl (or phenyl phenyl) ether sulfates (Na, NH 4, alkanolamine), polyoxyethylene, polyoxypropylene block polymer sulfates (Na, NH 4, sulfate-type surfactants such as alkanolamines), paraffins (alkanes) sulfonate (C 12 to 22, Na, Ca, alkanolamine), AOS (C 14 to 16, Na, alkanolamine), dialkyl sulfo Sa Shineto (C 8~12, Na, Ca, Mg), alkyl benzene sulphonate (C 12, Na, Ca, Mg, NH 4, alkyl amines, alkanol, amine, cyclohexylamine), mono- or di (C 3 to 6) naphthalene Sulfonate (Na, NH 4 , alkanolamine, Ca, Mg), naphthalene sulfonate-formalin condensate (Na, NH 4 ), alkyl (C 8-12 ) diphenyl ether disulfonate (Na, NH 4 ), lignin sulfonate (Na, Sulfonate type surfactants such as Ca), POE alkyl (C 8-12 ) phenyl ether sulfonate (Na), POE alkyl (C 12-18 ) ether sulfosuccinic acid half ester (Na), carboxylic acid type fatty acid salts (C 12 to 18, Na, K, NH 4, alkanolamine), - methyl - fatty acid sarcosinates (C 12~18, Na), resin acid salt (Na, K) such as POE alkyl (C 12 to 18) ether phosphate (Na, alkanolamine), POE mono- or dialkyl (C 8 to 12) Phenyl ether phosphate (Na, alkanolamine), POE benzyl (or styryl) phenyl (or phenylphenyl) ether phosphate (Na, alkanolamine), polyoxyethylene / polyoxypropylene block polymer (Na, alkanolamine), phosphatidylcholine Phosphatidylethanolimine (lecithin), phosphate surfactants such as alkyl (C 8-12 ) phosphate, cationic surfactants include alkyltrimethylammonium chloride (C 12-18 ), methyl Polyoxyethylene alkylammonium chloride (C 12-18 ), alkyl N-methylpyridium bromide (C 12-18 ), mono- or dialkyl (C 12-18 ) methylated ammonium chloride, alkyl (C 12-18 ) Ammonium type surfactants such as pentamethylpropylenediamine dichloride, benzalkonium type surfactants such as alkyldimethylbenzalkonium chloride (C 12-18 ), benzethonium chloride (octylphenoxyethoxyethyldimethylbenzylammonium chloride), amphoteric interfaces activator dialkyl (C 8 to 12) diamino ethyl betaine as, alkyl (C 12 to 18) betaine-type surfactants dimethylbenzyl betaine, dialkyl (C 8 to 12) diamino ethyl glycine, alkyl (C 12 to 18 It can be exemplified glycine type surfactants such as dimethyl benzyl glycine. These can be used individually by 1 type or in mixture of 2 or more types.
その他、通常の化学農薬や生物農薬の製剤に用いられる手法及び担体や界面活性剤、分散剤等の材料を用いて、例えば粉剤、水和剤、乳剤、フロアブル剤、粒剤等の形態とすることが出来る。 Other methods such as powders, wettable powders, emulsions, flowables, granules, etc. are used, for example, using conventional chemical pesticides and biological pesticides and materials such as carriers, surfactants, and dispersants. I can do it.
本発明の植物病害の防除方法は、上記本発明の植物病害防除剤組成物を用いて植物病害を防除する方法であれば、特に制限されるものではなく、本発明の植物病害防除剤組成物を通常の化学農薬と同様、各種農園芸作物等の植物体や土壌に散布処理等する方法を挙げることができる。また、特開２００１−３０２４０７記載のように、該製剤を施設内に送風する送風装置の送風口付近に設置し、送風口から送出される空気とともに農薬を散布することもできる。散布処理に当たっては、本発明の植物病害防除剤組成物を適当量の水等で希釈して使用することができ、散布量としては、バチルス属（Bacillus sp.）に属する菌体濃度に換算して、通常１×１０11 〜１×１０2 ｃｆｕ／ｍｌ、好ましくは１×１０9 〜１×１０4 ｃｆｕ／ｍｌの範囲とすることができる。The method for controlling plant diseases of the present invention is not particularly limited as long as it is a method for controlling plant diseases using the plant disease control composition of the present invention, and the plant disease control composition of the present invention. In the same manner as ordinary chemical pesticides, there can be mentioned a method of spraying on plants and soil such as various agricultural and horticultural crops. Further, as described in JP-A No. 2001-302407, it is possible to install the preparation in the vicinity of the air blowing port of a blower that blows the preparation into the facility, and spray the agricultural chemical together with the air sent from the air blowing port. In the spraying treatment, the plant disease control agent composition of the present invention can be used by diluting with an appropriate amount of water or the like, and the spraying amount is converted to the concentration of cells belonging to the genus Bacillus sp. In general, it can be in the range of 1 × 10 11 to 1 × 10 2 cfu / ml, preferably 1 × 10 9 to 1 × 10 4 cfu / ml.
本発明の市販のバチルス属製剤が十分な効果を示さない低温条件下とは具体的には、１０〜１５℃を示すことができる。また、本発明の特定の菌株に主として由来しそのごく一部の特性を変更させた菌株とは、自然的又は人為的に生じた変異体を選抜する方法、別の遺伝子を導入し形質を転換させた菌株を得る方法、 細胞融合のうち非対称融合により得られた菌株を得る方法等により得られた菌株を示すことができる。 Specifically, the low temperature condition in which the commercially available Bacillus genus preparation of the present invention does not have a sufficient effect can be 10 to 15 ° C. In addition, a strain mainly derived from a specific strain of the present invention and having only a small part of its characteristics changed is a method for selecting naturally or artificially generated mutants, and transforming a trait by introducing another gene. Strains obtained by a method for obtaining a strain obtained by the method, a method for obtaining a strain obtained by asymmetric fusion among cell fusion, and the like.
本発明の次のa〜ｄの連続した各工程からなる低温で高い活性を有するバチルス エスピー（Bacillus sp.）の選抜方法は具体的には次のような方法を示すことができる。
工程ａの.菌株分離源から菌体を採取する工程は通常の菌学的に知られている方法を適用できる。工程ｂの常温とは対象の植物病原菌が通常生育できる温度を示し、具体的には１６〜２５℃、好ましくは２０℃である。工程ｃの低温とは、対象の植物病原菌の増殖が停止する最低温度近辺を示し、具体的には８〜１２℃、好ましくは１０℃である。工程ｄの低温とは常温と工程ｃの低温との中間の温度を示し、具体的には１３〜１５℃、好ましくは１４℃である。The method for selecting Bacillus sp. Having high activity at low temperatures comprising the following successive steps a to d of the present invention can be specifically described as follows.
For the step of collecting the bacterial cells from the strain isolation source in step a, a usual method known from mycology can be applied. The normal temperature in step b indicates a temperature at which the target plant pathogen can normally grow, specifically 16 to 25 ° C, preferably 20 ° C. The low temperature in step c indicates the vicinity of the lowest temperature at which the growth of the target plant pathogen stops, specifically 8 to 12 ° C, preferably 10 ° C. The low temperature in step d indicates an intermediate temperature between the normal temperature and the low temperature in step c, specifically 13 to 15 ° C, preferably 14 ° C.
以下、本発明の植物病害防除剤組成物や、これを用いた植物病害防除方法を実施例により具体的に説明するが、本発明は以下の実施例に何ら限定されるものではなく、本発明の趣旨を逸脱しない範囲で、バチルス属（Bacillus sp.）の菌の種類、製剤の組成割合、剤型等を自由に変更することができる。また、以下の実施例では、バチルス属（Bacillus sp.）としてＦＥＲＭ ＢＰ−１０２４４、ＦＥＲＭ ＢＰ−１０２４５の２菌株を用いた。 Hereinafter, the plant disease control agent composition of the present invention and the plant disease control method using the same will be specifically described with reference to examples, but the present invention is not limited to the following examples. As long as it does not deviate from the above, the kind of bacteria of the genus Bacillus (Bacillus sp.), The composition ratio of the preparation, the dosage form and the like can be freely changed. Moreover, in the following Examples, two strains of FERM BP-10244 and FERM BP-10245 were used as Bacillus sp.
バチルス・ズブチリス菌の選抜
日本曹達株式会社榛原フィールドリサーチセンターの各圃場から採取した茶葉から、植物病害防除能を有する候補微生物を単離した。各圃場から採取した茶葉の葉面を少量の滅菌水内で洗浄し、その洗浄水を標準寒天培地（日水製薬社製）上に塗抹して、生育してきた微生物のコロニーを採取し、更に単一のコロニーとなるように分離・取得した。これらの微生物の中から、常温（２０℃）で灰色かび病（Botrytis cinerea）に対して防除効果を有する菌株を選抜した。選抜された菌株について低温（１０℃）で生育可能な菌株を更に選抜し、再度低温（１４℃）で灰色かび病に対して防除効果を有するバチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０、および、バチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株を見出した。Selection of Bacillus subtilis bacteria A candidate microorganism having the ability to control plant diseases was isolated from tea leaves collected from each field of Kashihara Field Research Center, Nippon Soda Co., Ltd. Wash the leaves of tea leaves collected from each field in a small amount of sterilized water, smear the washed water on a standard agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), collect colonies of the growing microorganisms, and It separated and acquired so that it might become a single colony. From these microorganisms, a strain having a controlling effect against gray mold (Botrytis cinerea) at room temperature (20 ° C.) was selected. A strain capable of growing at a low temperature (10 ° C.) is further selected from the selected strain, and Bacillus subtilis 4-5-1-1 having a control effect against gray mold at a low temperature (14 ° C.) again. Stock No. 30 and Bacillus sp. 4-5-21 0306 strains were found.
バチルス・ズブチリス菌体懸濁液の調製
３００ｍｌ容三角フラスコに標準液体培地（酵母エキス０．２５％，ペプトン０．５％，グルコース０．１％，ｐＨ７．０）１５０ｍｌを入れ、加熱滅菌後、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０、バチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株の前培養物０．１ｍｌをそれぞれ接種し、往復振盪機中２８℃、１００ｒｐｍで３日間培養した。培養液を水道水で洗浄する操作を３回繰り返し、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０、バチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株の菌体懸濁液を調製した。Preparation of Bacillus subtilis cell suspension A 300 ml Erlenmeyer flask was charged with 150 ml of a standard liquid medium (yeast extract 0.25%, peptone 0.5%, glucose 0.1%, pH 7.0). Bacillus subtilis 4-5-1-1 strain no. 30, 0.1 ml of a preculture of Bacillus sp. 4-5-21 0306 strain was inoculated and cultured in a reciprocating shaker at 28 ° C. and 100 rpm for 3 days. The operation of washing the culture solution with tap water was repeated three times, and Bacillus subtilis 4-5-1-1 strain No. 1 was obtained. 30, a cell suspension of Bacillus sp. 4-5-21 0306 strain was prepared.
植物病害防除水和剤組成物の製造
実施例２で調製したそれぞれのバチルス属菌体懸濁液を乾燥した。その乾燥物の生菌数は、それぞれ、６×１０10ｃｆｕ／ｇ、５．５×１０10ｃｆｕ／ｇであった。それぞれの菌体乾燥物１０重量部とラウリルサルフェートナトリウム塩２重量部、ナフタレンスルホン酸（ナトリウム）ホルムアルデヒド縮合物９重量部、硫酸カルシウム及びその水和物７９重量部を均一に混合し、水和剤Ａ，Ｂを得た。水和剤Ａ，Ｂは、それぞれバチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０、バチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株を有効成分とした組成物である。Manufacture of a plant disease control wettable composition Each Bacillus cell suspension prepared in Example 2 was dried. The viable cell counts of the dried product were 6 × 10 10 cfu / g and 5.5 × 10 10 cfu / g, respectively. 10 parts by weight of each dried cell body, 2 parts by weight of lauryl sulfate sodium salt, 9 parts by weight of naphthalenesulfonic acid (sodium) formaldehyde condensate, 79 parts by weight of calcium sulfate and its hydrate are uniformly mixed to obtain a wettable powder. A and B were obtained. The wettable powders A and B were respectively Bacillus subtilis 4-5-1-1 strain No. 30, a composition comprising Bacillus sp. 4-5-21 0306 strain as an active ingredient.
植物病害防除剤組成物の灰色かび病防除効果試験（室内試験）
実施例３で得られたそれぞれの水和剤Ａ，Ｂを水道水で１０００倍に希釈して処理液を調製した。また、比較例として市販製剤であるボトキラー水和剤（出光興産株式会社製）とインプレッション水和剤（株式会社エス・ディー・エスバイオテック製）をそれぞれ水道水で１０００倍及び５００倍に希釈して処理液を調製した。それぞれの処理液をインゲンの花器に散布した。その後、花器をインゲン葉の上に置き灰色かび病の発生温度の低温域である１５℃でインキュベートし、２４時間後灰色かび病の病原菌であるボトリシス・シネレア（Botrytis cinerea）の分生胞子懸濁液（５．０×１０5ｃｆｕ／ｍｌ）をインゲンの花器に散布した。散布処理後、１５℃で５日間インキュベートした。また、無処理を対照とし、ボトリシス・シネレア（Botrytis cinerea）の分生胞子懸濁液（５．０×１０5ｃｆｕ／ｍｌ）のみを散布処理した。調査は、インゲン葉に形成された病斑直径を計測した。Gray mold control effect test of plant disease control composition (in-house test)
Each wettable powder A and B obtained in Example 3 was diluted 1000 times with tap water to prepare a treatment solution. In addition, as comparative examples, commercially available preparations such as a water bottling wettable powder (manufactured by Idemitsu Kosan Co., Ltd.) and an impression wettable powder (manufactured by SDS Biotech Co., Ltd.) were diluted 1000 and 500 times with tap water, respectively. A treatment solution was prepared. Each treatment solution was sprayed on the flower vase. After that, a vase is placed on the kidney bean leaf and incubated at 15 ° C., which is the low temperature range of gray mold, and 24 hours later, a conidial spore suspension of Botrytis cinerea, the pathogen of gray mold. The liquid (5.0 × 10 5 cfu / ml) was sprayed on the bean vase. After spraying treatment, it was incubated at 15 ° C. for 5 days. In addition, using no treatment as a control, only a conidial spore suspension (5.0 × 10 5 cfu / ml) of Botrytis cinerea was sprayed. The survey measured the diameter of lesions formed on kidney beans.
表１から明らかなように、それぞれのバチルス属（Bacillus sp.）２菌株は、インゲンの葉に対して病斑は認められず、インゲン灰色かび病に対して防除効果を有することが認められた。それに対し、ボトキラー水和剤とインプレッション水和剤は、インゲン灰色かび病に対し、それぞれ１９．５，１９．８と低い防除価であった。As is clear from Table 1, each of the two strains of the genus Bacillus (Bacillus sp . ) Showed no lesions on the leaves of green beans and was found to have a controlling effect on green mildew. . In contrast, the bottom killer wettable powder and the impression wettable powder had low control values of 19.5 and 19.8, respectively, against bean gray mold.
植物病害防除水和剤組成物のインゲンの灰色かび病防除効果試験（ポット試験）
実施例３で得られたそれぞれの水和剤Ａ，Ｂを水道水で１０００倍に希釈して処理液を調製した。また、比較例としてボトキラー水和剤（出光興産株式会社）を水道水で１０００倍に希釈して処理液を調製した。それぞれの処理液をインゲンのポットに散布処理した。３日間隔で２回散布処理し、１４℃のインキュベーター内で栽培した。２回散布の翌日灰色かび病の病原菌であるボトリシス・シネレア（Botrytis cinerea）の分生胞子懸濁液（５．０×１０5ｃｆｕ／ｍｌ）をインゲンのポットに散布処理した。その後再び１４℃のインキュベーター内で栽培し、１週間後健全及び発病幼果数を計測した。Test of gray mold disease control effect of green beans of plant disease control wettable powder composition (pot test)
Each wettable powder A and B obtained in Example 3 was diluted 1000 times with tap water to prepare a treatment solution. As a comparative example, a bottling hydrate (Idemitsu Kosan Co., Ltd.) was diluted 1000 times with tap water to prepare a treatment solution. Each treatment solution was sprayed onto a bean pot. It was sprayed twice at intervals of 3 days and cultivated in a 14 ° C. incubator. The day after the second spraying, a conidial spore suspension (5.0 × 10 5 cfu / ml) of Botrytis cinerea, a pathogen of gray mold disease, was sprayed onto a bean pot. Thereafter, it was cultivated again in an incubator at 14 ° C., and after 1 week, the number of healthy and diseased young fruits was counted.
表２から明らかなように、それぞれのバチルス属（Bacillus sp.）２菌株は、インゲンの灰色かび病に対し、それぞれ８６．４及び８２．５と高い防除価が得られ、低温条件においてもインゲンの灰色かび病に対して高い防除効果を有することが認められた。それに対し、ボトキラー水和剤は、防除価１４．４と不充分であった。 As is clear from Table 2, each of the two Bacillus sp. Strains has a high control value of 86.4 and 82.5 against the gray bean disease of green beans, and even under low temperature conditions It was found to have a high control effect against gray mold disease. In contrast, the bottom hydrating agent was insufficient with a control value of 14.4.
植物病害防除水和剤組成物のカンキツ果実の灰色かび病防除効果試験（圃場試験）
実施例３で得られた水和剤Ａ、Ｂを水道水で１０００倍に希釈して処理液を調製した。また、比較例としてボトキラー水和剤（出光興産株式会社製）とインプレッション水和剤（株式会社エス・ディー・エスバイオテック製）を水道水で１０００倍及び５００倍に希釈して処理液を調製した。それぞれの処理液をビニルハウス内に植栽してあるナスに散布処理した。約７日間隔で２回散布処理し、最終散布７日後に灰色かび病発病果数と健全果数を調査し、防除果を算出した。なお、感染源としてボトリシス・シネレア（Botrytis cinerea）を感染させたカンキツ果実を試験ハウス内に点在させた。また、その試験期間中の気温を記録した。Gray mold disease control effect of citrus fruit of plant disease control wettable powder composition (field test)
Wet powders A and B obtained in Example 3 were diluted 1000 times with tap water to prepare a treatment solution. In addition, as a comparative example, a treatment liquid was prepared by diluting Botokiller wettable powder (manufactured by Idemitsu Kosan Co., Ltd.) and impression wettable powder (made by SDS Biotech Co., Ltd.) 1000 times and 500 times with tap water. did. Each treatment solution was sprayed on eggplants planted in a vinyl house. Spraying was performed twice at intervals of about 7 days, and the number of gray mold disease and healthy fruits was investigated 7 days after the final spraying, and the control results were calculated. Citrus fruits infected with Botrytis cinerea as an infection source were scattered in the test house. The temperature during the test period was recorded.
図１に示したようにこのときの気温変化を見てみると最低気温は１５℃を下回り、平均気温をとっても試験期間の半分以上は１５℃を下回っている。また、１０℃を下回る日も幾日か見受けられる。さらに最高気温を見ても試験初期は２５℃に近い気温を示したが、中盤からは１６〜１７℃、後半は１５℃を下回り、気温の低い状態が続いている状況であった。このような低気温条件の中、表３から明らかなように、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０とバチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株は、ナス灰色かび病に対し、それぞれ６８．１及び８０．４と高い防除価を示し、低気温条件に於いてもナス灰色かび病に対して高い防除効果を有することが認められた。それに対し、ボトキラー水和剤とインプレッション水和剤の防除価は、それぞれ５３．１及び５９．５と必ずしも十分とはいえないものであった。 As shown in FIG. 1, when looking at the temperature change at this time, the minimum temperature is below 15 ° C., and even if the average temperature is taken, more than half of the test period is below 15 ° C. There are also some days below 10 ° C. Further, even when looking at the maximum temperature, the initial temperature showed a temperature close to 25 ° C., but it was 16-17 ° C. from the middle stage and below 15 ° C. in the second half, and the temperature remained low. Under these low temperature conditions, as is apparent from Table 3, Bacillus subtilis 4-5-1-1 strain No. 30 and Bacillus sp. 4-5-21 0306 strains showed high control values of 68.1 and 80.4 against eggplant gray mold, respectively, and eggplant gray mold even under low temperature conditions. It was found to have a high control effect against the disease. On the other hand, the control values of the bottle killer wettable powder and the impression wettable powder were 53.1 and 59.5, respectively, which were not necessarily sufficient.
植物病害防除剤組成物の灰色かび病防除効果試験（室内試験）
実施例２と同様に培養・調製したバチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０ ＦＥＲＭ ＢＰ−１０２４４菌体懸濁液の凍結乾燥物（１．０×１０10ｃｆｕ／ｇ）１０％と昭和クレー４０％，塩化カリウム４０％，ニューカルゲンＰＳ−Ｐ１０％を均一に混合した後、粉砕して水和剤Cを得た。それを水道水で１０００倍，２０００倍，４０００倍に希釈して処理液を調製した。また、比較例として市販製剤であるボトキラー水和剤（出光興産株式会社製）とインプレッション水和剤（株式会社エス・ディー・エスバイオテック製）をそれぞれ水道水で１０００倍，２０００倍，４０００倍及び５００倍，１０００倍，２０００倍に希釈して処理液を調製した。それぞれの処理液をインゲンの花器に散布した。その後、花器をインゲン葉の上に置き灰色かび病の発生温度の低温域である１５℃でインキュベートし、２４時間後灰色かび病の病原菌であるボトリシス・シネレア（Botrytis cinerea）の分生胞子懸濁液（５．０×１０5ｃｆｕ／ｍｌ）をインゲンの花器に散布した。散布処理後、１５℃で５日間インキュベートした。また、無処理を対照とし、ボトリシス・シネレア（Botrytis cinerea）の分生胞子懸濁液（５．０×１０5ｃｆｕ／ｍｌ）のみを散布処理した。調査は、インゲン葉に形成された病斑直径を計測した。Gray mold control effect test of plant disease control composition (in-house test)
Bacillus subtilis 4-5-1-1 strain No. 1 cultured and prepared in the same manner as in Example 2. After 30% FERM BP-10244 cell suspension lyophilized product (1.0 × 10 10 cfu / g) 10%, Showa clay 40%, potassium chloride 40%, Newkalgen PS-
表４から明らかなように、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株は、ボトキラー水和剤やインプレッション水和剤に比べ、散布液中の菌体濃度が少なくても高い灰色かび病防除活性を示すことが認められた。
植物病害防除剤組成物のかんきつかいよう病菌防除効果試験（室内試験）
実施例７で使用した水和剤Ｃを水道水で１０００倍に希釈し、傷を付けたレモン切取葉を浸漬処理した。加湿状態で6時間静置後、傷口にかんきつかいよう病菌懸濁液（Ａ600＝ ０．５）を滴下した。２５℃で１０日間インキュベートした。また、無処理を対照とし、かんきつかいよう病菌懸濁液のみを滴下処理した。調査は、下記基準に基づいて発病度を求めた。
接種 1 週間後に発病度を
０：かいようが認められない
１：僅かに認められる
２：認められる
３：顕著に認められる
の４ 段階に分類し、防除価を求めた。Citrus wilt disease control effect test of plant disease control composition (in-house test)
The wettable powder C used in Example 7 was diluted 1000 times with tap water, and the cut lemon leaves were soaked. After leaving still for 6 hours in a humidified state, a suspicious fungus suspension (A 600 = 0.5) was dropped into the wound. Incubated at 25 ° C. for 10 days. In addition, with no treatment as a control, only the suspension of citrus disease was treated dropwise. In the survey, the severity was determined based on the following criteria.
One week after the inoculation, the disease severity was classified into four stages: 0: no irritation 1: slightly recognized 2: observed 3: remarkably recognized, and the control value was determined.
表５から明らかなように、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０は、カンキツかいよう病に対し、高い防除活性があることが認められることが明らかとなった。
植物病害防除剤組成物の軟腐病防除効果試験（室内試験）
実施例７で使用した水和剤Ｃを水道水で１０００倍に希釈し、レタス切片を浸漬処理した。加湿状態で６時間静置後、切片傷口に軟腐病菌懸濁液（Ａ600＝ ０．５）を滴下した。２５℃で７日間インキュベートした。また、無処理を対照とし、軟腐病菌懸濁液のみを滴下処理した。調査は、下記基準に基づいて発病度を求め、防除価を算出した。
処理７日後に発病度を
０：病変なし
１：病変が接種部付近に留まる
２：病変が中肋部全体に及ぶが外皮の内側に留まる
３：病変が外皮に及ぶが、左右の広がりが中肋部よりそれぞれ１ｃｍ未満
４：病変が外皮に及び、中肋部より１ｃｍ以上広がる
の 5 段階に分類し、防除価を求めた。Soft rot control effect test of plant disease control composition (in-house test)
The wettable powder C used in Example 7 was diluted 1000 times with tap water, and the lettuce slices were immersed. After leaving still for 6 hours in a humidified state, a soft rot suspension (A 600 = 0.5) was added dropwise to the cut wound. Incubated for 7 days at 25 ° C. Moreover, only the soft rot suspension was treated dropwise with no treatment as a control. In the survey, the disease severity was calculated based on the following criteria, and the control value was calculated.
7 days after treatment 0: No lesion 1: Lesion stays near the inoculation area 2: Lesions cover the entire middle but not inside the outer skin 3: Lesions reach the outer skin, but right and left spread Less than 1 cm each from the buttocks. 4: The lesion spreads to the outer skin and spreads more than 1 cm from the middle buttocks, and the control value was determined.
表６から明らかなように、バチルス ズブチリス（Bacillus subtilis）４−５−１−１株 Ｎｏ．３０は、野菜類の軟腐病に対し、防除効果が認められることが明らかとなった。
本発明の植物病害防除剤組成物は、各種農園芸作物等の植物の病害、特に灰色かび病や菌核病，うどんこ病，炭疽病，輪斑病，青かび病，緑かび病、軟腐病、かいよう病等種々様々な病害に対して、市販のバチルス属製剤が十分な効果を示さない低温条件下に於いても安定して優れた防除能を有する微生物を含有することにより長期に亘り病害防除能を保持することができ、また、一度に他種類の病害を防除することができる。かかる植物病害防除剤組成物を用いた本発明の植物病害の防除方法によれば、植物病害防除剤組成物を各種農園芸作物等の植物や、土壌に散布する等の方法により、植物の病害、特に灰色かび病や菌核病，うどんこ病，炭疽病，輪斑病，青かび病，緑かび病、軟腐病、かいよう病に対して効果的に防除することができる。 The plant disease control agent composition of the present invention comprises plant diseases such as various agricultural and horticultural crops, in particular, gray mold disease, mycorrhizal disease, powdery mildew disease, anthrax disease, ring spot disease, blue mold disease, green mold disease, soft rot disease. For various diseases such as scab, long-term disease by containing microorganisms with stable and excellent controlling ability even under low temperature conditions where commercial Bacillus preparations do not have sufficient effect Control ability can be maintained, and other kinds of diseases can be controlled at once. According to the plant disease control method of the present invention using such a plant disease control agent composition, the plant disease control method is applied to plants such as various agricultural and horticultural crops or to the soil by a method such as spraying the plant disease control composition. In particular, it can be effectively controlled against gray mold, mycorrhizal disease, powdery mildew, anthrax, ring spot, blue mold, green mold, soft rot, and mildew.
Claims (6)
バチルス エスピー（Bacillus sp.）４−５−２１ ０３０６株。(Claim 10 before amendment)
Bacillus sp. 4-5-21 0306 strain.
a.菌株分離源から菌体を採取する工程
b.常温（１８〜２５℃）で灰色かび病（Botrytis cinerea）に対して防除効果を有する菌株を選抜する工程
c.低温（８〜１２℃）で生育可能な菌株を選抜する工程
d.低温（１３〜１５℃）で灰色かび病（Botrytis cinerea）に対して防除効果を有する菌株を再選抜する工程A method for selecting a microorganism belonging to the genus Bacillus (Bacillus sp.) Having a disease control ability at 10 to 15 ° C., comprising the following successive steps a to d.
a. Collecting cells from strain isolation source
b. A step of selecting a strain having an effect of controlling against gray mold (Botrytis cinerea) at room temperature (18-25 ° C.)
c. Process for selecting strains that can grow at low temperature (8-12 ° C)
d. Reselection of a strain having a controlling effect against gray mold (Botrytis cinerea) at a low temperature (13 to 15 ° C.)
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| CN115011505B (en) * | 2022-04-24 | 2023-04-18 | 山东省农业科学院 | Bacillus subtilis and application thereof |
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