JP2001149066A - Method for producing sporangium of bacillus popilliae - Google Patents

Method for producing sporangium of bacillus popilliae

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Publication number
JP2001149066A
JP2001149066A JP33862899A JP33862899A JP2001149066A JP 2001149066 A JP2001149066 A JP 2001149066A JP 33862899 A JP33862899 A JP 33862899A JP 33862899 A JP33862899 A JP 33862899A JP 2001149066 A JP2001149066 A JP 2001149066A
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JP
Japan
Prior art keywords
sporangia
medium
bacillus
weight
spore
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
Application number
JP33862899A
Other languages
Japanese (ja)
Other versions
JP4472813B2 (en
Inventor
Masatoshi Kimura
雅敏 木村
Takeshi Ebara
岳 江原
Hideji Nishibashi
秀治 西橋
Azusa Fujiie
梓 藤家
Makoto Hasegawa
誠 長谷川
Masao Tanaka
正男 田中
Tomoko Yokoyama
とも子 横山
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chiba Prefectural Government
DIC Corp
Original Assignee
Chiba Prefectural Government
Dainippon Ink and Chemicals Co Ltd
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Priority to JP33862899A priority Critical patent/JP4472813B2/en
Publication of JP2001149066A publication Critical patent/JP2001149066A/en
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Publication of JP4472813B2 publication Critical patent/JP4472813B2/en
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Abstract

PROBLEM TO BE SOLVED: To provide an industrial method for producing a sporangium of Bacillus popilliae, capable of affording a large amount of sporangium of Bacillus popilliae containing a spore and a parasporal body by using an artificial medium not using insects of the family Scarabaeidae, to obtain an excellent controller for insects of the family Scarabaeidae, greatly damaging roots of lawn grass, agricultural and horticultural crops, trees, etc., comprising the sporangium as an active ingredient and to provide a method for controlling insects of the family Scarabaeidae. SOLUTION: This method for producing the sporangium of Bacillus popilliae containing a spore and a parasporal body is characterized in that a bacterium belonging to Bacillus popilliae is cultured in a medium containing 0.01 wt.% to 0.5 wt.% of activated carbon. This controller against insects of the family Scarabaeidae comprises the sporangium as an active ingredient. The method for controlling insects of the family Scarabaeidae uses the sporangium.

Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【0001】[0001]

【発明の属する技術分野】本発明は、バチルス・ポピリ
エに属する菌の人工培地での培養による胞子とパラスポ
ラルボディとを含むバチルス・ポピリエの胞子嚢の製造
方法と、該胞子嚢を有効成分とするコガネムシ科昆虫の
防除剤、及びコガネムシ科昆虫の防除方法に関する。
TECHNICAL FIELD The present invention relates to a method for producing a Bacillus popirie sporangia containing a spore and a parasporal body by culturing a bacterium belonging to the genus Bacillus popirie in an artificial medium, and using the sporangia as an active ingredient. And a method for controlling scarabidae insects.

【0002】[0002]

【従来の技術】コガネムシ科昆虫の幼虫は、芝や農園芸
作物や樹木等の広範囲な植物の根を食餌し、それらに多
大の被害を与える。これらの幼虫は地中に棲息すること
から、地上から散布する化学農薬が効きにくく、且つそ
の棲息場所が特定しにくいことから、十分な殺虫効果を
得る為には、広範囲に、多量の殺虫性農薬を散布して地
中に浸透させる必要があり、その結果、環境汚染を引き
起こし易い為に、事実上、その防除、駆除は殆ど困難で
あった。
2. Description of the Related Art Larvae of scarab beetles feed on the roots of a wide range of plants such as turf, agricultural and horticultural crops and trees, and cause great damage to them. Since these larvae live in the ground, chemical pesticides sprayed from the ground are difficult to work and their habitats are difficult to identify, so in order to obtain a sufficient insecticidal effect, a large amount of insecticidal Pesticides must be sprayed and penetrated into the ground. As a result, environmental pollution is likely to be caused, so that its control and control have been practically difficult.

【0003】一方、バチルス・ポピリエに属する菌は、
これらコガネムシ科昆虫の幼虫に寄生して乳化病を発病
させ、最終的にコガネムシ科昆虫を死に至らしめること
から、化学農薬が効きにくいコガネムシ科昆虫の駆除に
バチルス・ポピリエに属する菌を利用しようとする試み
が古くから行われてきた。
[0003] On the other hand, bacteria belonging to Bacillus popiliae are:
The parasites of these Scarabaeid insects parasitize and cause emulsification disease, eventually causing the Scarabaeidae insects to die.Therefore, an attempt is made to use bacteria belonging to Bacillus popirie for the control of Scarabaeidae insects, which are difficult to work with chemical pesticides. Attempts to do so have been made since ancient times.

【0004】また、バチルス・ポピリエに属する菌の培
養方法としては、寄生宿主であるコガネムシ科の生きた
幼虫を用いて菌を増殖させ胞子嚢を製造する方法が従来
から知られていた。
[0004] As a method for cultivating bacteria belonging to Bacillus popiliae, a method for producing sporangia by growing bacteria using living larvae of the family Scarabaeidae, which is a parasitic host, has been conventionally known.

【0005】即ち、バチルス・ポピリエの胞子嚢をコガ
ネムシ科昆虫の幼虫の存在する飼育培土に散布して、バ
チルス・ポピリエの胞子嚢を経口的にコガネムシ科昆虫
の幼虫に摂取させるか、又は体液中に注射により注入し
た後、幼虫を3週間〜4週間飼育し、該幼虫体内でバチ
ルス・ポピリエを増殖させ、その後、幼虫に孔を開ける
か切開して体液を採取し、得られた体液を遠心分離又は
濾過することにより、幼虫体内で増やした胞子嚢を得る
方法である。
That is, the sporangia of Bacillus popirie is sprayed on a breeding medium in which larvae of Scarabaeid insects are present, and the sporangia of Bacillus popirie is orally ingested by the larvae of Scarabaeid insects. After injection, the larvae are bred for 3 to 4 weeks, Bacillus populiae is propagated in the larvae, and then the bodily fluid is collected by piercing or incising the larvae, and the obtained bodily fluid is centrifuged. This is a method of obtaining sporangia increased in the larva by separating or filtering.

【0006】しかしながら、この方法では、コガネムシ
科昆虫の幼虫を大量に飼育する必要があり、結果的に大
量の胞子嚢を多量に、短時間に、且つ経済的に得ること
は困難であった。この為、コガネムシ科昆虫の幼虫を用
いない人工培地を用いての培養方法に関する多くの研究
が行われてきた。
However, according to this method, it is necessary to breed a large number of larvae of Scarabaeid insects, and as a result, it has been difficult to obtain a large amount of sporangia in a large amount in a short time and economically. For this reason, many studies have been conducted on a culture method using an artificial medium that does not use larvae of Scarabaeidae.

【0007】これらのコガネムシ科昆虫の幼虫を用いな
い人工培地での培養方法(以下、昆虫体外培養法と呼ぶ
ことがある)としては固体培養と液体培養がある。固体
培養ではFOSTER等(J.W.Foster et al.,1950年,J.Bacte
riol,59巻,463-470頁)、STEINKRAUS(Keith H.Steinkra
us,1957年, J.Bacteriol,74巻,625-632頁)の研究例
が、液体培養では、HAYNES等(W.C.Haynes et al.,Jour
nal of Bacteriology,June,1966年,91巻,No6,2270-2274
頁)の研究例がある。しかし、いずれの場合も、得られ
た胞子あるいは胞子嚢のコガネムシ科昆虫の幼虫に対す
る殺虫効果は明記されておらず、また商業生産にも繋が
っていない。
[0007] As a method of cultivating the artificial medium without using the larva of the insects of the order Scarabaeidae (hereinafter sometimes referred to as an insect extracorporeal culture method), there are solid culture and liquid culture. In solid culture, FOSTER et al. (JWFoster et al., 1950, J. Bacte
riol, 59, 463-470), STEINKRAUS (Keith H. Steinkra
US, 1957, J. Bacteriol, Vol. 74, pp. 625-632), but in liquid culture, HAYNES et al. (WCHaynes et al., Journey
nal of Bacteriology, June, 1966, 91, No6, 2270-2274
Page)). However, in any case, the insecticidal effect of the obtained spores or sporangia on the larvae of Scarabaeidae is neither specified nor led to commercial production.

【0008】米国特許(USP No.4824671)は人工培地を
用いた液体培養によりバチルス・ポピリエに属する菌を
培養している例である。しかし得られた胞子嚢に胞子は
有るが、パラスポラルボディは存在せず、幼虫体内で形
成された胞子嚢に比較してコガネムシ科昆虫の幼虫に対
する殺虫効果は弱い。また福原も人工培地から得た胞子
嚢では感染が起こらないと報告している(福原俊彦著
昆虫病理学 57頁)。
[0008] A US patent (USP No.4824671) is an example in which bacteria belonging to Bacillus popirie are cultured by liquid culture using an artificial medium. However, although the obtained sporangia has spores but no parasporal body, the insecticidal effect on the larvae of Scarabaeidae is weaker than that of the sporangia formed in the larva. Fukuhara also reported that infection did not occur in sporangia obtained from artificial media (by Toshihiko Fukuhara)
Insect pathology p. 57).

【0009】胞子嚢とは、図1に示す模式図の如く、胞
子とパラスポラルボディ(又は副胞子小体)と呼ばれる
小体を含む嚢であり、バチルス・ポピリエに属する菌が
産生する。バチルス・ポピリエの人工培地を用いた培養
方法に関する従来の文献では、胞子嚢と胞子とが明確な
区別なく用いられている場合が多く、文献中の胞子との
言葉が胞子のみを意味するのか、胞子は含むがパラスポ
ラルボディは含まない胞子嚢を意味するのか、又は胞子
とパラスポラルボディとを含む胞子嚢であるのかが不明
である場合が多い。
As shown in the schematic diagram of FIG. 1, a sporangia is a sac containing spores and a body called a parasporal body (or parasporium), and is produced by a bacterium belonging to Bacillus populiae. In the conventional literature on a culture method using an artificial medium of Bacillus populiae, sporangia and spores are often used without distinction, and the term spore in the literature means only spores, It is often unclear whether it refers to a sporangium containing spores but not a parasporal body, or a sporangium containing spores and a parasporal body.

【0010】従って、従来の文献に記載されている人工
培地での培養方法で得られたバチルス・ポピリエの胞子
が、胞子とパラスポラルボディを含む胞子嚢であるのか
は不明であり、更に該胞子もしくは胞子嚢のコガネムシ
科昆虫の幼虫に対する殺虫効果も明らかにされていない
が、後述する理由から、それらの多くは胞子とパラスポ
ラルボディとを含む胞子嚢ではなく、胞子のみ又は胞子
のみを含む胞子嚢であり、且つそのコガネムシ科昆虫に
対する殺虫効果は弱いと推定された。
Therefore, it is unclear whether the spores of Bacillus populiae obtained by the method of culturing in an artificial medium described in the conventional literature are spores containing spores and parasporal bodies. Although the insecticidal effect of spores or spores on the larvae of Scarabaeidae is not clear, many of them are not spores containing spores and parasporal bodies, but only spores or only spores. It was presumed that the insecticidal effect on Scarabaeidae insects was weak.

【0011】[0011]

【発明が解決しようとする課題】本発明が解決しようと
する課題は、コガネムシ科昆虫の幼虫を用いず、人工培
地を用いて胞子とパラスポラルボディとを含む多量のバ
チルス・ポピリエの胞子嚢を得るバチルス・ポピリエ胞
子嚢の工業的な製造方法、該胞子嚢を有効成分とする、
芝や農園芸作物や樹木等の根に多大な被害を与えるコガ
ネムシ科昆虫の優れた防除剤、及び防除方法を提供する
ことである。
SUMMARY OF THE INVENTION An object of the present invention is to provide a large amount of spores of Bacillus populiae containing spores and parasporal bodies using an artificial medium without using larvae of Scarabaeidae. An industrial method for producing a Bacillus popirie sporangia to obtain the sporangia as an active ingredient,
It is an object of the present invention to provide an excellent control agent and an excellent control method for insects of the order Scarabaeidae which cause great damage to the roots of turf, agricultural and horticultural crops, trees, and the like.

【0012】[0012]

【課題を解決するための手段】本発明者らは上記課題を
解決すべく鋭意研究を重ねた結果、コガネムシ科昆虫の
効果的な殺虫防除には、バチルス・ポピリエの胞子のみ
でなく、胞子とパラスポラルボディとを含む胞子嚢が必
要であることを見出した。更に、バチルス・ポピリエ菌
をコガネムシ科昆虫の幼虫体外で増殖させ、且つ胞子と
パラスポラルボディとを含む胞子嚢を形成させる為に
は、該菌の増殖に伴って菌体外に排出される胞子嚢形成
を阻害する代謝産物を除去する必要があること、また該
阻害代謝産物を除去する際に、胞子嚢の形成に必要な成
分までを除去しない必要があることを見出した。
Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, effective elimination of insects of the order Scarabaeidae is not limited to spores of Bacillus populiae but also spores. It has been found that a sporangia containing a parasporal body is required. Furthermore, in order to grow Bacillus popirie outside of the larva of Scarabaeidae, and to form a sporangium containing spores and a parasporal body, the bacterium is excreted out of the cell with the growth of the bacterium. It has been found that it is necessary to remove metabolites that inhibit sporangium formation, and that it is not necessary to remove components required for sporangial formation when removing the inhibitory metabolites.

【0013】従来の報告例においても、バチルス・ポピ
リエの人工培地での培養では、菌の生育の為に培養阻害
物質を除去することが必要であり、活性炭や可溶性デン
プン等の吸着剤を人工培地に添加している例が見られ
た。しかしながら、本発明者らは、阻害物質の除去材で
あるこれらの活性炭の添加量を制限して培養を行わね
ば、胞子とパラスポラルボディとを含む胞子嚢が形成さ
れないことを見出した。
[0013] Even in the conventional reports, in the cultivation of Bacillus populiae in an artificial medium, it is necessary to remove a culture inhibitor for the growth of the bacterium, and an adsorbent such as activated carbon or soluble starch is added to the artificial medium. Was added. However, the present inventors have found that a sporangium containing spores and a parasporal body is not formed unless the culture is carried out with a limited amount of the activated carbon as a material for removing an inhibitory substance.

【0014】即ち、本発明者らは、バチルス・ポピリエ
の人工培地への活性炭の添加量が0.5重量%を越える
と、胞子嚢を形成するのに必要な栄養分までも除去して
しまう為か、胞子とパラスポラルボディとを含む胞子嚢
形成が急激に減少することを見出した。一方、従来報告
されている阻害物質吸着剤の添加量は、培地に対してい
ずれも0.6重量%以上であり、且つコガネムシ科昆虫
の殺虫効果を発揮する胞子又は胞子嚢の形成程度は報告
されていなかった。
That is, the present inventors have found that if the amount of activated carbon added to the artificial medium of Bacillus popirie exceeds 0.5% by weight, even nutrients necessary for forming sporangia are removed. Alternatively, sporangia formation including spores and parasporal bodies was found to be sharply reduced. On the other hand, the amount of the conventionally added inhibitor adsorbent is 0.6% by weight or more with respect to the medium, and the degree of formation of spores or spores that exhibit the insecticidal effect of Scarabaeidae insects is reported. Had not been.

【0015】本発明者らは、従来報告された0.6重量
%以上の活性炭を含む人工培地での培養では、菌(栄養
細胞)自体の生育は良好であるものの、胞子とパラスポ
ラルボディとを含む胞子嚢の生産量は極めて少なく、コ
ガネムシ科昆虫の殺虫効果を発揮するのに十分な胞子と
パラスポラルボディとを含む胞子嚢が得られないこと、
更に人工培地中にグルコースを一定量以上含有している
場合には、更に胞子嚢を得にくいことも併せて見いだし
た。
The inventors of the present invention have reported that culturing in an artificial medium containing 0.6% by weight or more of activated carbon, which has been reported so far, allows the growth of the bacterium (vegetative cell) itself to be good, but the spores and the parasporal body The production amount of sporangia containing spores is extremely small, and a sporangium containing sufficient spores and a parasporal body to exhibit an insecticidal effect of a scarabid insect is not obtained,
Furthermore, it was also found that when the artificial medium contains a certain amount of glucose or more, it is more difficult to obtain sporangia.

【0016】従って、従来報告された活性炭を含有する
人工培地でのバチルス・ポピリエの培養では、胞子とパ
ラスポラルボディとを含む胞子嚢が十分に得られず、明
確なコガネムシ科昆虫の幼虫の殺虫効果が確認できなか
った為に、その殺虫効果の報告がなかったものと推定さ
れる。
[0016] Therefore, the conventionally reported cultivation of Bacillus populiae in an artificial medium containing activated charcoal does not provide sufficient spores containing spores and parasporal bodies, and the larvae of the definite insects of the order Scarabaeidae cannot be obtained. It is highly probable that no insecticidal effect was reported because the insecticidal effect could not be confirmed.

【0017】これらから本発明者らは人工培地中の活性
炭量及びグルコース量を制限することにより、コガネム
シ科に属する昆虫に対して殺虫性を有する胞子とパラス
ポラルボディとを含む多数の胞子嚢を有するバチルス・
ポピリエの培養方法及び、該培養方法によって得られる
胞子嚢を有効成分とするコガネムシ科昆虫の防除剤を完
成するに至った。
[0017] From these, the present inventors restricted the amount of activated carbon and glucose in the artificial medium to thereby produce a large number of sporangia containing insecticidal spores and parasporal bodies against insects belonging to the family Scarabaeidae. Bacillus with
A method for cultivating poppyri and a control agent for Scarabaeidae comprising a sporangia obtained by the culturing method as an active ingredient have been completed.

【0018】即ち、本発明は、(1)バチルス・ポピリ
エに属する菌を活性炭0.01重量%〜0.5重量%を
含む培地で培養することを特徴とする、胞子とパラスポ
ラルボデイとを含むバチルス・ポピリエの胞子嚢の製造
方法と、
That is, the present invention provides (1) spores and a parasporal body, wherein a bacterium belonging to Bacillus popiliae is cultured in a medium containing 0.01% to 0.5% by weight of activated carbon. A method for producing a Bacillus popirie sporangia comprising:

【0019】(2)式1の胞子嚢化率で示される、胞子
嚢数の全菌数当たりの割合が0.1%以上であることを
特徴とする(1)に記載の製造方法と、
(2) The method according to (1), wherein the ratio of the number of sporangia to the total number of bacteria is 0.1% or more, which is represented by the sporangialization rate of the formula 1,

【0020】[0020]

【式1】 胞子嚢化率(%)=(胞子嚢数)/(全菌数)×100[Formula 1] Sporulation rate (%) = (number of sporangia) / (total number of bacteria) × 100

【0021】(3)培地中に含まれるグルコース濃度が
0.01重量%以下であることを特徴とする(1)又は
(2)に記載の製造方法と、(4)胞子とパラスポラル
ボディとを含む胞子嚢がコガネムシ科の昆虫に対して殺
虫性を有することを特徴とする(1)〜(3)の何れか
1つに記載の製造方法と、
(3) The production method according to (1) or (2), wherein the concentration of glucose contained in the medium is 0.01% by weight or less, and (4) spores and a parasporal body. The method according to any one of (1) to (3), wherein the sporangia comprising: has insecticidal activity against insects of the order Scarabaeidae; and

【0022】(5)コガネムシ科の昆虫がドウガネブイ
ブイである(4)に記載の製造方法と、(6)培養が固
体培養である(1)〜(5)の何れか1つに記載の製造
方法と、
(5) The production method according to (4), wherein the insect of the family Scarabaeidae is Scutellariae, and (6) the production method according to any one of (1) to (5), wherein the culture is a solid culture. When,

【0023】(7)培地中に、寒天、ポリペプトン、酵
母エキス及びリン酸塩を含むことを特徴とする(6)に
記載の製造方法と、(8)バチルス・ポピリエに属する
菌がバチルス・ポピリエ・セマダラ(FERM P−1
6818)である(1)〜(7)の何れか1つに記載の
製造方法と、
(7) The production method according to (6), wherein the medium contains agar, polypeptone, yeast extract and phosphate, and (8) the bacterium belonging to Bacillus popiliae is・ Semadara (FERM P-1
6818), the method according to any one of (1) to (7),

【0024】(9)上記の(1)〜(8)の何れか1つ
に記載の製造方法により製造された、胞子とパラスポラ
ルボデイとを含むバチルス・ポピリエの胞子嚢を有効成
分とするコガネムシ科昆虫の防除剤と、
(9) The sporangium of Bacillus popirie containing spores and parasporal body, which is produced by the production method according to any one of the above (1) to (8), is used as an active ingredient. An insecticide for scarabidae,

【0025】(10)上記の(1)〜(8)の何れか1
つに記載の製造方法により製造された胞子とパラスポラ
ルボディとを含むバチルス・ポピリエの胞子嚢をコガネ
ムシ科昆虫に作用させるコガネムシ科昆虫の防除方法と
を含むものである。
(10) Any one of the above (1) to (8)
And a method for controlling Scarabaeidae insects in which a spore bacillus of Bacillus popirie containing a spore and a parasporal body produced by the production method described in (1) is applied to Scarabaeidae.

【0026】[0026]

【発明の実施の形態】本発明で用いられるバチルス・ポ
ピリエに属する菌とは、バチルス属ポピリエ種(Bacill
us popilliae)に属する菌である。バチルス属ポピリエ
種の細菌学的性質は、バージェイズ・マニュアル・オブ
・デターミネイティブ・バクテリオロジー(Bargeys Ma
nual of Determinative Baceriology)によれば、形態
的性質は長さが1.3〜5.2μm、幅が0.5〜0.
8μmのグラム陰性桿菌、生育温度は20〜35℃で胞
子嚢の中にパラスポラルボディを持っている。
BEST MODE FOR CARRYING OUT THE INVENTION The bacterium belonging to the genus Bacillus populiae used in the present invention is Bacillus sp.
us popilliae). The bacteriological properties of the Bacillus spp. Species are described in the Bargeys Manual of Deterministic Bacteriology (Bargeys Ma
According to the Natural of Determinative Baceriology), the morphological properties are 1.3-5.2 μm in length and 0.5-0.5 μm in width.
8 μm gram-negative bacilli, growth temperature 20-35 ° C., with a parasporal body in the sporangia.

【0027】バチルス属ポピリエ種(Bacillus popilli
ae)に属する菌には、バチルス・ポピリエ・セマダラ株
(Bacillus popilliae semadara)(平成10年5月2
1日から通商産業省工業技術院生命工学工業技術研究所
に受託番号 FERM P−16818として寄託され
ている)、バチルス・ポピリエ・デュトキ株(Bacillus
popilliae dutky)(American Type Culture Collecti
on No.14706、以下ATCC 14706と称
す)、バチルス・ポピリエ・メロロンサ(Bacilluspopi
lliae melolontha)等が挙げられる。
Bacillus popilli
The bacteria belonging to ae) include Bacillus popilliae semadara (Bacillus popilliae semadara) (May 2, 1998).
The Bacillus popirie dutoki strain (deposited with the Accession No. FERM P-16818 at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry)
popilliae dutky) (American Type Culture Collecti
on No. 14706, hereafter referred to as ATCC 14706), Bacillus popirie meloronsa (Bacilluspopi)
lliae melolontha) and the like.

【0028】以下に本発明によるバチルス・ポピリエ胞
子嚢の製造方法について述べる。該微生物の培養方法と
しては通常の固体培養で行うことができる。固体培養の
基材としては寒天・ジェランガム・カードラン・キサン
タンガム等の多糖類が使用できるが、寒天を用いた培養
が特に好ましい。
Hereinafter, a method for producing a Bacillus populiae sporangia according to the present invention will be described. As a method for culturing the microorganism, ordinary solid culture can be used. Polysaccharides such as agar, gellan gum, curdlan, and xanthan gum can be used as a substrate for solid culture, but culture using agar is particularly preferred.

【0029】一般に微生物の良好な増殖を促すには培地
中に糖類などの炭素源が加えられる。本発明に用いるバ
チルス・ポピリエに属する菌も、グルコース等の糖源を
添加することにより菌体の増殖が促進される。しかしな
がら胞子とパラスポラルボデイとを含む胞子嚢を形成さ
せるためには、グルコースの添加は好ましくなく、培地
中に含まれるグルコース濃度は0.01重量%以下にす
ることが好ましい。
In general, a carbon source such as a saccharide is added to the medium to promote good growth of microorganisms. Bacteria belonging to the genus Bacillus popillier used in the present invention are also promoted by the addition of a sugar source such as glucose. However, in order to form a sporangia containing spores and parasporal body, the addition of glucose is not preferable, and the concentration of glucose contained in the medium is preferably 0.01% by weight or less.

【0030】培地に用いる窒素源としてはポリペプト
ン、乾燥酵母エキス等の有機性窒素が使用できる。これ
らの濃度は0.1重量%〜3.0重量%、好ましくは
0.25重量%〜1.5重量%の範囲である。それらの
窒素源には、しばしばグルコースが含まれている場合が
あるが、培地中のグルコース濃度が0.01重量%を越
えないように、窒素源を選択し使用することが好まし
い。
As the nitrogen source used for the medium, organic nitrogen such as polypeptone and dried yeast extract can be used. These concentrations range from 0.1% to 3.0% by weight, preferably 0.25% to 1.5% by weight. These nitrogen sources often contain glucose, but it is preferable to select and use a nitrogen source so that the glucose concentration in the medium does not exceed 0.01% by weight.

【0031】培地に用いるリン酸塩としては、リン酸カ
リウム、リン酸水素二カリウム等のリン酸塩又はそのナ
トリウム塩等の無機塩が使用できる。これらの濃度は
0.05重量%〜1.0重量%の範囲で用いることが望
ましい。固体培地とするために加えられる寒天、ジェラ
ンガム等の多糖類の濃度は0.5重量%〜5.0重量
%、好ましくは1.0重量%〜3.0重量%である。
As the phosphate used in the medium, phosphates such as potassium phosphate and dipotassium hydrogen phosphate or inorganic salts such as sodium salts thereof can be used. These concentrations are desirably used in the range of 0.05% by weight to 1.0% by weight. The concentration of polysaccharides such as agar and gellan gum added to make a solid medium is 0.5% by weight to 5.0% by weight, preferably 1.0% by weight to 3.0% by weight.

【0032】培地中には活性炭が添加されるが、添加さ
れる活性炭の種類は粉末状、粒状、シート状等のいかな
る種類の活性炭でもよく、特に制限はないが、粉末状の
活性炭が好ましい。培地中の活性炭濃度は、本発明の重
要な要件の一つであり、0.01重量%〜0.5重量%
である必要がある。培地中の活性炭濃度が0.01重量
%以下では、胞子とパラスポラルボデイとを含む胞子嚢
の形成を阻害する物質の除去効果が十分でなく、また
0.5重量%を越えると、胞子とパラスポラルボデイと
を含む胞子嚢の形成が急激に減少する。
Activated carbon is added to the medium, and the type of activated carbon to be added may be any type of activated carbon such as powder, granule, sheet and the like. There is no particular limitation, but powdered activated carbon is preferred. The activated carbon concentration in the medium is one of the important requirements of the present invention, and is 0.01% to 0.5% by weight.
Needs to be When the concentration of activated carbon in the medium is 0.01% by weight or less, the effect of removing substances that inhibit the formation of sporangia including spores and parasporal body is not sufficient. And the formation of sporangia including parasporal body is sharply reduced.

【0033】活性炭の添加方法としては殺菌前の培地中
に添加しても良いし、殺菌後の培地に添加しても良い
が、活性炭が十分に殺菌され他の微生物に汚染されてい
ないことが必要である。培養温度は25℃〜35℃、好
ましくは27℃〜30℃である。培養時間は培養温度に
よって異なるが、27℃〜30℃の場合で通常10〜1
5日である。
The activated carbon may be added to the medium before sterilization or may be added to the medium after sterilization. However, it is important that the activated carbon is sufficiently sterilized and is not contaminated by other microorganisms. is necessary. The culturing temperature is 25 ° C to 35 ° C, preferably 27 ° C to 30 ° C. The culturing time varies depending on the culturing temperature, but is usually 10 to 1 at 27 ° C to 30 ° C.
5 days.

【0034】培養終了後、培養物から胞子嚢を取得する
方法としては、胞子嚢を含んだ菌体が固体培養基の表面
にあることから、水あるいはリン酸緩衝液、Tris-HCl等
の緩衝液を用いて菌体及び胞子嚢を懸濁させて洗い流
し、その後、遠心分離や濾過等の一般の分離方法で菌体
及び胞子嚢を取得する。
After completion of the culture, a method for obtaining sporangia from the culture is as follows. Since the cells containing the sporangium are on the surface of the solid culture medium, a buffer solution such as water or a phosphate buffer or Tris-HCl is used. The cells and the sporangia are suspended and washed off by using, and then the cells and the sporangia are obtained by a general separation method such as centrifugation or filtration.

【0035】0.6重量%以上の活性炭を含有する従来
の人工培地での培養では、胞子とパラスポラルボデイと
を含むバチルス・ポピリエの胞子嚢は殆ど得られず、式
1の胞子嚢化率で示される、全菌数当たりの胞子嚢数の
割合は、通常0.05重量%未満である。
In cultivation in a conventional artificial medium containing at least 0.6% by weight of activated carbon, almost no spores of Bacillus popirie containing spores and parasporal body are obtained, and sporangia of Formula 1 is obtained. The ratio of the number of sporangia to the total number of bacteria, expressed as a percentage, is usually less than 0.05% by weight.

【0036】[0036]

【式1】 胞子嚢化率(%)=(胞子嚢数)/(全菌数)×100[Formula 1] Sporulation rate (%) = (number of sporangia) / (total number of bacteria) × 100

【0037】これに対して、本発明のバチルス・ポピリ
エの胞子嚢の製造方法により得られる胞子嚢数は、従来
の培養方法で得られる胞子嚢数よりも遥かに多い。
On the other hand, the number of sporangia obtained by the method for producing sporangia of Bacillus populiae of the present invention is much larger than the number of sporangia obtained by the conventional culture method.

【0038】取得した菌体及び胞子嚢は、それらを懸濁
した緩衝液のまま、コガネムシ科昆虫の防除剤として用
いてもよく、あるいは乾燥して粉末にして用いても良
い。また乾燥した後、水あるいは緩衝液の懸濁液として
使用しても良い。更にこれらの胞子嚢は農薬に用いられ
る公知慣用の賦形剤、担体、栄養剤等の成分と混合して
使用することもできる。また本発明の胞子と他の微生物
製剤を混合して使用することも可能である。
The obtained bacterial cells and sporangia may be used as a control agent for insects of the order Scarabaeidae in the form of a buffer in which they are suspended, or may be dried and used as a powder. After drying, it may be used as a suspension of water or a buffer solution. Further, these sporangia may be used in admixture with components such as known and commonly used excipients, carriers and nutrients used for agricultural chemicals. It is also possible to use the spores of the present invention in combination with other microbial preparations.

【0039】本発明のバチルス・ポピリエの胞子嚢を有
効成分とするコガネムシ科昆虫の防除剤は、芝や農作物
の害虫としてよく知られているコガネムシ科昆虫のドウ
ガネブイブイ(Anomala cuprea)、セマダラコガネ(Bl
itopertha orientalis)、マメコガネ(Popillia japo
nica)、ウスチャコガネ(phyllopertha diversa )、
チャイロコガネ(Adoretus tenuimaculatus)、ヒメコ
ガネ(Anomala rufocuprea)等に対して、いずれも優れ
た殺虫性、防除効果を示し、中でも比較的大型なコガネ
ムシであるドウガネブイブイやセマダラコガネの幼虫に
対して優れた殺虫、防除効果を示す。
The insecticide of the present invention, which comprises the sporangia of Bacillus popiliae as an active ingredient, is an insect of the order Scarabaeidae, which is a well-known insect pest of lawns and agricultural crops.
itopertha orientalis, Bean beetle (Popillia japo)
nica), phyllopertha diversa,
Both exhibit excellent insecticidal and control effects on Chinese locust bean (Adoretus tenuimaculatus) and Japanese corn beetle (Anomala rufocuprea), and are particularly excellent against the larvae of relatively large beetles, Douganebuibui and Semarakogane. Show the effect.

【0040】[0040]

【実施例】以下、実施例及び試験例により本発明を更に
具体的に説明するが、本発明の範囲はこれらに限定され
るものではない。
The present invention will be described more specifically with reference to the following examples and test examples, but the scope of the present invention is not limited to these examples.

【0041】(実施例1)(培地の製造) 300ml容量の三角フラスコ2本それぞれに蒸留水1
00mlを入れ、トリプトン0.5重量%(DIFCO
社製)、イーストエクストラクト1.5重量%(オキゾ
イド社製)、リン酸水素二カリウム0.3重量%(和光
純薬社製特級)、寒天2.0重量%(和光純薬社製特
級)を添加し混合した。(以下、これら培地成分を含有
する培地を培地Aと称する。)
(Example 1) (Preparation of culture medium) Two 300 ml Erlenmeyer flasks each containing distilled water 1
00 ml, tryptone 0.5% by weight (DIFCO
), 1.5% by weight of yeast extract (manufactured by Oxoid), 0.3% by weight of dipotassium hydrogen phosphate (special grade manufactured by Wako Pure Chemical Industries), 2.0% by weight of agar (special grade manufactured by Wako Pure Chemical Industries) ) Was added and mixed. (Hereinafter, a medium containing these medium components is referred to as medium A.)

【0042】培地Aに和光純薬社製特級粉末活性炭を、
無添加、0.1重量%及び1.0重量%となるように加
えた。その後121℃、20分のオートクレーブ殺菌を
行い寒天が固化しないうちに十分撹拌して、直径9cm
のプラスチックシャーレにて20ml添加して平板培地
を作成した。活性炭含有量が異なるそれら平板培地を用
いて、バチルス・ポピリエ・セマダラ株及びバチルス・
ポピリエ・デュトキ(ATCC 14706)株を培養
した。
Medium A contains a special-grade powdered activated carbon manufactured by Wako Pure Chemical Industries, Ltd.
It was added so as to be 0.1% by weight and 1.0% by weight. Thereafter, the mixture was sterilized in an autoclave at 121 ° C. for 20 minutes and sufficiently stirred before the agar was solidified, and the diameter was 9 cm.
Was added in a plastic petri dish to prepare a plate medium. Using those plate media having different activated carbon contents, Bacillus popirie semadala strain and Bacillus
Popilier dutoki (ATCC 14706) strain was cultured.

【0043】セマダラ株(FERM P−16818)
は、乳化病コガネムシ感染幼虫から採取した体液中の胞
子嚢を用いた。胞子嚢数を胞子顕微鏡による直接検境法
により計測し1×104個/mlとなるように調製しエ
ッペンチューブに0.5ml取り、ヒートブロックにて
70℃、20分の加熱処理後、そのうちの50μlを上
記で調製した平板培地に塗布した。
Semadara strain (FERM P-16818)
Used sporangia in bodily fluids collected from larvae infected with scarab beetles infected with emulsified disease. The number of sporangia was measured by a direct detection method using a spore microscope, adjusted to 1 × 10 4 cells / ml, taken in an eppen tube 0.5 ml, and heated at 70 ° C. for 20 minutes in a heat block. Was applied to the plate medium prepared above.

【0044】デュトキ株は凍結乾燥保存されていた菌体
(胞子及び胞子嚢は含まず)を、DIFCO社製トリプ
トン0.5重量%、オキゾイド社製イーストエクストラ
クト1.5重量%、和光純薬社製特級リン酸水素二カリ
ウム0.3重量%、和光純薬社製特級粉末活性炭0.1
重量%を添加して121℃で20分の殺菌処理した液体
培地を用いて、30℃のインキュベータ内にて120r
pmの撹拌条件により1週間培養した。その培養液の
0.1mlを無菌的に寒天培地に塗布した。以上の菌体
を塗布した平板培地を30℃のインキュベータ内にて1
4日間培養した。
The Dutoki strain was prepared by lyophilizing and storing cells (excluding spores and sporangia) by 0.5% by weight of tryptone manufactured by DIFCO, 1.5% by weight of yeast extract manufactured by Oxoid, and Wako Pure Chemical Industries, Ltd. 0.3% by weight of special grade dipotassium hydrogen phosphate manufactured by Wako Pure Chemical Industries, Ltd.
Using a liquid medium that had been sterilized at 121 ° C. for 20 minutes with the addition of 20% by weight, and in a 30 ° C. incubator for 120 r.
The cells were cultured for one week under the stirring conditions of pm. 0.1 ml of the culture was aseptically applied to an agar medium. The plate medium coated with the above cells was placed in a 30 ° C. incubator for 1 hour.
Cultured for 4 days.

【0045】培養終了後、シャーレに無菌的に蒸留水2
mlを添加して発生したコロニーを懸濁させた後、菌体
及び胞子嚢を取得した。取得した溶液中の単位容積当た
りの胞子嚢数、及び全菌数を顕微鏡による直接検鏡によ
り計測し、式1により胞子嚢化率を算出した。表1にセ
マダラ株を用いた場合の試験結果を示す。活性炭添加濃
度が0.1重量%の場合においてのみ胞子嚢が形成し
た。
After completion of the culture, distilled water 2 is aseptically added to a petri dish.
After the resulting colonies were suspended by adding ml, bacterial cells and sporangia were obtained. The number of sporangia per unit volume in the obtained solution and the total number of bacteria were measured by direct microscopy with a microscope, and the sporangialization rate was calculated by Equation 1. Table 1 shows the test results in the case where the Semadara strain was used. Only when the activated carbon addition concentration was 0.1% by weight, sporangia was formed.

【0046】[0046]

【式1】 胞子嚢化率(%)=(胞子嚢数)/(全菌数)×100[Formula 1] Sporulation rate (%) = (number of sporangia) / (total number of bacteria) × 100

【0047】活性炭添加濃度が0.1重量%の場合に得
られたセマダラ株の胞子嚢の顕微鏡写真を図2に示す。
顕微鏡写真は株式会社ニコン製の光学顕微鏡ECLIPSE E6
00に株式会社ソニーのカラービデオカメラモジュールXC
-003を接続して撮影した(倍率:×3800)。胞子嚢
中に1つの胞子と1つのパラスポラルボデイとが含まれ
ることが判る。
FIG. 2 shows a micrograph of the sporangium of the semaphora strain obtained when the activated carbon addition concentration was 0.1% by weight.
The micrograph is of ECLIPSE E6, an optical microscope manufactured by Nikon Corporation.
00 to Sony Color Video Camera Module XC
-003 was connected and photographed (magnification: × 3800). It can be seen that the sporangium contains one spore and one parasporal body.

【0048】表2にデュトキ株を用いた場合の試験結果
を示した。同様に活性炭添加濃度が0.1重量%の場合
においてのみ胞子嚢が形成した。表1にセマダラ株によ
る胞子嚢の形成を、表2にデュトキ株による胞子嚢の形
成状況を示した。
Table 2 shows the test results when the Dutoki strain was used. Similarly, sporangia was formed only when the activated carbon concentration was 0.1% by weight. Table 1 shows the formation of sporangia by the Semadara strain, and Table 2 shows the formation status of sporangia by the Dutoki strain.

【0049】[0049]

【表1】 [Table 1]

【0050】[0050]

【表2】 [Table 2]

【0051】(実施例2)培地中の活性炭量の検討を行
った。300ml容量の三角フラスコ7本それぞれに培
地Aの成分を添加し、そこに和光純薬社製特級粉末活性
炭を、無添加、0.05重量%、0.1重量%、0.2
5重量%、0.5重量%、1.0重量%、2.0重量%
含むように調製した。その後121℃、20分のオート
クレーブ殺菌を行い実施例1と同様に平板培地を作成し
た。
(Example 2) The amount of activated carbon in the medium was examined. The components of the medium A were added to each of seven 300-ml Erlenmeyer flasks, and no special powdered activated carbon manufactured by Wako Pure Chemical Industries, Ltd. was added thereto, and 0.05% by weight, 0.1% by weight, and 0.2% by weight.
5 wt%, 0.5 wt%, 1.0 wt%, 2.0 wt%
It was prepared to contain. Thereafter, autoclave sterilization was performed at 121 ° C. for 20 minutes to prepare a plate medium in the same manner as in Example 1.

【0052】セマダラ株は実施例1において活性炭添加
濃度0.1重量%にて培養し取得した胞子嚢を含む菌体
を使用した。取得液を無菌的に蒸留水にて希釈して1×
10 5個/mlとなるように調製した。その液をエッペ
ンチューブに0.5ml取り、ヒートブロックにて70
℃、20分の加熱処理後、50μlを取り上記で調製し
た活性炭添加量の異なる平板培地にそれぞれ塗布した。
菌体を塗布した上記の平板培地を30℃のインキュベー
タ内にて14日間培養した。
The sedara strain was prepared by adding activated carbon in Example 1.
Cells containing sporangia obtained by culturing at a concentration of 0.1% by weight
It was used. The obtained solution is aseptically diluted with distilled water and 1 ×
10 FiveIt was prepared so that the number of cells / ml. Eppe the solution
0.5 ml into a heat tube and 70 with a heat block.
After heating at ℃ for 20 minutes, take 50μl and prepare as above
The activated carbon was applied to plate media with different amounts of activated carbon.
The plate medium coated with the cells was incubated at 30 ° C.
The cells were cultured for 14 days in a container.

【0053】培養終了後、シャーレに無菌的に蒸留水2
mlを添加して発生したコロニーを懸濁させた後、胞子
嚢及び菌体を取得した。取得した溶液中の単位容積当た
りの胞子嚢数及び全菌数を顕微鏡による直接検鏡により
計測し、式1により胞子嚢化率を算出した。図3に培地
中の活性炭濃度と胞子嚢化率の関係を示す。縦軸は胞子
嚢化率(%)を、横軸は培地中の活性炭濃度(重量%)
を表す。活性炭濃度が0.5重量%以下で良好な胞子嚢
形成が見られる。
After completion of the culture, distilled water 2 is aseptically added to a petri dish.
After the colonies generated were suspended by adding ml, sporangia and bacterial cells were obtained. The number of sporangia per unit volume and the total number of bacteria in the obtained solution were measured by direct microscopy with a microscope, and the sporangialization rate was calculated by Equation 1. FIG. 3 shows the relationship between the activated carbon concentration in the medium and the sporangialization rate. The vertical axis shows the sporangialization rate (%), and the horizontal axis shows the activated carbon concentration (% by weight) in the medium.
Represents When the activated carbon concentration is 0.5% by weight or less, good sporangia formation is observed.

【0054】(実施例3)培地中のグルコース濃度が胞
子嚢形成に与える影響を検討した。培地Aの活性炭濃度
が0.1重量%となるように調製したものと、培地Aに
グルコース濃度が0.2重量%、活性炭濃度が0.1重
量%含まれるように調製した平板培地を作成した(以
下、それぞれ平板培地P、平板培地Qと称す)。
Example 3 The influence of the concentration of glucose in the medium on sporangia formation was examined. A plate medium was prepared so that the activated carbon concentration of the medium A was 0.1% by weight, and a plate medium prepared such that the glucose concentration was 0.2% by weight and the activated carbon concentration was 0.1% by weight in the medium A. (Hereinafter, referred to as plate medium P and plate medium Q, respectively).

【0055】また培地Aの成分中のトリプトン及びイー
ストエキストラクト濃度をそれぞれ2倍にした培地に活
性炭濃度が0.1重量%となるように調製し平板を作成
した(以下、平板培地Rと称す)。
A plate was prepared by adjusting the concentration of activated carbon to 0.1% by weight in a medium in which the concentrations of tryptone and yeast extract in the components of the medium A were each doubled (hereinafter referred to as a plate medium R). ).

【0056】更に培地成分をDIFCO社製トリプトン
1.0重量%、オキゾイド社製イーストエクストラクト
0.6重量%、和光純薬社製特級リン酸水素二カリウム
0.3重量%、和光純薬社製特級寒天1.5重量%、和
光純薬社製特級溶性デンプン1.0重量%、和光純薬製
特級グルコース0.1重量%、和光純薬製特級フラクト
ース0.1重量%、和光純薬製特級シュークロース0.
1重量%、和光純薬製特級マルトース0.1重量%、和
光純薬製特級マンノース0.1重量%、和光純薬製特級
サリシン0.1重量%として和光純薬社製特級粉末活性
炭を0.6重量%とした平板培地(平板培地Sと称す)
を作成した。
Further, the medium components were 1.0% by weight of tryptone manufactured by DIFCO, 0.6% by weight of yeast extract manufactured by Oxoid, 0.3% by weight of special grade dipotassium hydrogen phosphate manufactured by Wako Pure Chemical, and Wako Pure Chemical. 1.5% by weight of special grade agar manufactured, 1.0% by weight of special grade starch made by Wako Pure Chemical Co., 0.1% by weight of special grade glucose made by Wako Pure Chemical, 0.1% by weight of special grade fructose made by Wako Pure Chemical, Wako Pure Chemical Special grade sucrose 0.
1 wt%, Wako Pure Chemical Special Grade Maltose 0.1 wt%, Wako Pure Chemical Special Grade Mannose 0.1 wt%, Wako Pure Chemical Special Grade Salicin 0.1 wt% Plate medium with 0.6% by weight (referred to as plate medium S)
It was created.

【0057】それら培地中のグルコース濃度を比較する
ために培地成分A中に含まれるグルコース濃度を測定し
た。培地A成分から寒天を除いた溶液を調製し、ベーリ
ンガー・マンハイム社のFキットを用いて酵素法により
グルコース濃度を測定した。この結果得られたグルコー
ス濃度は38mg/l(0.0038重量%)であっ
た。
The glucose concentration in the medium component A was measured to compare the glucose concentration in the medium. A solution was prepared by removing agar from the medium A component, and the glucose concentration was measured by an enzymatic method using an F kit from Boehringer Mannheim. The resulting glucose concentration was 38 mg / l (0.0038% by weight).

【0058】以上により各平板培地中のグルコース濃度
は、平板培地Pが0.0038重量%、平板培地Qが
0.2重量%、平板培地Rが0.0076重量%、平板
培地Sが0.1重量%である。尚、平板培地Rについて
はトリプトン・イーストエクストラクトの濃度が2倍で
あることから、平板Pの測定値を2倍した。また平板培
地QとRについてはそれぞれグルコースが添加されてい
るので、存在するグルコース濃度は添加グルコース濃度
に等しい。
As described above, the glucose concentration in each plate medium was 0.0038% by weight for the plate medium P, 0.2% by weight for the plate medium Q, 0.0076% by weight for the plate medium R, and 0.1% for the plate medium S. 1% by weight. The measured value of the plate P was doubled because the concentration of the tryptone / yeast extract was doubled in the plate medium R. Since glucose is added to each of the plate media Q and R, the concentration of the existing glucose is equal to the added glucose concentration.

【0059】上記の4種類の平板罰にてバチルス・ポピ
リエ・セマダラ株を培養した。用いた培養方法は実施例
1と同様である。また実施例1と同様の方法により、胞
子嚢化率を算出した。表3に培地中のグルコース濃度と
胞子嚢化率とを示す。グルコース濃度0.01重量%以
下の場合に胞子嚢化率が良好であることがわかる。
The Bacillus popirie semadara strain was cultured using the above four types of plate punishment. The culture method used is the same as in Example 1. The sporangialization rate was calculated in the same manner as in Example 1. Table 3 shows the glucose concentration in the medium and the sporangialization rate. It can be seen that the sporangialization rate is good when the glucose concentration is 0.01% by weight or less.

【0060】[0060]

【表3】 [Table 3]

【0061】(実施例4)本発明の製造方法により得ら
れた胞子嚢によるコガネムシ科昆虫の殺虫試験を行っ
た。直径6cmのプラスチックカップ10個に腐葉土2
0gづつを入れた。そのうちの5個には、胞子嚢数が1
×108個/カップとなるよう胞子嚢を散布した。散布
した胞子嚢は実施例3に示した平板Pと同様の組成の平
板を作成し、同じ条件にて培養して取得した胞子嚢を使
用した。
Example 4 An insecticidal test was conducted on a beetle insect using the sporangia obtained by the production method of the present invention. Humic soil 2 in 10 plastic cups 6cm in diameter
0 g was added at a time. Five of them had 1 sporangia.
The sporangia was sprayed so as to give × 10 8 / cup. For the sporangial spores sprayed, a spore sac obtained by preparing a plate having the same composition as the plate P shown in Example 3 and culturing it under the same conditions was used.

【0062】残りの5個はコントロールとして、胞子嚢
無添加で試験した。それぞれのカップにセマダラコガネ
3令幼虫を1頭ずつ入れ、25℃のインキュベータ内で
40日間飼育し、死亡個体数を調べると共に死亡体内に
乳化病胞子が形成されているかを顕微鏡で調べた。
The remaining five were tested as controls without sporangia. In each cup, one third-stage larva of the moss, Semaphora kogane, was placed one by one, bred in an incubator at 25 ° C. for 40 days, the number of dead individuals was examined, and the presence of emulsified disease spores in the dead bodies was examined with a microscope.

【0063】表4にセマダラコガネに対する昆虫体外形
成胞子嚢の殺虫活性の結果を示す。30日目までに全て
の幼虫が死亡し、死亡した幼虫5頭の内、2頭が体内に
乳化病の胞子嚢を形成していた。
Table 4 shows the results of the insecticidal activity of the extracorporeal sporangia of the insect against Scutellaria cricket. By day 30, all larvae had died, and two of the five larvae that had died formed spores of emulsified disease in the body.

【0064】[0064]

【表4】 [Table 4]

【0065】(実施例5)実施例4と同様にして調製し
たカップを3個づつ用意した。そこにドウガネブイブイ
2令幼虫を1頭ずつ入れ25℃のインキュベータ内で4
0日間飼育し、死亡個体数を調べると共に死亡体内に乳
化病胞子が形成されているかを顕微鏡で調べた。本発明
の製造方法により製造した胞子嚢のドウガネブイブイに
対する殺虫活性試験の結果を表5に示した。30日目ま
でに67%の幼虫が死亡し、死亡した幼虫2頭のうち1
頭が体内に乳化病の胞子嚢を形成していた。
Example 5 Three cups prepared in the same manner as in Example 4 were prepared. Then put 2nd instar larvae of Douganebuui in the incubator at 25 ° C.
After breeding for 0 days, the number of dead animals was examined, and the presence of emulsified disease spores in the dead bodies was examined under a microscope. Table 5 shows the results of the insecticidal activity test of the sporangia produced by the production method of the present invention with respect to Douganebuui. By day 30, 67% of the larvae had died, and one out of two dead larvae
The head had formed a sporocyst of emulsified disease in the body.

【0066】[0066]

【表5】 [Table 5]

【0067】[0067]

【発明の効果】本発明は、コガネムシ科昆虫の幼虫を用
いることなく、人工培地を用いて胞子とパラスポラルボ
ディとを含む多量のバチルス・ポピリエの胞子 嚢を短
期間で得るバチルス・ポピリエ胞子嚢の工業的な製造方
法、該胞子嚢を有効成分とする芝や農園芸作物や樹木等
の根に多大な被害を与えるコガネムシ科昆虫の優れた防
除剤、及び防除方法を提供することができる。
Industrial Applicability The present invention provides a Bacillus popirie spore that can obtain a large amount of Bacillus popirie spores containing spores and parasporal bodies using an artificial medium without using larvae of Scarabaeidae. It is possible to provide an industrial method for producing a sac, an excellent control agent for a scarabid insect that causes a great deal of damage to the roots of turf, agricultural and horticultural crops, trees, and the like, and a method for controlling the sac as an active ingredient. .

【図面の簡単な説明】[Brief description of the drawings]

【図1】 胞子とパラスポラルボディとを含むバチルス
・ポピリエの胞子嚢の模式図である。
FIG. 1 is a schematic diagram of a Bacillus popirie sporangia containing spores and a parasporal body.

【図2】 実施例1で、活性炭添加濃度が0.1重量%
の場合に得られたセマダラ株の胞子嚢の顕微鏡写真であ
る。
FIG. 2 shows that in Example 1, the concentration of activated carbon added was 0.1% by weight.
5 is a photomicrograph of a sporangium of a semaphora strain obtained in the case of FIG.

【図3】 培地中の活性炭濃度と胞子嚢化率の関係を示
す図である。縦軸は胞子嚢化率(%)、横軸は培地中の
活性炭濃度(重量%)を表す。
FIG. 3 is a graph showing the relationship between the activated carbon concentration in a medium and the sporangialization rate. The vertical axis represents the sporangialization rate (%), and the horizontal axis represents the activated carbon concentration (% by weight) in the medium.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) //(C12N 1/20 (C12N 1/20 C12R 1:07) C12R 1:07) (72)発明者 西橋 秀治 千葉県佐倉市大崎台2−23−9 (72)発明者 藤家 梓 千葉県長尾郡睦沢町上市場256−3 (72)発明者 長谷川 誠 千葉県千葉市緑区椎名崎町486−E−503 (72)発明者 田中 正男 千葉県千葉市美浜区高浜6−11−12 (72)発明者 横山 とも子 千葉県千葉市美浜区磯部5−16−1−501 Fターム(参考) 4B065 AA15X BB02 BB15 BB23 BB26 BB29 BC13 BC41 CA48 CA60 4H011 AC01 BB21 DC11 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 (C12N 1/20 C12R 1:07) C12R 1:07) (72) Inventor Hideharu Nishihashi 2-23-9 Osakidai, Sakura City, Chiba Prefecture (72) Inventor Azusa Fujiya 256-3, Kamiichichi, Mutsawa-machi, Nagao-gun, Chiba Prefecture (72) Inventor Makoto Hasegawa 486-E, Shiinazakicho, Midori-ku, Chiba-shi, Chiba Prefecture −503 (72) Masao Tanaka, Inventor 6-11-12 Takahama, Mihama-ku, Chiba-shi, Chiba Pref. (72) Tomoko Yokoyama 5-16-1-501, Isobe, Mihama-ku, Chiba-shi, Chiba F-term (reference) 4B065 AA15X BB02 BB15 BB23 BB26 BB29 BC13 BC41 CA48 CA60 4H011 AC01 BB21 DC11

Claims (10)

【特許請求の範囲】[Claims] 【請求項1】 バチルス・ポピリエに属する菌を活性炭
0.01重量%〜0.5重量%を含む培地で培養するこ
とを特徴とする、胞子とパラスポラルボディとを含むバ
チルス・ポピリエの胞子嚢の製造方法。
1. A spore of Bacillus popirie containing a spore and a parasporal body, wherein the bacterium belonging to Bacillus popirie is cultured in a medium containing 0.01% to 0.5% by weight of activated carbon. How to make the capsule.
【請求項2】 式1の胞子嚢化率で示される、胞子嚢数
の全菌数当たりの割合が0.1%以上であることを特徴
とする請求項1に記載の製造方法。 (式1) 胞子嚢化率(%)=(胞子嚢数)/(全菌数)×100
2. The method according to claim 1, wherein the ratio of the number of sporangia to the total number of bacteria, which is represented by the sporangialization rate of the formula 1, is 0.1% or more. (Formula 1) Sporulation rate (%) = (spore number) / (total number of bacteria) × 100
【請求項3】 培地中に含まれるグルコース濃度が0.
01重量%以下であることを特徴とする請求項1又は2
に記載の製造方法。
3. The method according to claim 1, wherein the concentration of glucose contained in the medium is 0.5.
3. The composition according to claim 1, wherein the content is not more than 01% by weight.
The production method described in 1.
【請求項4】 胞子とパラスポラルボディとを含む胞子
嚢がコガネムシ科の昆虫に対して殺虫性を有することを
特徴とする請求項1〜3の何れか1つに記載の製造方
法。
4. The method according to claim 1, wherein the sporangia containing the spores and the parasporal body has insecticidal properties against insects of the order Scarabaeidae.
【請求項5】 コガネムシ科の昆虫がドウガネブイブイ
である請求項4に記載の製造方法。
5. The production method according to claim 4, wherein the insect of the family Scarabaeidae is Scutellaria cruzi.
【請求項6】 培養が、固体培養である請求項1〜5の
何れか1つに記載の製造方法。
6. The production method according to claim 1, wherein the culture is a solid culture.
【請求項7】 培地中に、寒天、ポリペプトン、酵母エ
キス及びリン酸塩を含むことを特徴とする請求項6に記
載の製造方法。
7. The method according to claim 6, wherein the medium contains agar, polypeptone, yeast extract and phosphate.
【請求項8】 バチルス・ポピリエに属する菌が、バチ
ルス・ポピリエ・セマダラ(FERM P−1681
8)である請求項1〜7の何れか1つに記載の製造方
法。
8. The bacterium belonging to Bacillus popiliae is Bacillus popiriae semadara (FERM P-1681).
8) The method according to any one of claims 1 to 7, wherein
【請求項9】 請求項1〜8の何れか1つに記載の製造
方法により製造された、胞子とパラスポラルボデイとを
含むバチルス・ポピリエの胞子嚢を有効成分とするコガ
ネムシ科昆虫の防除剤。
9. A control of an insect of the order Scarabaeidae, comprising a spore of Bacillus popirie containing a spore and a parasporal body produced by the method of any one of claims 1 to 8 as an active ingredient. Agent.
【請求項10】 請求項1〜8の何れか1つに記載の製
造方法により製造された胞子とパラスポラルボディとを
含むバチルス・ポピリエの胞子嚢をコガネムシ科昆虫に
作用させるコガネムシ科昆虫の防除方法。
10. A Scarabaeid insect, wherein a Bacillus popirie sporangia containing a spore and a parasporal body produced by the method according to any one of claims 1 to 8 is allowed to act on a Scarabaeid insect. Control method.
JP33862899A 1999-11-29 1999-11-29 Method for producing Bacillus popilie sporangia Expired - Lifetime JP4472813B2 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7087423B2 (en) 2003-12-23 2006-08-08 Dainippon Ink And Chemicals, Inc. Process for producing sporangia of Bacillus popilliae
US7319028B2 (en) 2003-12-09 2008-01-15 Dainippon Ink And Chemicals, Inc. Process for producing sporangia of Bacillus popilliae, control agent, and controlling method

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002291468A (en) * 2001-03-30 2002-10-08 Chiba Prefecture Method for producing sporangium of bacillus popilliae, agent for controlling insect in scarabaeidae and method for controlling the insect
JP2002291467A (en) * 2001-03-30 2002-10-08 Chiba Prefecture Method for producing sporangium of bacillus popilliae, agent for controlling insect in scarabaeidae and method for controlling the insect

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7319028B2 (en) 2003-12-09 2008-01-15 Dainippon Ink And Chemicals, Inc. Process for producing sporangia of Bacillus popilliae, control agent, and controlling method
US7087423B2 (en) 2003-12-23 2006-08-08 Dainippon Ink And Chemicals, Inc. Process for producing sporangia of Bacillus popilliae

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