JP2708686B2 - Selection medium - Google Patents
Selection mediumInfo
- Publication number
- JP2708686B2 JP2708686B2 JP34708892A JP34708892A JP2708686B2 JP 2708686 B2 JP2708686 B2 JP 2708686B2 JP 34708892 A JP34708892 A JP 34708892A JP 34708892 A JP34708892 A JP 34708892A JP 2708686 B2 JP2708686 B2 JP 2708686B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- drigalski
- soft rot
- bacteria
- soft
- 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.)
- Expired - Fee Related
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、学名エルビニア・カロ
トボーラ サブスピ カロトボーラ（Ｅｒｗｉｎｉａ
ｃａｒｏｔｏｖｏｒａ ｓｕｂｓｐ． ｃａｒｏｔｏｖ
ｏｒａ）に属する、植物病原細菌の主要な一つである、
いわゆる軟腐病菌を検出するための選択培地に関するも
のである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the scientific name Erwinia carotobola
carotovora subsp. carotov
ora), which is one of the main plant pathogenic bacteria,
The present invention relates to a selective medium for detecting so-called soft rot bacteria.
【０００２】[0002]
【従来技術およびその問題点】エルビニア・カロトボー
ラ細菌は、多くの野菜に軟腐病を引き起こす植物病原細
菌である。この軟腐病菌の菌濃度を希釈平板法により検
出するための選択培地として、変法ドリガルスキー培地
（津山博之（１９６２）、東北大農研彙報１３巻、２２
１−３４５ページ）もしくはドリガルスキー改良培地
（市販品）やＭＭＳ培地（Ｍｏｄｉｆｉｅｄ Ｍｉｌｌ
ｅｒ−Ｓｃｈｒｏｔｈ培地：Ｋｌｏｅｐｐｅｒ，Ｊ．
Ｗ．（１９８３） Ｐｈｙｔｏｐａｔｈｏｌｏｇｙ ７
３巻，２１７−２１９ページ）などが提案されている。
これらの培地は、いずれも軟腐病菌がラクトースを資化
し、生じた酸性物質を色素であるブロモチモールブルー
により黄色のコロニーとして検出するものである。BACKGROUND OF THE INVENTION Erwinia carotobola bacteria are phytopathogenic bacteria that cause soft rot on many vegetables. As a selective medium for detecting the bacterial concentration of this soft rot fungus by the dilution plate method, a modified Drigalsky medium (Hiroyuki Tsuyama (1962), Tohoku Univ.
1-345) or improved Drigalski medium (commercially available) or MMS medium (Modified Mill)
er-Schroth medium: Kloepper, J. et al.
W. (1983) Phytopathology 7
3, pp. 217-219).
In each of these media, soft rot fungus assimilates lactose, and the resulting acidic substance is detected as a yellow colony using bromothymol blue as a pigment.
【０００３】しかしながら、これらの培地を用いて土壌
中や作物上の軟腐病菌濃度を検出しようとしても、共雑
菌（軟腐病菌以外の菌をいう。以下同じ。）が多い場合
には検出感度が低く、軟腐病菌の検出にはまだ不十分と
言わざるをえず、実用的には問題がある。また、ＭＭＳ
培地については成分数が多く、作成に手間がかかるとい
う欠点を有する。[0003] However, even if an attempt is made to detect the concentration of soft rot bacteria in soil or crops using these media, the detection sensitivity is low when there are many commensal bacteria (fungi other than soft rot fungi; the same applies hereinafter). However, it cannot be said that it is still insufficient for the detection of soft rot bacteria, and there is a problem in practical use. Also, MMS
The medium has the disadvantage that the number of components is large and the preparation is troublesome.
【０００４】[0004]
【問題点を解決するための手段】本発明者らは、軟腐病
菌の検出の回収率が高く、かつ土壌および作物中の共雑
菌が増殖しにくい培地成分について鋭意検討を行なった
結果、変法ドリガルスキー培地またはドリガルスキー改
良培地にタリウムの塩を加えることにより、軟腐病菌の
増殖を抑制することなく、軟腐病菌以外の共雑菌の増殖
を著しく減少させ得ることを見出し、さらに、これらの
成分に、抗生物質であるエリスロマイシンを加えること
で一層の軟腐病菌検出の選択性が得られることを見出
し、本発明に到達した。Means for Solving the Problems The present inventors have conducted intensive studies on a medium component which has a high recovery rate of soft rot bacteria and hardly grows common germs in soil and crops. By adding thallium salt to the Drigalski medium or the Drigalski improved medium, it was found that the growth of soft rot fungi could be significantly reduced without suppressing the growth of soft rot bacteria. The present inventors have found that the addition of an antibiotic, erythromycin, can further enhance the selectivity for detecting soft-rot fungi, and arrived at the present invention.
【０００５】すなわち本発明は、変法ドリガルスキー培
地もしくはドリガルスキー改良培地にタリウムの塩を加
えることを特徴とする軟腐病菌の選択培地、さらには変
法ドリガルスキー培地もしくはドリガルスキー改良培地
にタリウムの塩およびエリスロマイシンを加えることを
特徴とする軟腐病菌の選択培地である。[0005] That is, the present invention provides a selective medium for soft rot fungus characterized by adding a thallium salt to a modified Drigalski medium or a Drigalski improved medium, and a thalium is added to a modified Drigalski medium or a Drigalski improved medium. It is a selective medium for soft rot fungi, characterized by adding salt and erythromycin.
【０００６】さて、軟腐病菌は、普遍的に土壌に存在し
ていることが報告されている。５年以上この菌の宿主と
なる作物を栽培していない畑でも軟腐病の発生が観察さ
れる場合がある。この菌の生態は次の様に考えられてい
る（津山博之、植物防疫 第３４巻 ２９４−２９８ペ
ージ，１９８０年）。例えば、白菜の場合には播種後、
４０日位から根部の周囲でこの細菌が増殖し、根圏土
壌、葉部などほとんどあらゆる箇所に存在が認められる
ようになる。台風や昆虫、および作業などにより白菜に
傷がつくと、そこから細菌が侵入し、気候条件さえ整え
ば一晩のうちに病原菌濃度が上昇し病斑が認められるこ
とになる。ところが、こうした普遍的に土壌に存在する
軟腐病菌でも作物を栽培していない土壌中や作物の生育
が初期の土壌中もしくは作物上の菌濃度は非常に低い。
したがって、菌濃度を測定しようとしても、軟腐病菌に
対して相対的に共雑菌が多いために通常用いられる希釈
平板法では検出が困難である。[0006] It has been reported that soft rot fungi are universally present in soil. Occurrence of soft rot may be observed even in a field in which a crop serving as a host of this fungus has not been cultivated for 5 years or more. The ecology of this fungus is considered as follows (Hiroyuki Tsuyama, Plant Protection, 34: 294-298, 1980). For example, in the case of Chinese cabbage, after sowing,
From about 40 days, this bacterium grows around the root, and its presence is found in almost all places such as rhizosphere soil and leaves. If the Chinese cabbage is damaged by typhoons, insects, or work, bacteria will invade from it, and if the climatic conditions are met, the concentration of pathogenic bacteria will rise overnight and lesions will be observed. However, even with such soft rot fungi that are generally present in the soil, the bacterial concentration in the soil where no crop is cultivated or in the soil in the early stage of crop growth or on the crop is extremely low.
Therefore, even if an attempt is made to measure the bacterial concentration, it is difficult to detect the bacterial concentration by a commonly used dilution plate method because there are many common bacteria relative to the soft rot bacteria.
【０００７】本発明は、従来測定が困難であった低い軟
腐病菌の菌濃度を希釈平板法を用いて簡単に測定するた
めの選択培地を提供するものである。以下、本発明の構
成について詳しく記述する。[0007] The present invention provides a selective medium for simply measuring the concentration of low soft rot bacteria, which has been difficult to measure conventionally, using a dilution plate method. Hereinafter, the configuration of the present invention will be described in detail.
【０００８】本発明の選択培地は、通常、希釈平板法に
より軟腐病菌の菌濃度を検出するために使用される変法
ドリガルスキー培地もしくはドリガルスキー改良培地に
タリウムの塩、または、タリウムの塩とエリスロマイシ
ンを加えたものである。[0008] The selective medium of the present invention is usually prepared by adding a thallium salt or a thallium salt to a modified Drigalski medium or a Drigalski improved medium used for detecting the concentration of soft rot bacteria by a dilution plate method. Erythromycin is added.
【０００９】これらの選択培地は当然軟腐病菌の検出に
ついては変法ドリガルスキー培地もしくはドリガルスキ
ー改良培地と同等の良好な回収率で検出することができ
る。それに加えて、共雑菌が多い試料についても、土壌
中または作物上に存在する種々の雑菌は増殖し難いとい
う特性を有するため、良好な軟腐病菌の検出が可能とな
る。[0009] These selective culture media can naturally detect soft rot fungi with the same good recovery as the modified Drigalski medium or the improved Drigalski medium. In addition, even with respect to a sample containing many common bacteria, various bacteria existing in the soil or on the crop have a characteristic that it is difficult to proliferate, so that it is possible to detect good soft rot bacteria.
【００１０】変法ドリガルスキー培地はつぎの様な組成
である。 肉エキス 10g ペプトン 10g ラクトース 10g 塩化ナトリウム 5g ブロモチモールブルー 0.08g クリスタルバイオレット 0.04g 寒天 15g 水 1L pH 7.0〜7.2 また、ドリガルスキー改良培地はつぎの様な組成であ
る。The modified Drigalski medium has the following composition. Meat extract 10g Peptone 10g Lactose 10g Sodium chloride 5g Bromothymol blue 0.08g Crystal violet 0.04g Agar 15g Water 1L pH 7.0-7.2 In addition, the improved Drigalski medium has the following composition.
【００１１】肉エキス 4g ペプトン 10g ラクトース 10g ブロモチモールブルー 0.04g 寒天 15g 水 1L pH 7.0〜7.2 これらはいずれも当業者が容易に改変しうる範囲で組成
を変更することは、当然可能である。4 g of meat extract 10 g of peptone 10 g of lactose 10 g of bromothymol blue 0.04 g of agar 15 g of water 1 L pH 7.0 to 7.2 It is of course possible to change the composition of any of these in a range that can be easily modified by those skilled in the art.
【００１２】本発明においては、タリウムの塩の量は寒
天培地１リットル当り、１ｍｇから１００ｍｇ好ましく
は５ｍｇから３０ｍｇが良い。エリスロマイシンは５ｍ
ｇから１００ｍｇの範囲で加えるのがよく、より好まし
くは５ｍｇから３０ｍｇである。いずれの成分について
加減量を満たさない場合には共雑菌の排除効果が減じ、
検出感度が下がり、一方、上限値を越える場合には、軟
腐病菌の繁殖に悪影響を及ぼすことがあり、いずれも好
ましくない。In the present invention, the amount of thallium salt is 1 mg to 100 mg, preferably 5 mg to 30 mg per liter of agar medium. Erythromycin is 5m
The amount is preferably in the range of g to 100 mg, and more preferably 5 mg to 30 mg. If any component does not satisfy the weight loss, the effect of eliminating common bacteria decreases,
If the detection sensitivity is lowered and exceeds the upper limit, the reproduction of soft rot bacteria may be adversely affected, and both are not preferable.
【００１３】本発明においてタリウムの塩という場合、
タリウムを含有し水溶性を呈するものをいい、１価また
は３価のタリウムの、ハロゲン化物（塩化物、臭化物、
沃化物）、硝酸塩、硫酸塩、燐酸塩、炭酸塩などの無機
塩またはこれらの酸性塩、複塩もしくはアンモニウムと
の複塩、酢酸、しゅう酸などの有機酸塩などならびに、
水酸化物、酸化物をも包含するものである。通常の場
合、硝酸第一タリウムが入手し易いのでこれを用いるの
が好ましい。In the present invention, when referring to a thallium salt,
Thallium-containing and water-soluble ones, monovalent or trivalent thallium halides (chloride, bromide,
Iodides), nitrates, sulfates, phosphates, carbonates and other inorganic salts or their acidic salts, double salts or double salts with ammonium, acetic acid, organic acid salts such as oxalic acid,
It also includes hydroxides and oxides. In the normal case, thallous nitrate is easily available and is preferably used.
【００１４】また、本発明においてはシクロヘキシミド
などの抗生物質などを添加することも可能である。本発
明の選択培地は土壌や作物中の軟腐病菌が低菌濃度の場
合にも、軟腐病菌を検出することができ、畑の軟腐病発
病の予想がし易くなり農薬散布等の的確な防除が可能と
なる。また、本発明で示す選択培地は病原性のない軟腐
病菌に対しても検出ができることから、微生物農薬とし
ての非病原性軟腐病菌の残留性を検定する選択培地とし
ても用いることができる。In the present invention, it is also possible to add an antibiotic such as cycloheximide. The selective medium of the present invention can detect soft-rot fungi even when the soft-rot fungus in soil or crops has a low bacterial concentration, making it easy to predict the onset of soft-rot in the field, making it possible to accurately control pesticide application and the like. It becomes possible. Further, since the selective medium shown in the present invention can detect even non-pathogenic soft rot bacteria, it can be used as a selective medium for testing the persistence of non-pathogenic soft rot bacteria as a microbial pesticide.
【００１５】[0015]
【実施例】以下実施例により本発明をより詳細に説明す
るが、これらの実施例によって限定されるものではな
い。The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
【００１６】調製例 前述した変法ドリガルスキー培地（津山博之（１９６
２）、東北大農研彙報１３巻、２２１−３４５ページに
従って調製した。）およびドリガルスキー改良培地（栄
研化学（株）製、パールコアドリガルスキー改良培地：
栄研）を基本培地として、それに硝酸第一タリウムまた
は硝酸第一タリウムとエリスロマイシンを加えた次の組
成を有する培地を調製し、それぞれの培地をシャーレに
固定した。Preparation Example A modified Drigalski medium described above (Hiroyuki Tsuyama (196)
2), prepared according to Tohoku Univ. ) And Drigalski improved medium (Eiken Chemical Co., Ltd., Pearl Core Drigalski improved medium:
Using Eiken) as a basic medium, a thallium nitrate or a medium having the following composition obtained by adding thallous nitrate and erythromycin was prepared, and each medium was fixed on a petri dish.
【００１７】調製例１ 変法ドリガルスキー培地に硝酸第一タリウムを１７．５
ｍｇ加えた培地 調製例２ 変法ドリガルスキー培地に硝酸第一タリウムを１７．５
ｍｇおよびエリスロマイシン１０ｍｇ加えた培地 調製例３ ドリガルスキー改良培地に硝酸第一タリウムを１７．５
ｍｇ加えた培地 調製例４ ドリガルスキー改良培地に硝酸第一タリウムを１７．５
ｍｇおよびエリスロマイシン１０ｍｇ加えた培地 実施例１ 約3×103cfu/mlの軟腐病菌縣濁液の１００マイクロリッ
トルを寒天培地に塗布したのち、２８℃、２日間培養し
た後軟腐病菌に特有な黄色のコロニー数を数えた。表１
に軟腐病菌の回収率を示す。変法ドリガルスキー培地も
しくはドリガルスキー改良培地へ硝酸第一タリウムまた
は硝酸第一タリウムとエリスロマイシンを加えた培地の
軟腐病菌の検出は良好であることがわかる。なお、ここ
ではドリガルスキー改良培地の回収率を１００％とし
た。Preparation Example 1 Modified Drigalski medium was supplemented with 17.5 thallium nitrate.
Preparation Example 2 Modified Drigalski medium supplemented with 17.5 thallium nitrate
Medium and 10 mg of erythromycin Preparative Example 3 17.5 thallium nitrate was added to a modified Drigalski medium.
Preparative Example 4 Medium supplemented with Drigalsky 17.5 thallium nitrate
Example 1 A medium containing 10 mg of erythromycin and 100 μl of a suspension of about 3 × 10 3 cfu / ml of soft rot bacteria were applied to an agar medium, and cultured at 28 ° C. for 2 days. Counted the number. Table 1
Shows the recovery rate of soft rot fungi. It can be seen that the detection of soft rot bacteria in a medium obtained by adding thallous nitrate or thallous nitrate and erythromycin to a modified Drigalski medium or a modified Drigalski medium is good. Here, the recovery rate of the improved Drigalski medium was set to 100%.
【００１８】[0018]
【表１】 [Table 1]
【００１９】実施例２ 土壌３０グラムを取り、２７０ミリリットルの滅菌水を
加え攪拌した。次に、この土壌縣濁液１０ミリリットル
を１０倍および１００倍に希釈した。これらの土壌縣濁
液の１００マイクロリットルをそれぞれの寒天培地に塗
布し、２８℃、２日間培養した後プレートに現れた雑菌
のコロニー数を数えた。表２に示す。Example 2 Thirty grams of soil was taken, 270 ml of sterile water was added, and the mixture was stirred. Next, 10 ml of this soil suspension was diluted 10 times and 100 times. One hundred microliters of these soil suspensions were applied to each agar medium and cultured at 28 ° C. for 2 days, after which the number of bacterial colonies that appeared on the plate was counted. It is shown in Table 2.
【００２０】[0020]
【表２】 [Table 2]
【００２１】実施例３ ハクサイ畑のハクサイ根圏の土壌を採取した後、土壌３
０グラムを取り、２７０ミリリットルの滅菌水を加え攪
拌した。次に、この土壌縣濁液１０ミリリットルを１０
倍および１００倍に希釈した。これらの土壌縣濁液の１
００マイクロリットルをそれぞれの寒天培地に塗布し、
２８℃、２日間培養した後軟腐病菌に特有な黄色のコロ
ニー数を数えた。表３に軟腐病菌の回収率を示す。Example 3 After collecting soil in the Chinese cabbage rhizosphere of a Chinese cabbage field,
0 g was taken, 270 ml of sterile water was added and stirred. Next, 10 ml of this soil suspension was added to 10
1: and 1: 100 dilution. One of these soil suspensions
Apply 00 microliters to each agar medium,
After culturing at 28 ° C. for 2 days, the number of yellow colonies unique to the soft-rot fungus was counted. Table 3 shows the recovery rate of soft rot fungi.
【００２２】[0022]
【表３】 [Table 3]
【００２３】実施例４ ハクサイ畑のハクサイを採取し、ハクサイ１００グラム
を取り、９００ミリリットルの滅菌水を加えミキサーで
粉砕した後にろ紙で固形物を取除いた。次に、このハク
サイ抽出液１０ミリリットルを１０倍および１００倍に
希釈した。これらのハクサイ抽出液の１００マイクロリ
ットルをそれぞれの寒天培地に塗布し、２８℃、２日間
培養した後軟腐病菌に特有な黄色のコロニー数を数え
た。表４に軟腐病菌のコロニー数を示す。Example 4 Chinese cabbage was collected from a Chinese cabbage field, 100 g of Chinese cabbage was taken, 900 ml of sterilized water was added, the mixture was pulverized by a mixer, and solid matter was removed with a filter paper. Next, 10 ml of the Chinese cabbage extract was diluted 10-fold and 100-fold. One hundred microliters of these Chinese cabbage extracts were applied to each agar medium and cultured at 28 ° C. for 2 days, after which the number of yellow colonies unique to the soft-rot fungus was counted. Table 4 shows the number of soft rot colonies.
【００２４】[0024]
【表４】 [Table 4]
【００２５】[0025]
【発明の効果】本発明により、従来検出方法が困難とさ
れてきた植物細菌病の主要な一つである軟腐病の病原菌
である軟腐病菌を効果的に定量することが可能となっ
た。本発明の選択培地は簡単な組成でありながら軟腐病
菌の存在を高精度で検定することができるという効果を
奏する。Industrial Applicability According to the present invention, it has become possible to effectively quantify soft rot bacteria which are pathogenic bacteria of soft rot which is one of the major plant bacterial diseases which have been conventionally difficult to detect. The selective medium of the present invention has an effect that the presence of soft rot fungus can be assayed with high accuracy while having a simple composition.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 平４−179475（ＪＰ，Ａ) ＰＬＡＮＴ ＤＩＳＥＡＳＥ ＲＥＰ ＯＲＴＥＲ，62〜２！（1978−２）, Ｐ．167−169 ＰＬＡＮＴ ＤＩＳＥＡＳＥ，70〜 ６！（1986−６），Ｐ．575−７ ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-4-179475 (JP, A) PLANT DISSEASE REP ORTER, 62-2! (1978-2), p. 167-169 PLANT DISEASE, 70-6! (1986-6), p. 575-7
Claims (2)
スキー改良培地にタリウムの塩を加えたことを特徴とす
る軟腐病菌の選択培地。1. A selective medium for soft rot fungi, characterized by adding a thallium salt to a modified Drigalski medium or an improved Drigalski medium.
スキー改良培地にタリウムの塩およびエリスロマイシン
を加えたことを特徴とする軟腐病菌の選択培地。2. A selective medium for soft rot fungi, characterized by adding a salt of thallium and erythromycin to a modified Drigalski medium or an improved Drigalski medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34708892A JP2708686B2 (en) | 1992-12-25 | 1992-12-25 | Selection medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34708892A JP2708686B2 (en) | 1992-12-25 | 1992-12-25 | Selection medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06189748A JPH06189748A (en) | 1994-07-12 |
| JP2708686B2 true JP2708686B2 (en) | 1998-02-04 |
Family
ID=18387832
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP34708892A Expired - Fee Related JP2708686B2 (en) | 1992-12-25 | 1992-12-25 | Selection medium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2708686B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5493387B2 (en) * | 2009-02-25 | 2014-05-14 | 国立大学法人 東京大学 | Method for producing selective medium and use thereof |
-
1992
- 1992-12-25 JP JP34708892A patent/JP2708686B2/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| ＰＬＡＮＴ ＤＩＳＥＡＳＥ ＲＥＰＯＲＴＥＲ，６２〜２！（１９７８−２），Ｐ．１６７−１６９ |
| ＰＬＡＮＴ ＤＩＳＥＡＳＥ，７０〜６！（１９８６−６），Ｐ．５７５−７ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH06189748A (en) | 1994-07-12 |
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