CN102443544B - A kind of Isolation and purification method of sesame wilt germs - Google Patents
A kind of Isolation and purification method of sesame wilt germs Download PDFInfo
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- CN102443544B CN102443544B CN201010505951.6A CN201010505951A CN102443544B CN 102443544 B CN102443544 B CN 102443544B CN 201010505951 A CN201010505951 A CN 201010505951A CN 102443544 B CN102443544 B CN 102443544B
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Abstract
The invention discloses a kind of Isolation and purification method of sesame wilt germs, belong to biological technical field.The method adopts without living contaminants isolation technique and single spore separation purification technique, is separated to the sesame wilt germs (sickle-like bacteria) without living contaminants.The present invention both efficiently solved living contaminants, improve bacterium colony purifying rate, guaranteed again to obtain reliable sesame withered pathogenic bacteria.Employing the method, in busier production season, can gather sesame blight diseased plant in different time, area, through unified drying, separation, purifying, improve working efficiency; In bacterium colony purge process, adopt little PDA bacterium block prepare finite concentration conidial suspension and be coated with slat chain conveyor, can simply, quick picking monospore bacterium colony, avoid purifying thoroughly with by the inefficiencies of microscopic examination picking colony.The method efficiently and accurately abstraction and purification goes out sesame wilt germs, is also applicable to the wilt that other vascular bundle diseases of plant causes simultaneously and is separated and purifying.
Description
Technical field
The present invention relates to a kind of method of abstraction and purification wilt from dry sesame withers diseased plant material, belong to biological technical field.
Background technology
Sesame blight is a kind of sesame vascular bundle diseases, and its cause of disease wilt (sickle-like bacteria) is that a kind of root infects fungi.Directly be separated about sesame wilt germs separation many employings live body root, cane segment at present, because the non-vascular bundle miscellaneous bacteria such as xylem, phloem exists, cause very large difficulty to the separation of wilt.Mostly the purifying of wilt is again to inoculate way of purification at colony edge picking bacterium block, or under microscopical help, carries out single spore separation operation, so, is not easy to obtain the wilt bacterial strain of Economical Purification and operational difficulty, loaded down with trivial details, efficiency is low.Current urgent need is inquired into one and effectively can be solved living contaminants, improve bacterium colony purification efficiency, can guarantee again the method obtaining reliable sesame withered pathogenic bacteria, for the research and development of sesame wilt germs lays the foundation.
Summary of the invention
The object of the invention is the Isolation and purification method providing a kind of efficient sesame wilt germs without living contaminants.
For realizing the object of the invention, take following technical scheme:
1, the separation of wilt
1) sesame blight diseased plant is collected: the dry sesame cane having infected blight in the cool;
2) cane close to blight sesame stalk root is cut into 2-3cm segment, with 70% mass percent alcohol to its surface sterilization 1 minute, with aseptic water washing 3-4 time;
3) with aseptic cutters cane segment from middle part rip cutting into two, marrow vascular tissue is revealed;
4) with tweezers tweezer central bundle tissue, be put on the PDA flat board containing 100 μ g/ml Streptomycin sulphates;
5) containing after cultivating 72h at dull and stereotyped 25 DEG C of vascular tissue PDA, fungal colony is had to occur, i.e. the wilt bacterial strain that obtains without living contaminants of separation.
Above 2), 3) and 4) step all operations on Bechtop.
2, the single spore separation purification technique of wilt
1) isolated strains is transferred on another PDA flat board containing 100 μ g/ml Streptomycin sulphates, after 96h, according to colony colour white, red-purple, blueness with have the biological characteristicses such as large and small two kinds of conidiums, preliminary evaluation cause of disease is sesame wilt germs (sickle-like bacteria);
2) be after wilt through Pathogen Identification, cut one 0.4 × 0.8cm PDA bacterium block at PDA plating place, be positioned in 1.5ml centrifuge tube;
3) add 800 μ l sterilized waters in centrifuge tube, thermal agitation 1min, makes the conidium on bacterium block fully be released in water, is prepared into conidial suspension;
4) draw conidial suspension 15 μ l, measure conidium concentration with Hematocyte Counter;
5) take out PDA bacterium block in centrifuge tube and spore suspension is mixed with the conidial suspension that concentration is 100-1000/ml;
6) drawing 100 μ l conidial suspensions is spread evenly across on PDA flat board;
7) at 25 DEG C after slat chain conveyor 60h, growing multiple monospore bacterium colony, select part sporangium and fall within PDA test tube slant or another PDA flat board, for preserving, research, namely completing the single spore separation purifying of sesame wilt germs.
This separation method utilizes cause of disease to cause vascular bundle diseases, when cane is dry, vascular tissue is easily separated with xylem, direct picking vascular tissue is separated, the peripheral xylem of diseased plant etc. isolates other non-wilt as the natural cover for defense, reduces the living contaminantses such as cane surface, phloem and xylem non-virulent sickle-like bacteria; And this separation bacterium colony obtains from the sesame cane vascular tissue having manifest symptom, ensure that the high incidence of this bacterium inoculation sesame and this cause of disease are sesame wilt germs.
The invention has the advantages that: the present invention both efficiently solved living contaminants, improve bacterium colony purifying rate, guarantee again to obtain reliable sesame withered pathogenic bacteria, is an important achievement in sesame wilt germs research.Employing the method, in busier production season, can gather sesame blight diseased plant in different time, area, through unified drying, separation, purifying, improve working efficiency; In bacterium colony purge process, adopt little PDA bacterium block prepare finite concentration conidial suspension and be coated with slat chain conveyor, can simply, quick picking monospore bacterium colony, avoid purifying thoroughly with by the inefficiencies of microscopic examination picking colony.The method efficiently and accurately abstraction and purification goes out sesame wilt germs, is also applicable to the wilt that other vascular bundle diseases of plant causes simultaneously and is separated and purifying.
Accompanying drawing explanation
Fig. 1 is the stem section close to blight sesame stalk root cane 2-3cm;
Fig. 2 is the vascular tissue in the middle part of blight sesame stalk root cane after rip cutting;
Fig. 3 is the vascular tissue of tweezer for blight sesame stalk root cane pathogen separation;
Fig. 4 is bacterium colony after the vascular tissue of blight sesame stalk root cane pathogen separation of the present invention inoculates 96h on PDA substratum;
Fig. 5 is large and small two kinds of conidiums of the separation and purification of the present invention of examining under a microscope;
Fig. 6 is the single spore separation bacterium colony that the present invention 100 μ l concentration produces for 60h after 100/ml conidial suspension coated plate;
Fig. 7 is the single spore separation bacterium colony that the present invention 100 μ l concentration produces for 60h after 1000/ml conidial suspension coated plate.
Embodiment
For better illustrating the present invention, give an actual example as follows:
The separation of embodiment 1.09FO-027 sesame wilt germs and purification process thereof
1) sesame blight diseased plant is collected: get in the cool the fully kept dry blight sesame cane of 3 months, with the dust impurity on its surface of aseptic water washing;
2) cane close to blight sesame stalk root is cut into 2-3cm segment, with 70% mass percent alcohol to its surface sterilization 1 minute, with aseptic water washing 3-4 time; See accompanying drawing 1.
3) with aseptic cutters cane segment from middle part rip cutting (as accompanying drawing 2) into two, marrow vascular tissue is revealed;
4) with tweezers tweezer central bundle tissue (as accompanying drawing 3) gently, be put on the PDA flat board containing 100 μ g/ml Streptomycin sulphates;
5) there is fungal colony to occur containing at dull and stereotyped 25 DEG C of vascular tissue PDA after 72h, be namely separated the wilt obtained without living contaminants.
Above 2), 3) and 4) step all operations on Bechtop.
6) isolated strains is transferred on the new PDA flat board containing 100 μ g/ml Streptomycin sulphates, after 96h, according to bacterium colony superficial white, slightly redness is had in inoculation center bottom colourless look, there is the biological characteristicses such as large and small two kinds of conidiums, determine that cause of disease is wilt 09FO-027 bacterial strain, (as accompanying drawing 4);
7) cut one 0.4 × 0.8cm PDA bacterium block at PDA plating place, be positioned in 1.5ml centrifuge tube;
8) add 800 μ l sterilized waters in centrifuge tube, thermal agitation 1min, makes the conidium on bacterium block fully be released in water, is prepared into conidial suspension;
9) draw conidial suspension 15 μ l, measure conidium concentration with Hematocyte Counter;
10) above-mentioned conidial suspension is mixed with the conidial suspension that concentration is 100-1000 conidium/ml, mixing;
11) drawing 100 μ l concentration is that 1000/ml conidial suspension is spread evenly across PDA flat board;
12) after flat board is positioned over 25 DEG C of cultivation 60h, grows multiple monospore bacterium colony, select part sporangium and fall within PDA test tube slant or new PDA flat board, for preserving, studying.
Namely the single spore separation purifying of sesame wilt germs is completed.Just obtain 09FO-027 sesame wilt germs monospore bacterium colony, (as accompanying drawing 7).
Claims (1)
1. an Isolation and purification method for sesame wilt germs, is characterized in that, is realized by following steps:
The separation of A, wilt
1) sesame blight diseased plant is collected: the dry blight sesame cane infected in the cool;
2) cane close to blight sesame stalk root is cut into 2-3cm segment, with 70% mass percent alcohol to its surface sterilization 1 minute, with aseptic water washing 3-4 time;
3) with aseptic cutters cane segment from middle part rip cutting into two, marrow vascular tissue is revealed;
4) with tweezers tweezer central bundle tissue, be put on the PDA flat board containing 100 μ g/ml Streptomycin sulphates;
5) containing after cultivating 72h at dull and stereotyped 25 DEG C of vascular tissue PDA, fungal colony is had to occur, i.e. the wilt bacterial strain that obtains without living contaminants of separation;
Above 2), 3) and 4) step all operations on Bechtop;
The single spore separation purifying of B, wilt
1) above-mentioned steps is separated the bacterial strain obtained to be transferred on another PDA flat board containing 100 μ g/ml Streptomycin sulphates, after 96h, obtains sesame wilt germs;
2) cut one 0.4 × 0.8cm PDA bacterium block at PDA plating place, be positioned in centrifuge tube;
3) add sterilized water in centrifuge tube, thermal agitation, the conidium on bacterium block is fully released in water, is prepared into conidial suspension;
4) draw conidial suspension, measure conidium concentration with Hematocyte Counter;
5) spore suspension is mixed with the conidial suspension that concentration is 100-1000/ml;
6) drawing 100 μ l conidial suspensions is spread evenly across on PDA flat board;
7), at 25 DEG C after slat chain conveyor 60h, grow multiple monospore bacterium colony, select part sporangium and fall within PDA test tube slant or another PDA flat board, namely complete the single spore separation purifying of sesame wilt germs.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010505951.6A CN102443544B (en) | 2010-10-14 | 2010-10-14 | A kind of Isolation and purification method of sesame wilt germs |
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| CN201010505951.6A CN102443544B (en) | 2010-10-14 | 2010-10-14 | A kind of Isolation and purification method of sesame wilt germs |
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| CN105925488A (en) * | 2016-05-16 | 2016-09-07 | 安徽省农业科学院园艺研究所 | Preparation method of strawberry verticillium dahlia spore suspension |
| CN112941007B (en) * | 2021-04-19 | 2022-06-17 | 广西大学 | Single spore separation method of banana fusarium wilt |
| CN114058508B (en) * | 2021-12-09 | 2024-03-15 | 中国农业大学 | Method for rapidly separating and purifying cercospora and detecting drug resistance |
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Non-Patent Citations (6)
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| Evaluation of rocket resistance to Fusarium wilt;chen guo-kang 等;《湖北植保》;20020131(第1期);33-41 * |
| 山西省重要作物萎蔫病病原镰刀菌(Fusarium)种类的鉴定;贺冰 等;《山西农业大学学报》;20060531;第5卷(第6期);第20页左栏第1－2段 * |
| 罗富成 等.草地有害生物及其防治.《草业科学实践教学指导书》.云南科技出版社,2008,93. * |
| 芝麻枯萎病病原茵分离和纯化方法研究;苏银玲 等;《河南农业科学》;20120131;第41卷(第1期);92-95 * |
| 芝麻枯萎病的发生与防治建议;董朱霞 等;《河北农业科学》;19900731(第7期);13-14 * |
| 香蕉枯萎病菌的培养性状和致病性研究;谢艺贤 等;《植物保护》;20050430;第31卷(第4期);71-73 * |
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