CN100352938C - Method for rapid selection of plant disease-resistant fungi marine microorganism - Google Patents
Method for rapid selection of plant disease-resistant fungi marine microorganism Download PDFInfo
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- CN100352938C CN100352938C CNB031338798A CN03133879A CN100352938C CN 100352938 C CN100352938 C CN 100352938C CN B031338798 A CNB031338798 A CN B031338798A CN 03133879 A CN03133879 A CN 03133879A CN 100352938 C CN100352938 C CN 100352938C
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Abstract
The present invention relates to sieving of fungi marine microorganisms for resisting plant diseases, particularly to a method for rapidly sieving fungi marine microorganisms for resisting plant diseases. The method comprises the steps that (1) the parallel bacteriostasis experiment of corrugated Candida strains and plant disease fungi is carried out by antifungal antibiotic, and the corrugated Candida strain which can be used as an indicating bacterium is selected; (2) the corrugated Candida strain obtained in step (1) is used as the indicating bacterium, and marine microorganisms having the inhibiting action on the indicating bacterium are sieved. The present invention has the following advantages of high efficiency, simple operation and reliable result, and furthermore, the work efficiency of sieving antifungal resources from microorganisms is promoted, and the existing relevant measurement technology is simplified.
Description
Technical field
The present invention relates to the screening of anti-Plant diseases fungi marine microorganism, the method for the anti-Plant diseases fungi of a kind of specifically rapid screening marine microorganism.
Background technology
In agriculture production, some fungies make agriculture production be subjected to very big harm to doing the effect of deposits yields disease; Produce toxin as purple Penicillium notatum soybeans they grow is produced toxic action, make the soybean underproduction, cause very big loss.In the prior art, at harmful fungoid, people constantly seek at occurring in nature and can kill or the microorganism of antagonism harmful fungoid and the active substance of its generation, harmful fungoid is used as the process that the target bacterium is participated in screening and detects often, but most harmful fungoids are because of the characteristic of poor growth (48-72hr), easy diffusion (spore that generation is dispersed etc.), in actual detected, waste time and energy, efficient is low, sometimes also influence the timeliness of detected result and cause laboratory pollution, and troubling, the limitation of its method and advanced weak point are apparent.
Summary of the invention
In order to overcome the deficiency in the conventional art, the purpose of this invention is to provide the method for a kind of simple to operate, reliable results, the anti-Plant diseases fungi of rapid screening marine microorganism that efficient is high.
To achieve these goals, the technical solution used in the present invention is:
1) does standard unit with fold candida bacterial strain and Plant diseases fungi with antifungal antibiotic, carry out parallel bacteriostatic test, select the fold candida bacterial strain that can be used as indicator;
2) the fold candida bacterial strain that obtains with step 1) is done indicator, and screening has inhibiting marine microorganism to it.
The antibacterial time in the screening process is generally 10～24 hours; The cell concentration of fold candida bacterium in substratum preferably answers 〉=10 in the antibacterial screening process
6Individual/milliliter; Antibacterial screening is generally carried out under 26～32 ℃; Antifungal antibiotic can be nystatin, KETOKONAZOL or amphotericin etc.; The general bacteriostatic test plate that adopts is operated in the screening process; The piece method of digging, cylinder plate method, punch method or filter paper method that bacteriostatic test plate is commonly used.
The present invention has following advantage:
1. efficient height.The present invention is to be both fungi but the bacteroid fold candida of the class of fast growth replaces phytopathogen to measure anti-microbial activity, and the result is (10-24hr) fast.
2. simple to operate.Used harmless, nontoxic, the no spore of fold candida of the present invention disperses, colonial morphology exactly likes bacterium, measure in the method for anti-mycotic activity at microorganism biological, replace poor growth, easily diffusion, it is clear to detect antibacterial blur margin, and the pathogenic fungi that is easy to pollute is as the target bacterium, and is pollution-free, antibacterial edge clear, method is simple.
3. reliable results.The used indicator of the present invention screens from multiple harmless yeast, has the marine microorganism of bacteriostatic activity that the Plant diseases fungi is also had restraining effect to it, and is accurately stable.
In a word, the present invention may be used on the microorganism in various sources in principle and the anti-microbial activity of other antimycotic material is measured, adopt double-deck culture medium culturing detection or plate to be coated with bacterium and cultivate the method that detects, can make things convenient for the anti-microbial activity of the microorganism in rapid determination marine microorganism (mainly being marine actinomycete) and other source, promote the working efficiency of the antimycotic resource of screening from microorganism, and simplified existing related assays technology.
Embodiment
Embodiment 1
1. the rapid screening indicator chooses
Choose the purple Penicillium notatum (Penicillum purpurogenum) of harmless, fast growth fold candida bacterium 28 strains and three strain Plant diseases fungies, sickle-like bacteria (Fusarium solani) and botrytis (Botrytis fuckeliana), carry out parallel bacteriostatic test with nystatin (also can be other antifungal drug), determine the reaction and the responsive dependency of Plant diseases fungi and harmless yeast antibiont matter; Wherein there are 3 strain fold candida bacterial strains nystatin to be had identical reaction and (the fold candida bacterial strain carried out parallel bacteriostatic test with the Plant diseases fungi with antifungal antibiotic with purple Penicillium notatum, they can occur inhibition zone simultaneously), thus select their fold candida bacterial strains as the rapid screening indicator;
1) preparation of indicator (detecting bacterium cultivates)
Get the saccharomycetic cultivation agar slant seed of a ring and be inoculated in the test tube that the 10ml liquid nutrient medium is housed, 28 ℃ cultivate 24 hours standby;
Select for use conventional microzyme culture medium to get final product, liquid nutrient medium consists of: glucose 2%, yeast extract paste 1%, peptone 0.2～0.3%, transfer PH 6.5-7.0, and sterilized 20 minutes for 115 ℃; Solid medium both added 2% agar in the liquid medium within;
2) preparation of double-layer plate:
The indicator of newly cultivating is made bacteria suspension: get 28 ℃ of bacterium liquid 0.3ml that cultivate 24 hours with transfer pipet, add and be equipped with in the triangular flask of the liquid nutrient medium of 50ml temperature between 50～55 ℃, getting 5ml after shaking up adds and to contain the 15ml that has an appointment and solidify substratum and do promptly to get double-layer plate in the plate of lower floor.
2. the rapid screening of anti-Plant diseases marine microorganism
The fungistatic effect that marine streptomyces that the piece method sees that branch is pure and marine bacteria produce the fold candida bacterial strain of selecting is dug in employing, thereby rapid screening goes out Plant diseases is had inhibiting marine microorganism;
Be specially: put into the long agar block that test organisms is arranged on the double-layer plate making, cultivated 10～24 hours, can obtain its anti-microbial activity result.
1) preparation of test organisms
Obtain marine streptomyces 941 strains from coastal area of china sampling separation, will divide pure marine streptomyces to be applied on its solid plate that is suitable for growing, cultivate after 7 days standby for 28 ℃;
2) detect double-deck the cultivation: the fluidized solid substratum falls bottom platform, what will be incubated in 54 ℃ of water-baths after the condensation is mixed with saccharomycetic solid medium (every 50ml substratum adds the 0.3ml liquid culture) again, fall upper panel, after solidifying, put into marine streptomyces sample agar block to be detected in the plate, 28 ℃ leave standstill cultivation 10-24hr, obtain to have marine streptomyces 38 strains of anti-microbial activity.
Embodiment 2
Difference from Example 1 is, the rapid screening of anti-Plant diseases marine microorganism,
1) preparation of test organisms
Obtain 71 strains of ocean sporeformer from coastal area of china sampling separation, will divide pure ocean sporeformer to be applied on its solid plate that is suitable for growing, cultivate after 1 day standby for 28 ℃;
Detect double-deck the cultivation: liquefaction Y76 solid medium 200ml falls bottom (wooden frame or stainless steel framework), what will be incubated in 54 ℃ of water-baths after the condensation is mixed with the saccharomycetic solid Y76 of Y76 substratum (every 50ml substratum adds the 0.3ml liquid culture) again, fall the upper strata, after waiting to coagulate, put into detected sample ocean sporeformer agar block, 28 ℃ leave standstill cultivation 10-24hr, obtain to have ocean sporeformer 9 strains of anti-microbial activity.
Embodiment 3
Filter out the detection of bacterial strain:
Obtain the marine bacteria and marine streptomyces 47 strains of antagonistic action for the screening of target bacterium with the candiyeast, replicate(determination) is to the bacteriostatic activity of purple Penicillium notatum and candiyeast, determine target bacterium and purple Penicillium notatum reaction and the responsive dependency to screening bacterial strain, measurement result shows that the bacterial strain that filters out has restraining effect equally to purple Penicillium notatum; With the bacterial strain that filters out plant pathogen sickle-like bacteria and botrytis are carried out bacteriostatic test, the result is effective equally.
Wherein there is the effect of a strain fold candida bacterial strain Y76 fabulous.
Claims (7)
1. the method for the marine microorganism of the anti-Plant diseases fungi of rapid screening is characterized in that:
1) the fold candida bacterial strain is carried out parallel bacteriostatic test with the Plant diseases fungi with antifungal antibiotic, select the fold candida bacterial strain that can be used as indicator;
2) the fold candida bacterial strain that obtains with step 1) is done indicator, and screening has inhibiting marine microorganism to it.
2. according to the method for the marine microorganism of the anti-Plant diseases fungi of the described rapid screening of claim 1, it is characterized in that: the antibacterial time in the screening process is 10～24 hours.
3. according to the method for the marine microorganism of the anti-Plant diseases fungi of the described rapid screening of claim 1, it is characterized in that: cell concentration 〉=10 of fold candida bacterium in substratum in the antibacterial screening process
6Individual/milliliter.
4. according to the method for the marine microorganism of the anti-Plant diseases fungi of the described rapid screening of claim 1, it is characterized in that: antibacterial screening is carried out under 26～32 ℃.
5. according to the method for the marine microorganism of the anti-Plant diseases fungi of the described rapid screening of claim 1, it is characterized in that: described antifungal antibiotic is nystatin, KETOKONAZOL or amphotericin.
6. according to the method for the marine microorganism of the anti-Plant diseases fungi of the described rapid screening of claim 1, it is characterized in that: adopt bacteriostatic test plate to operate in the screening process.
7. according to the method for the marine microorganism of the anti-Plant diseases fungi of the described rapid screening of claim 6, it is characterized in that: described bacteriostatic test plate is for digging piece method, cylinder plate method, punch method or filter paper method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031338798A CN100352938C (en) | 2003-07-09 | 2003-07-09 | Method for rapid selection of plant disease-resistant fungi marine microorganism |
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| CNB031338798A CN100352938C (en) | 2003-07-09 | 2003-07-09 | Method for rapid selection of plant disease-resistant fungi marine microorganism |
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| CN1566359A CN1566359A (en) | 2005-01-19 |
| CN100352938C true CN100352938C (en) | 2007-12-05 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09127097A (en) * | 1995-11-02 | 1997-05-16 | Kyokuto Seiyaku Kogyo Kk | Microplate for testing sensitivity to antifungal agent, test method for sensitivity and kit therefor |
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2003
- 2003-07-09 CN CNB031338798A patent/CN100352938C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09127097A (en) * | 1995-11-02 | 1997-05-16 | Kyokuto Seiyaku Kogyo Kk | Microplate for testing sensitivity to antifungal agent, test method for sensitivity and kit therefor |
Non-Patent Citations (2)
| Title |
|---|
| 抗生素筛选模型的建立 路福平等.华南理工大学学报，第25卷第8期 1997 * |
| 海洋霉菌抗真菌作用的研究I具广谱抗菌活性的海洋霉菌M182的分离 李淑彬等.海洋通报，第19卷第6期 2000 * |
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