CN101624571A - Rapid separation and culture method of apple scab germs - Google Patents
Rapid separation and culture method of apple scab germs Download PDFInfo
- Publication number
- CN101624571A CN101624571A CN200910164721A CN200910164721A CN101624571A CN 101624571 A CN101624571 A CN 101624571A CN 200910164721 A CN200910164721 A CN 200910164721A CN 200910164721 A CN200910164721 A CN 200910164721A CN 101624571 A CN101624571 A CN 101624571A
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- Prior art keywords
- apple
- culture
- substratum
- apple scab
- separation
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- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 206010039509 Scab Diseases 0.000 title claims abstract description 9
- 244000052616 bacterial pathogen Species 0.000 title abstract description 7
- 238000012136 culture method Methods 0.000 title abstract 4
- 239000011521 glass Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 16
- 241000894006 Bacteria Species 0.000 claims description 14
- 241000228452 Venturia inaequalis Species 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims 1
- 244000000004 fungal plant pathogen Species 0.000 abstract description 4
- 239000000758 substrate Substances 0.000 abstract 3
- 238000012258 culturing Methods 0.000 abstract 1
- 230000002073 mitogenetic effect Effects 0.000 abstract 1
- 238000002955 isolation Methods 0.000 description 6
- 238000005192 partition Methods 0.000 description 6
- 238000003113 dilution method Methods 0.000 description 4
- 238000012364 cultivation method Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000581835 Monodora junodii Species 0.000 description 1
- 241001256872 Perula Species 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a rapid separation and culture method of apple scab germs, comprising a germ monospore separation method and an in-substrate culture method. The monospore separation method comprises the following steps: rubbing a fresh apple scab with a glass slide with a dry and clean surface; and using a hair to stick single mitogenetic spore of the apple scab germs and transfer to culture in a substrate under the conditions that a microscope has a 15-fold eye lens and a 10-fold objective lens. In addition, the invention also provides culture conditions and culture substrate suitable for the apple scab germs. The separation and culture method not only can rapidly and effectively separate and culture the apple scab germs, but also can be used for rapidly and effectively separating and culturing other plant pathogenic fungi capable of generating spores.
Description
Technical field:
The present invention relates to germ and separate and the cultivation field, be specifically related to a kind of method of separation and Culture apple black star bacteria, comprise that the germ monospore separates and cultivates in substratum.
Technical background:
Scab of apple is one of the important disease in each apple producing region, the world, apple black star bacteria (Venturia inaequalis (Cke.) Wint.) can infect apple blade, petiole, flower, sepal, bennet and fruit, also can infect young shoot and perula etc., the cracking of fruit deformity of being injured, cause fallen leaves, shedding when serious, directly influence output, quality and the commodity value thereof of apple.Scab of apple mainly occurs in Shaanxi, Liaoning, Xinjiang, Heilungkiang 4 provinces in China, in Jilin, there is distribution in province partial area such as Henan, Gansu, Ningxia, Shandong, Shanxi, Sichuan, Yunnan.In recent years, scab of apple has the trend that increases the weight of year by year in China some areas, is seriously threatening the development of apple industry.In non-irrigated plateau apple producing region, north, the eugenic district-Weihe of China's apple, scab of apple is respectively at 1997,1998,1999, be very popular in 2004 and 2005, caused serious economy loss, major cause is to lack the research of scab of apple in regularty of epidemic of China and the heritable variation rule of pathogenic bacteria etc., not effectively prophylactico-therapeutic measures targetedly, the research of these problems all be unable to do without separation of scab of apple pathogenic bacteria and cultivation, and apple black star bacteria is slow because of its speed of growth on substratum, contaminated easily, be one of domestic pathogenic micro-organism that is difficult to for a long time to separate and cultivates.
Summary of the invention:
The invention provides a kind of method of separation and Culture apple black star bacteria, the monospore that comprises germ separates and cultivates in substratum.
The monospore separation method that is wherein adopted is the slide glass friction with fresh scab of scab of apple and surface drying cleaning, at microscopical eyepiece is 15 times, object lens are under 10 times the condition, get single apple black star bacteria conidium with hairline is sticking, are transferred in the substratum and cultivate.
The preparation method of used substratum is: get apple blade 40g and peeling potato 40g, in 500mL distilled water, boil 30min respectively, filtering the back mixes, add the 15g agar powder, be settled to 1000mL with distilled water, 121 ℃ of sterilization 20min are when treating that substratum is cooled to 55 ℃, add Vetstrep and make its final concentration reach 100mg/L, promptly get substratum of the present invention.
In addition, the present invention also provides suitable germ mycelial growth and has produced the culture condition of spore, is 15-20 ℃ in temperature promptly, and the pH value is 5.0-6.5, and intensity of illumination is 600lx, and the photoperiod is to cultivate under the 12h condition, and above-mentioned conditions favouring is with the germ mycelial growth and produce spore.
Isolation cultivation method of the present invention not only can fast and effeciently carry out the separation and Culture of apple black star bacteria, and also can be used for other simultaneously can the effective fast of spore-bearing plant pathogenic fungi separate and cultivation.
Specific embodiment:
Embodiment 1: comparison test
2004 we (kind is a loud, high-pitched sound in 8 years green apple gardens from Tai Cun township, Xunyi, Shaanxi, the pollinizers kind is Qin Guan) take the scab of apple blade standard specimen of falling ill, take back the laboratory, adopt tissue block partition method, isolation by dilution method and monospore partition method to be divided into respectively from 234 wares (table 1).Tissue block partition method surface sterilization condition is 70% alcohol 5sec, 0.1% mercuric chloride 3min, and aseptic water washing 3 times is divided into from 147 wares, has 5 wares to succeed, and success ratio only is 3.4%.Adopt isolation by dilution method to be divided into from 20 wares, have 2 wares to obtain success, and very easily polluted by other assorted bacterium, success ratio is 10.0%.Adopt monospore partition method of the present invention to make 67 wares altogether, have 49 wares to obtain pure isolate, success ratio is 73.1%, is higher than tissue block partition method and isolation by dilution method far away, is a kind of separation method rapidly and efficiently that is suitable for apple black star bacteria.
Table 1 apple black star bacteria separation method relatively
This shows tissue block partition method and the isolation by dilution method of monospore separation method of the present invention compared to routine, fast and also success ratio high.
Above-mentioned in addition monospore separation method can also be used substratum of the present invention, further isolating monospore is cultivated under culture condition of the present invention.Monospore separation method of the present invention, medium preparation method and culture condition are the effective separation and Culture matching methods of apple black star bacteria, are better than the isolation cultivation method of conventional plant pathogenic fungi.Simultaneously, the monospore separation method that the present invention set up also is suitable for other and can the effective fast of spore-bearing plant pathogenic fungi separates.
Embodiment 2: the preparation of substratum
Get apple blade 40g and peeling potato 40g, in 500mL distilled water, boil 30min respectively, filtering the back mixes, add the 15g agar powder, be settled to 1000mL with distilled water, 121 ℃ of sterilization 20min are when treating that substratum is cooled to 55 ℃, add Vetstrep and make its final concentration reach 100mg/L, promptly get substratum of the present invention.Be poured in the culture dish substratum that makes standby.
Embodiment 3: cultivate
Slide glass friction with fresh scab of scab of apple and surface drying cleaning, at microscopical eyepiece is 15 times, object lens are under 10 times the condition, get single apple black star bacteria conidium with hairline is sticking, being transferred in the prepared substratum of embodiment 2, is 15-20 ℃ in temperature, and the pH value is 5.0-6.5, intensity of illumination is 600lx, and the photoperiod is to cultivate under the 12h condition.
Claims (3)
1, a kind of method of separation and Culture apple black star bacteria, the monospore that comprises germ separates and cultivates in substratum, wherein said germ monospore separating process is the slide glass friction with fresh scab of scab of apple and surface drying cleaning, at microscopical eyepiece is 15 times, object lens are under 10 times the condition, get single apple black star bacteria conidium with hairline is sticking, be transferred in the substratum and cultivate.
2, method according to claim 1, wherein said substratum prepares by the following method: get apple blade 40g and peeling potato 40g, in 500mL distilled water, boil 30min respectively, filter the back and mix, add the 15g agar powder, be settled to 1000mL with distilled water, 121 ℃ of sterilization 20min, when treating that substratum is cooled to 55 ℃, add Vetstrep and make its final concentration reach 100mg/L, promptly get described substratum.
3, according to claim 1 or 2 each described methods, wherein said cultivation is to carry out at following culture condition, and temperature is 15-20 ℃, the pH value is 5.0-6.5, intensity of illumination is 600lx, and the photoperiod is 12h, and above-mentioned conditions favouring is with the germ mycelial growth and produce spore.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910164721A CN101624571A (en) | 2009-07-16 | 2009-07-16 | Rapid separation and culture method of apple scab germs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910164721A CN101624571A (en) | 2009-07-16 | 2009-07-16 | Rapid separation and culture method of apple scab germs |
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| Publication Number | Publication Date |
|---|---|
| CN101624571A true CN101624571A (en) | 2010-01-13 |
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| CN200910164721A Pending CN101624571A (en) | 2009-07-16 | 2009-07-16 | Rapid separation and culture method of apple scab germs |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651240A (en) * | 2015-02-13 | 2015-05-27 | 四川农业大学 | Method for separating monospores of plasmodiophoromycetes and method for establishing a monospore line by host propagation |
| CN106032523A (en) * | 2015-03-17 | 2016-10-19 | 李保华 | Simple method for separating and purifying apple ring spot bacteria |
-
2009
- 2009-07-16 CN CN200910164721A patent/CN101624571A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651240A (en) * | 2015-02-13 | 2015-05-27 | 四川农业大学 | Method for separating monospores of plasmodiophoromycetes and method for establishing a monospore line by host propagation |
| CN106032523A (en) * | 2015-03-17 | 2016-10-19 | 李保华 | Simple method for separating and purifying apple ring spot bacteria |
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Owner name: XIBEI UNIV. OF AGRICULTURAL + FOREST SCIENCE + TEC Free format text: FORMER OWNER: HU XIAOPING Effective date: 20091218 |
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Effective date of registration: 20091218 Address after: No.3, Taiheng Road, Yangling Demonstration Zone, Shaanxi Province Applicant after: Northwest Agriculture and Forestry University Address before: Plant Protection College, Northwest Agriculture and Forestry University, No. 22 West Nong Road, Yangling demonstration area, Shaanxi Applicant before: Hu Xiao Ping Co-applicant before: Yang Jiarong Co-applicant before: Wang Baotong |
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Application publication date: 20100113 |