CN105176842A - Separation method of tomato gray leaf spot pathogen - Google Patents
Separation method of tomato gray leaf spot pathogen Download PDFInfo
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- CN105176842A CN105176842A CN201510661795.5A CN201510661795A CN105176842A CN 105176842 A CN105176842 A CN 105176842A CN 201510661795 A CN201510661795 A CN 201510661795A CN 105176842 A CN105176842 A CN 105176842A
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- leaf spot
- gray leaf
- tomato gray
- pathogenic bacteria
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Abstract
The invention discloses a separation method of tomato gray leaf spot pathogen. The separation method comprises the following steps: (1) a disease spot part of a tomato gray leaf spot specimen is taken, and impurities of dust and the like at the disease spot part are blown with a rubber pipette bulb; (2) the disease spot part of the dust-removed tomato gray leaf spot specimen is placed under an integrated type microscope, the amplification factor is adjusted so as to clearly observe pathogen hyphae, an inoculating hook is fired on an alcohol lamp until the inoculating hook is reddish and then cooled, and the pathogen hyphae at the disease part is picked directly and inoculated to a PDA slop culture medium; (3) the inoculated PDA slop culture medium is cultured in a dark condition with the temperature of 24 DEG C to 26 DEG C, and bacterial colony edge mycelium is picked for transit culture after a bacterial colony grows. According to the method, the separation steps are easy to operate, all specimens are separated successfully by one time, the separation success rate reaches 100%, and the separated pathogen contamination rate is low, no infectious microbe exists, and purification is easy.
Description
Technical field
The invention belongs to phytopathogen separation technology field, the separation method of especially a kind of Tomato gray leaf spot pathogenic bacteria.
Background technology
Tomato gray leaf spot is a kind of fungal disease that Stemphylium (StemphyliumWallroth) causes, this genus fungi often parasitizes on the leaf of various plants, seed or fruit, can cause vegetables and other plurality of plant diseases such as garlic, clover, lettuce, tomato, wild cabbage, French beans, capsicum.At present, nearly 70 kinds of tomato in China disease, and more rare in the past for the domestic report of Tomato gray leaf spot generation.But, in recent years along with the replacing of the continuous expansion of greenhouse tomato cultivated area, frequently Introduced From Abroad kind and main breed, such disease is laid particular stress on year by year in some tomato planting bases popular causing harm.This disease also started in Chifeng Area, Inner Mongolia to occur in recent years, caused tomato producing region, many ground to drop in production over a large area, seriously govern tomato production.Therefore, the research of Tomato gray leaf spot is very important, and the separation of pathogenic bacteria is the basis and prerequisite carrying out studying, this research is studied for the separation method of Tomato gray leaf spot pathogenic bacteria, be intended to the separation method finding a kind of new pathogenic bacteria, for follow-up test lays a good foundation.
In prior art, the ordinary method of separation and Extraction Tomato gray leaf spot pathogenic bacteria is: be good for from the disease of site of pathological change the tissue block that intersection cuts 5mm × 5mm, through 75% ethanol disinfection, after rinsing 3 times, tissue block is moved on in PDA flat board in sterilized water, cultivate under 25 DEG C of dark conditions, after growing bacterium colony, by the method for picking colony edge mycelium or single spore separation, purifying and switching cultivation are carried out to pathogenic bacteria.The shortcoming of this separation method is: Tomato gray leaf spot bacterium can not disposable separation success, some samples need to carry out secondary even separating for several times just can isolate object bacteria, pollution rate is high, just will can obtain pure target bacterium colony through repeatedly purifying.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide the separation method of a kind of Tomato gray leaf spot pathogenic bacteria, the method separating step is simple to operation, disposable separation success, and isolated pathogen contamination rate is low, without miscellaneous bacteria, and easy purifying.
Technical scheme of the present invention is as follows: the separation method of a kind of Tomato gray leaf spot pathogenic bacteria, it comprises the steps:
(1) get the scab position of Tomato gray leaf spot sample, blow away the impurity such as the dust at scab position with rubber pipette bulb;
(2) under the scab position of the Tomato gray leaf spot sample after dedusting being placed in Stereo microscope, adjustment magnification can be clear that pathogenic bacteria mycelia, inoculation be hooked on spirit lamp and burn to general red rear cooling, the pathogenic bacteria mycelia in direct picking disease portion, is seeded on PDA slant medium;
(3) cultivated under 24 ~ 26 DEG C of dark conditions by the PDA slant medium of inoculation, after growing bacterium colony, picking colony edge mycelium carries out switching and cultivates.
Further, PDA slant medium used in the present invention is made up of the raw material of following weight part: potato 180 ~ 220 parts, glucose 18 ~ 22 parts, 15 ~ 20 parts, agar, 1000 parts, water.
Further, each Tomato gray leaf spot specimen inoculation 10 pipe PDA slant medium.
Advantage of the present invention is:
(1) directly picking pathogenic bacteria mycelia is separated, and operation is more direct, and have more specific aim, Success rate of virus isolation can reach 100%;
(2) separating step is simple, and easy to operate, the bacterial isolate bacterium time used is short, reduces the probability that miscellaneous bacteria infects;
(3), when method of the present invention is separated, due to only picking pathogenic bacteria mycelia, eliminate the possibility that disease portion tissue carries other miscellaneous bacterias, the isolate obtained is purer.
(4) adopt method of the present invention to be separated pathogenic bacteria, pure object bacteria can be obtained in the short period of time.
Embodiment
Below by embodiment, the present invention is illustrated.
Embodiment: the separation method of a kind of Tomato gray leaf spot pathogenic bacteria, it comprises the steps:
(1) PDA slant medium is prepared: in 1000ml distilled water, add potato 200g, glucose 20g, agar 18g; Through boiling, dissolve, filter and being dispensed in vitro obtained PDA slant medium.
(2) get the scab position of Tomato gray leaf spot sample, blow away the impurity such as the dust at scab position with rubber pipette bulb;
(3) under the scab position of the Tomato gray leaf spot sample after dedusting being placed in Stereo microscope, adjustment magnification can be clear that pathogenic bacteria mycelia, inoculation being hooked on spirit lamp burns to general red rear cooling, the pathogenic bacteria mycelia in direct picking disease portion, be seeded on PDA slant medium, every specimen inoculation 10 pipe PDA slant medium;
(4) cultivated under 25 DEG C of dark conditions by the PDA slant medium of inoculation, after growing bacterium colony, picking colony edge mycelium carries out switching and cultivates.
10 the Tomato gray leaf spot samples adopted back going to the countryside by the method for the present embodiment have carried out the separation of pathogenic bacteria, result shows, adopt method bacterial isolate bacterium of the present invention, separating step is simple to operation, all samples are disposable separation success, and Success rate of virus isolation reaches 100%, and isolated pathogen contamination rate is low, without miscellaneous bacteria, easy purifying.
Claims (3)
1. a separation method for Tomato gray leaf spot pathogenic bacteria, it comprises the steps:
(1) get the scab position of Tomato gray leaf spot sample, blow away the impurity such as the dust at scab position with rubber pipette bulb;
(2) under the scab position of the Tomato gray leaf spot sample after dedusting being placed in Stereo microscope, adjustment magnification can be clear that pathogenic bacteria mycelia, inoculation be hooked on spirit lamp and burn to general red rear cooling, the pathogenic bacteria mycelia in direct picking disease portion, is seeded on PDA slant medium;
(3) cultivated under 24 ~ 26 DEG C of dark conditions by the PDA slant medium of inoculation, after growing bacterium colony, picking colony edge mycelium carries out switching and cultivates.
2. the separation method of Tomato gray leaf spot pathogenic bacteria according to claim 1, it is characterized in that: described PDA slant medium is made up of the raw material of following weight part: potato 180 ~ 220 parts, glucose 18 ~ 22 parts, 15 ~ 20 parts, agar, 1000 parts, water.
3. the separation method of Tomato gray leaf spot pathogenic bacteria according to claim 1 and 2, is characterized in that: each Tomato gray leaf spot specimen inoculation 10 pipe PDA slant medium.
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| CN201510661795.5A CN105176842A (en) | 2015-10-12 | 2015-10-12 | Separation method of tomato gray leaf spot pathogen |
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| CN201510661795.5A CN105176842A (en) | 2015-10-12 | 2015-10-12 | Separation method of tomato gray leaf spot pathogen |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974512A (en) * | 2017-12-12 | 2018-05-01 | 东北农业大学 | A kind of primer and method for detecting Tomato gray leaf spot bacterium |
| CN109006013A (en) * | 2018-08-16 | 2018-12-18 | 辽宁省农业科学院 | A kind of Tomato gray leaf spot artificial inoculation technique and disease-resistant material screening methodologies |
| CN109609589A (en) * | 2018-12-20 | 2019-04-12 | 东北农业大学 | A kind of method that Tomato gray leaf spot bacterium conidium stablizes sprouting and identifies for artificial inoculation on seedling |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667080A (en) * | 2013-12-04 | 2014-03-26 | 中国农业科学院油料作物研究所 | Method for isolating plant pathogenic fungus |
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2015
- 2015-10-12 CN CN201510661795.5A patent/CN105176842A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667080A (en) * | 2013-12-04 | 2014-03-26 | 中国农业科学院油料作物研究所 | Method for isolating plant pathogenic fungus |
Non-Patent Citations (2)
| Title |
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| 赵丽萍等: "番茄灰叶斑病研究进展", 《江苏农业学报》 * |
| 韩长志: "番茄灰叶斑病病原鉴定及其生物学特性研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107974512A (en) * | 2017-12-12 | 2018-05-01 | 东北农业大学 | A kind of primer and method for detecting Tomato gray leaf spot bacterium |
| CN109006013A (en) * | 2018-08-16 | 2018-12-18 | 辽宁省农业科学院 | A kind of Tomato gray leaf spot artificial inoculation technique and disease-resistant material screening methodologies |
| CN109609589A (en) * | 2018-12-20 | 2019-04-12 | 东北农业大学 | A kind of method that Tomato gray leaf spot bacterium conidium stablizes sprouting and identifies for artificial inoculation on seedling |
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