CN106967669A - A kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria - Google Patents
A kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria Download PDFInfo
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- CN106967669A CN106967669A CN201710369047.9A CN201710369047A CN106967669A CN 106967669 A CN106967669 A CN 106967669A CN 201710369047 A CN201710369047 A CN 201710369047A CN 106967669 A CN106967669 A CN 106967669A
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Abstract
The present invention discloses a kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria, belongs to technical field of plant protection.This method includes：Cabbage heart anthrax bacteria is separated and culture；Cabbage heart anthrax bacteria is induced to produce conidium；Obtain a large amount of cabbage heart anthrax bacteria conidiums.The method of the present invention has：Largely produce conidial time short：It is 10~15d from time of the induction cabbage heart anthrax bacteria generation conidium to needed for largely obtaining conidium；Conidium yield is big：The cabbage heart anthrax bacteria conidial suspension obtained with induction is coated in being cultivated on PDA culture medium flat board, and seldom, what culture was produced is substantially conidium to the mycelium that it is produced, and sporulation quantity is big；It is easy to operate：It is easy to operate to conidial whole flow process is largely obtained from separation, induction, purifying culture, and superclean bench, constant incubator, autoclave are only needed, without special instrument and equipment, common laboratory can be achieved.
Description
Technical field
The invention belongs to technical field of plant protection, it is related to a kind of quick, a large amount of acquisition cabbage heart anthrax bacteria conidium
Cultural method.
Background technology
Cabbage heart is one of most characteristic vegetable crop in Guangdong Province or even South China.It is many in Guangdong and South China
Large-scale Vegetable Base is all to produce based on cabbage heart.By cabbage heart anthrax bacteria (namely John Higgins anthrax-bacilus Colletotrichum
Higginsanum cabbage heart anthracnose caused by) infecting is the upper main disease of cabbage heart production, and Brassica campestris leaves of causing harm cause " fiber crops
Point ", so that cabbage heart loses commodity value.The disease measure is prevented and treated to be mainly monitoring cabbage heart anthrax bacteria pathogenicity dynamic, plant
Plant disease-resistant cabbage heart kind and early stage sprays medicament prevention and control, and evaluate the disease resistance level of cabbage heart kind, screen high activity
The application studies such as the pathogenicity difference of bactericide and monitoring diseased plant bacterium, are required to obtain the substantial amounts of mitogenetic spore of cabbage heart anthrax bacteria
Son.
At present, the preparation method of plant anthrax bacteria conidium mainly has 3 kinds.(1) plant tissue or pan-fried juice culture
Anthracnose mycelia, for example, be connected to the fruit face of dry beanpod by base Fiber differentiation, and beanpod outside can produce mitogenetic after culture a period of time
Spore.This method is the main method for inducing plant anthrax bacteria to produce conidium, and the production spore time is shorter, and the scope of application is wider,
But want to obtain the more plant tissue (such as dry beanpod) of a large amount of conidiums needs, operate loaded down with trivial details.(2) extension pathogen training
The time of supporting.Plant anthrax bacteria silk is placed in PDA culture medium and cultivates for a long time or culture medium is gradually dried, charcoal can be promoted
Subcutaneous ulcer germ produces spore.This method is easy to operate, but incubation time is long, generally requires the time of culture more than 1 month.(3) damage
Hinder mycelia Fiber differentiation.Plant anthrax bacteria is inoculated into PDA culture medium, after mycelia covers with culture dish, with sterilizing
Scalpel scratches the bacterium colony of planar surface, mycelium is sustained damage, then through cultivating after a while, the mycelium week of damage
Conidium can be produced by enclosing.This method is easy to operate, but acquisition conidial time is also longer, typically also takes more than 20 days,
And the conidium amount obtained is also limited.
The content of the invention
In order to overcome, the sporulation quantity of prior art is few, incubation time length shortcoming and deficiency, and it is an object of the invention to carry
For a kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria.This method can be obtained largely in a short time
Conidium, the disease resistance level for evaluating cabbage heart kind, screening high activity bactericide and monitoring cabbage heart are inoculated with for indoors artificial
The application studies such as anthrax bacteria pathogenicity difference.
The purpose of the present invention is achieved through the following technical solutions：
A kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria, comprises the following steps：
(1) separation of cabbage heart anthrax bacteria and culture；
(2) induction cabbage heart anthrax bacteria produces conidium：The mycelia block for purifying culture is connected to cowpea that is sterile, drying
In pod tissue block, it is then inserted on water agar and cultivates；Microscopy cowpea pod tissue block, if can be organized in cowpea pod
Block epidermis microscopy is to substantial amounts of oblong conidium, you can primarily determine that the conidium for obtaining cabbage heart anthrax bacteria；
The conidium of acquisition is inoculated with cabbage heart, confirms that it causes a disease to cabbage heart and causes typical anthrax disease symptoms；Then monospore is carried out
Separation and purifying culture, so as to obtain cabbage heart anthrax bacteria；
(3) a large amount of cabbage heart anthrax bacteria conidiums are obtained：Step (2) is taken to confirm as the conidium of cabbage heart anthrax bacteria
Spore suspension is configured to, spore suspension is coated on PDA culture medium flat board, is positioned on superclean bench and dries up；Put
5~10d is cultivated in 27 ± 2 DEG C；Then conidium is washed down with sterilized water, that is, obtains substantial amounts of cabbage heart anthrax bacteria mitogenetic
Spore.
The separation of cabbage heart anthrax bacteria and culture described in step (1), comprise the following steps：
Cabbage heart anthracnose sample is gathered, the tissue with typical scab is chosen, is rinsed well with clear water, is cut in the strong intersection of disease
Take tissue block；After being disinfected successively in 75% alcohol and 1% liquor natrii hypochloritis, rinsed 3 times in sterilized water；With
The filter paper of sterilizing sucks unnecessary moisture, it is to be dried after be placed on PDA culture medium flat board, cultivated in 27 ± 2 DEG C of incubators；Treat
Grow after mycelia, picking edge mycelia block carries out purifying culture.
Described tissue block is the tissue block of 0.3cm × 0.5cm sizes.
Described disinfects the time for 15s~45s, preferably 30s.
Condition of culture described in step (2) is 27 ± 2 DEG C of cultures 4d~7d, preferably 27 ± 2 DEG C culture 5d.
Cowpea pod tissue block described in step (2) is to dry the tissue block for removing seed.
Spore suspension concentration described in step (3) is 1 × 104Individual/mL~1 × 106Individual/mL.
The present invention has the following advantages and effect relative to prior art：
(1) conidial time is largely produced short：Conidium is produced to largely obtaining from induction cabbage heart anthrax bacteria
Time needed for conidium is 10d~15d.
(2) conidium yield is big：The cabbage heart anthrax bacteria conidial suspension obtained with induction is coated in PDA
Cultivated on culture medium flat plate, seldom, what culture was produced is substantially conidium to the mycelium that it is produced, and sporulation quantity is big.
(3) it is easy to operate：It is easy to operate to conidial whole flow process is largely obtained from separation, induction, purifying culture,
And superclean bench, constant incubator, autoclave are only needed, without special instrument and equipment, common laboratory can be real
It is existing.
Brief description of the drawings
Fig. 1 is a large amount of acquisition conidial operational flowcharts of cabbage heart anthrax bacteria.
Fig. 2 is production spore culture figure of the cabbage heart anthrax bacteria in cowpea pod tissue block.
Colonial morphology figure when Fig. 3 is cabbage heart anthrax bacteria big volume production spore.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Raw material and specification：
Cowpea pod：Slightly well-done cowpea purchased in market, is rinsed well, 40~50 DEG C of drying, is then cut into 3~5cm length, is removed
It is standby after seed.
Material or instrument required for experimental implementation：Common superclean bench, autoclave, incubator, culture dish, liquid relief
Device, coating glass bar, PDA culture medium, distilled water, 75% alcohol and 1% liquor natrii hypochloritis etc..
Described SNA (synthesis low nutrition agar medium) culture medium：1g/L KH2PO4, 1g/L KNO3, 0.5g/L
MgSO4·7H2O, 0.5g/L KCl, 0.2g/L glucose, 0.2g/L sucrose, 15g/L agar powders；120 DEG C of sterilizing 20min standby
With.
Described water agar：It is standby after 15g/L agar powders, 120 DEG C of sterilizing 20min.
It is a large amount of to obtain the conidial operational flowchart of cabbage heart anthrax bacteria, as shown in Figure 1.
1 pair of embodiment derives from germ separation and production spore effect in the cabbage heart anthracnose sample of field
(1) the typical fresh sick sample of cabbage heart anthracnose is gathered in field, clear water rinses the rear strong intersection group of clip disease well
Block (about 0.3cm × 0.5cm) is knitted, 30s is respectively sterilized with 75% alcohol and 1% liquor natrii hypochloritis respectively on superclean bench,
Dried after aseptic water washing three times；Then move on PDA culture medium flat board, 27 DEG C of culture 5d, picking edge mycelia block carries out pure
Change culture.
(2) mycelia block is connected in cowpea pod tissue block (removing seed) that is sterile, drying, is then inserted into water fine jade
Infiltrated on fat culture medium；Microscopy cowpea pod tissue block after 27 DEG C of culture 5d.If can be in cowpea pod tissue block epidermis microscopy to largely
Oblong conidium, that is, primarily determine that as cabbage heart anthrax bacteria (John Higgins anthrax-bacilus) conidium.By point of acquisition
Raw spore inoculating cabbage heart, confirms that it causes a disease to cabbage heart and causes typical anthrax disease symptoms；Then single spore separation and purifying are carried out
Culture, so as to obtain cabbage heart anthrax bacteria.Fig. 2 is shown in production spore culture of the cabbage heart anthrax bacteria in cowpea pod tissue block.
(3) cabbage heart anthrax bacteria conidium is collected, it is 1 × 10 to prepare spore concentration5Individual spore/mL spore suspension
Liquid, draws 100 μ L spore suspensions with liquid-transfering gun and is coated on a diameter of 9cm PDA culture medium flat board, on superclean bench
Drying, 27 DEG C of culture 5d；Then conidium under the sterile washings of 50mL is used, three layers of sterile gauze filtering collect filtering bacterium solution simultaneously
The dilution of 10 times of gradients is prepared, the dilution for drawing 1 μ L with liquid-transfering gun is observed under 10 times of mirror visuals field and divided as on slide
Sporogenic quantity.When the quantity of conidium in the dilution for observing 1 μ L is 10~30, samples 20 times, calculate 1
Conidium average in μ L dilutions；It is then convert into the concentration of every milliliter of conidial suspension.As a result show, using this
Method, to the anthrax-bacilus from field cabbage heart anthracnose sample, obtains the concentration of conidial suspension up to 1.5 × 106It is individual
Spore/mL.Colonial morphology during the big volume production spore of cabbage heart anthrax bacteria is shown in Fig. 3.
Embodiment 2 produces spore effect to the cabbage heart anthrax bacteria of medium-term and long-term preservation
(1) the cabbage heart anthrax bacteria that 10 DEG C preserve 3 years in SNA culture mediums is taken, 27 DEG C of cultures on PDA culture medium flat board
5d；After mycelia is grown, mycelia block is connected in cowpea pod tissue block (removing seed) that is sterile, drying, is then inserted into
Infiltrated on to water agar, 27 DEG C of culture 5d；With the conidium of cowpea pod tissue block epidermis under sterile washing.
(2) cabbage heart anthrax bacteria conidium eluate is collected, it is 1 × 10 to prepare spore concentration5Individual spore/mL spore
Suspension.Using the step of embodiment 1 operating method of (3), obtain conidium liquid and to calculate conidial suspension dense
Degree.As a result show, using this method, to the cabbage heart anthrax bacteria of medium-term and long-term preservation, the concentration for obtaining conidial suspension can
Up to 1.2 × 106Individual spore/mL.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (6)
1. a kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria, it is characterised in that comprise the following steps：
(1) separation of cabbage heart anthrax bacteria and culture；
(2) induction cabbage heart anthrax bacteria produces conidium：The mycelia block for purifying culture is connected to cowpea pod group that is sterile, drying
Knit in block, be then inserted on water agar and cultivate；Microscopy cowpea pod tissue block, if can be in cowpea pod tissue block table
Skin microscopy primarily determines that the conidium for obtaining cabbage heart anthrax bacteria to substantial amounts of oblong conidium；It will obtain
Conidium inoculation cabbage heart, confirm that it causes a disease to cabbage heart and causes typical anthrax disease symptoms；Then carry out single spore separation and
Purifying culture, so as to obtain cabbage heart anthrax bacteria；
(3) a large amount of cabbage heart anthrax bacteria conidiums are obtained：The conidium for taking step (2) to confirm as cabbage heart anthrax bacteria is prepared
Into spore suspension, spore suspension is coated on PDA culture medium flat board, is positioned on superclean bench and dries up；It is placed in 27
± 2 DEG C of 5~10d of culture；Then conidium is washed down with sterilized water, that is, obtains the substantial amounts of mitogenetic spore of cabbage heart anthrax bacteria
Son.
2. quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria according to claim 1, its feature exists
In：
The separation of cabbage heart anthrax bacteria and culture described in step (1), comprise the following steps：
Cabbage heart anthracnose sample is gathered, the tissue with typical scab is chosen, is rinsed well with clear water, in the strong intersection clip group of disease
Knit block；After being disinfected successively in 75% alcohol and 1% liquor natrii hypochloritis, rinsed 3 times in sterilized water；With sterilizing
Filter paper suck unnecessary moisture, it is to be dried after be placed on PDA culture medium flat board, cultivated in 27 ± 2 DEG C of incubators；Wait to grow
After mycelia, picking edge mycelia block carries out purifying culture.
3. quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria according to claim 2, its feature exists
In：The described time disinfected is 15s~45s.
4. quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria according to claim 1, its feature exists
In：The condition of culture described in step (2) is 27 ± 2 DEG C of culture 4d~7d.
5. quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria according to claim 1, its feature exists
In：Cowpea pod tissue block described in step (2) is to dry the tissue block for removing seed.
6. quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria according to claim 1, its feature exists
In：
Spore suspension concentration described in step (3) is 1 × 104Individual/mL~1 × 106Individual/mL.
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| CN201710369047.9A CN106967669A (en) | 2017-05-23 | 2017-05-23 | A kind of quick, a large amount of acquisition conidial cultural method of cabbage heart anthrax bacteria |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384725A (en) * | 2018-04-25 | 2018-08-10 | 河南科技大学 | A kind of separation method of black root of tobacco bacterium |
| CN110257316A (en) * | 2019-07-02 | 2019-09-20 | 广东省农业科学院植物保护研究所 | A kind of plant anthrax bacteria conidium rapid separation and purification method |
| CN112375728A (en) * | 2020-10-23 | 2021-02-19 | 浙江省农业科学院 | New application of nano titanium dioxide |
-
2017
- 2017-05-23 CN CN201710369047.9A patent/CN106967669A/en active Pending
Non-Patent Citations (3)
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| 张丽勍等: "草莓炭疽病菌鉴定技术规程", 《上海农业科技》 * |
| 楼兵干等: "一种新大豆豆荚炭疽病症状类型及其病原鉴定", 《植物保护学报》 * |
| 王晨芳: "化学诱导剂在诱导黄瓜抗病性中的作用", 《中国优秀博硕士学位论文全文数据库 (硕士)农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384725A (en) * | 2018-04-25 | 2018-08-10 | 河南科技大学 | A kind of separation method of black root of tobacco bacterium |
| CN110257316A (en) * | 2019-07-02 | 2019-09-20 | 广东省农业科学院植物保护研究所 | A kind of plant anthrax bacteria conidium rapid separation and purification method |
| CN112375728A (en) * | 2020-10-23 | 2021-02-19 | 浙江省农业科学院 | New application of nano titanium dioxide |
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Application publication date: 20170721 |
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