CN110029083A - A method of rape plasmodiophora brassicae monospore is separated based on methylene blue agarose method - Google Patents
A method of rape plasmodiophora brassicae monospore is separated based on methylene blue agarose method Download PDFInfo
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Abstract
The present invention provides a kind of method based on methylene blue agarose method separation rape plasmodiophora brassicae monospore.The following steps are included: preparing concentration first is 5 × 103A spore mL‑1Rape clubroot resting spores of bacteria suspension；1.3% agarose solution is added in 0.01% methylene blue staining liquid, methylene blue agarose glass slide is prepared and coagulates layer；It takes 2 μ L rape clubroot resting spores of bacteria uniform suspensions to be coated on solidifying layer surface, observes under the microscope, when determining only one spore under the visual field, separate the solidifying layer containing monospore, be buckled at cruciferous sprouts root hair, cultivation obtains rape plasmodiophora brassicae monospore system.The present invention carries out rape plasmodiophora brassicae single spore separation using methylene blue agarose method, and background is in slight blue, and free from admixture and bubble can clearly distinguish rape plasmodiophora brassicae monospore.The method process is simple, easy to operate, substantially increases rape plasmodiophora brassicae single spore separation efficiency and accuracy rate.
Description
Technical field
The invention belongs to single spore separation fields, and in particular to one kind is based on methylene blue staining liquid combination agarose method
Rape plasmodiophora brassicae single spore separation method.
Background technique
Cruciferae clubroot (Clubroot disease) is by rape plasmodiophora brassicae (Plasmodiophora
Brassicae Woron.) a kind of caused worldwide disease of obligatory parasitism is infected, it is known as the title of " Cruciferae cancer ", is appointed
The susceptible variety of what crucifer can be infected, and the abnormal division of parenchyma cell is caused to increase, and form warty daetylorhiz.
Clubroot resting spores of bacteria is survived in the soil of noninductive virus vector plant up to 20 years as long as, and soil will be fitted no longer once polluting
The cultivation of suitable crucifer.In recent years, clubroot throughout our country spread rapidly by area, wherein southwest, northeast, East China etc.
Area becomes the severely afflicated area of clubroot.It is counted according to " Chinese agriculture yearbook ", the warp of crop in cruciferae caused by being endangered as clubroot
Ji loss is more than 4,000,000,000 yuan, and in the before, during and after related influence of industrial chain, causing economic loss is more than 10,000,000,000 yuan.
Selecting disease-resistant variety is the effective way for preventing and treating Cruciferae clubroot.Since there are physiology under rape knee strain
Microspecies, often occur that Cruciferae Host Cultivars are immune to pathogen some or a small number of biological strains or highly resistance in production, and right
Other biological strains then hyperinfection the phenomenon that.It is big in production since disease resistance is for some or certain biological strains
When area solely promotes the kind with such resistance, be easy to cause the biological strain for infecting it rise to dominant races and
It is infected, superseded, thus disease resistance cannot be stablized and persistently.The rape plasmodiophora brassicae monospore system for obtaining purifying is that development physiology is small
The basis of many research work such as kind identification, pathogen hereditary variation, and stabilization, permanent disease-resistant breeding.
Rape plasmodiophora brassicae belongs to stringent obligate parasite, not can be carried out and is manually separately cultured, and can only plant from living body host
Nutrition is absorbed in strain and is bred, and leaving host organism cannot survive, therefore is difficult separation and obtains pure bacterial strain monospore system.
The method of plasmodiophora brassicae single spore separation mainly has following four both at home and abroad at present:
First method is water agar block method separation plasmodiophora brassicae monospore, and spore suspension is dripped in the load glass containing water agar block
On piece disposes miniature punch accurately to cut a spore on optical microscopy Nosepiece and is put in host root.It is this
Method has the following problems: bubble is also easy to produce in water agar block preparation process, and more containing impurity, and rape plasmodiophora brassicae
Resting spore is colourless, very small, and 4.6~6.0 × 1.6~4.6 μm of monospore size, water agar block is observed under the microscope to be difficult
Spore, bubble or impurity are distinguished, tends not to whether accurate judgement contains rape plasmodiophora brassicae monospore, leads to the monospore mesh cut
Block is marked without or with multiple rape plasmodiophora brassicae monospores.
Second method is that (Li Qian etc., 2012, rape plasmodiophora brassicae (Plasmodiophora brassicae) is single for micro-pipe method
Spore separation inoculation and the identification plant protection of biological strain, 2012,38 (3): 95-101), by micro-pipe on water agar block
It accurately draws a spore and is put in host root.This method is removed there are water agar block bubble and impurity are more, and be not easily distinguishable rape root
Outside the problem of swollen bacterium monospore, there is also easily occur spore when drawing suspension repeatedly using micro-pipe and be stained on micro-pipe wall to cause to make
The problem of not observing, this method efficiency are lower.
The third method be freeze box bacterination process (Liu Yifan etc., 2017, rape plasmodiophora brassicae single spore separation technical system structure
Build and the identification gardening journal of areas of Shenyang plasmodiophora brassicae, 2017,44 (12): 2383-2390), by acupuncture needle in water agar
Accurate one spore of picking is accurately put in host root on block, is incubated at 81 hole freeze box.This method is also in traditional water agar
One monospore of acupuncture needle picking is utilized on block under an optical microscope, it may be bubble or miscellaneous for equally existing picking target spore
The problem of matter, furthermore acupuncture needle can make conidial cell wall broken during picking, directly result in spore death, influence disease incidence, together
When this method time-consuming in picking monospore it is more long.
Fourth method is so that suspension is reached 1 μ L by diluting rape clubroot resting spores of bacteria suspension and contain one
Then the concentration of resting spore is observed on glass slide repeatedly with liquid-transfering gun, connect after determining only one spore
Kind.This method is since resting spore suspension miospore is unevenly distributed, and the monospore suspension that may be inoculated with is without containing spore or not
It is monospore.
Above 4 kinds of prior arts exist in agar block there are bubble or impurity, and spore and background difference are unobvious, no
The problems such as easily distinguishing monospore causes picking monospore time-consuming, laborious, and success rate is lower.
Summary of the invention
To solve the above problems existing in the prior art, the purpose of the present invention is to provide one kind to be based on methylene blue staining
The rape plasmodiophora brassicae single spore separation method that liquid is combined with agarose method.Water agar method is substituted with agarose method, can effectively be avoided
There is the problem of bubble or impurity interfere in water agar；Methylene blue staining liquid is added in agarose, agarose background presents light
Micro- blue, and rape clubroot resting spores of bacteria is colourless, it is easier to monospore and agarose background are distinguished, solves rape plasmodiophora brassicae
Monospore objective fuzzy is not easy the disadvantages of picking.Operation of the present invention is simple, can greatly improve the accuracy of picking monospore, improve
A possibility that monospore system is obtained after inoculation.
Rape plasmodiophora brassicae single spore separation method provided by the present invention based on methylene blue staining liquid combination agarose method,
Include: preparation rape plasmodiophora brassicae spore suspension, prepares methylene blue agarose solution, prepares methylene blue agarose load glass
Piece coagulates layer, picking rape plasmodiophora brassicae monospore.
Specifically comprise the following steps:
1) Cruciferae clubroot incidence tissue is broken into homogenate, filtered, abandon precipitating, preparation concentration is 5 × 102A spore
Sub- mL-1~5 × 104A spore mL-1Rape plasmodiophora brassicae spore suspension, 4 DEG C~8 DEG C save backup, best to save temperature
Degree is 4 DEG C；
2) it prepares quality concentration expressed in percentage by volume and is the agarose solution of 1.3%-1.5%, and sterilize；Prepare quality volume hundred
Dividing concentration is the methylene blue staining liquid of 0.005%-0.01%；
By the agarose solution after the methylene blue staining liquid and sterilizing according to the ratio of 100 μ L:30mL of volume ratio
Example mixing mixes, and obtains methylene blue staining liquid agarose solution；
3) clean glass slide is taken, is dipped in step 2) the methylene blue staining liquid agarose solution, makes to carry glass
Piece surface forms solidifying layer；
4) rape clubroot resting spores of bacteria uniform suspension described in step 1) is coated on methylene blue staining liquid agar
The solidifying layer surface of sugar, is observed under the microscope, and when determining only one spore under the visual field, separation contains the rape plasmodiophora brassicae monospore
Methylene blue staining liquid agarose coagulate layer, be buckled at the seedling root hair of crucifer, cultivation obtain rape plasmodiophora brassicae
Monospore system.
Above method step 1) and 2) -3) between without sequencing, can also carry out simultaneously.
In above method step 1), the mode of the filtering is using eight layers of filtered through gauze.
In above method step 2), the solvent in the agarose solution and methylene blue staining liquid is sterile water；Institute
State the condition of sterilizing are as follows: 121 DEG C of sterilizing 20min.
In above method step 2), the agarose solution after sterilizing is melted and is cooled to 50 DEG C -60 DEG C, then plus
Enter the methylene blue staining liquid to be added in the agarose solution after the sterilizing.
In above method step 4), the dosage of the rape clubroot resting spores of bacteria suspension is 1 μ of μ L~5 L.
In above method step 4), the microscopical maximum amplification is not less than 1000 times, specially 400 times.
The methylene blue staining liquid agarose of the separation containing the rape plasmodiophora brassicae monospore coagulates the mode of layer are as follows: with operation
Knife cuts about 1mm2Methylene blue staining liquid agarose containing monospore coagulates layer.
In above method step 4), concretely chrysanthemum heart Chinese cabbage (is purchased from middle vegetable kind industry science and technology to the crucifer
(Beijing) Co., Ltd).The condition of the cultivation are as follows: 25 DEG C of temperature, humidity 80%, dark culture are for 24 hours.
After the present invention carries out dyeing processing to agarose using methylene blue, agarose coagulates layer background and slight blue is presented,
And on rape clubroot resting spores of bacteria vigor without influence, therefore can more clearly judge rape plasmodiophora brassicae monospore, it solves breakthroughly
Traditional water agar method background of having determined is impure and bubble is more, be not easily distinguishable rape plasmodiophora brassicae monospore the problems such as, substantially increase
The speed and accuracy rate for obtaining plasmodiophora brassicae monospore improve the efficiency for obtaining monospore system.
Detailed description of the invention
Fig. 1 is a large amount of spores of rape plasmodiophora brassicae and monospore photo of different single spore separation methods preparation；Wherein A, B difference
For a large amount of spores of methylene blue agarose method rape plasmodiophora brassicae and monospore photo, methylene blue agarose background is in slight blue,
And free from admixture and bubble, easily distinguishable rape plasmodiophora brassicae monospore；C, D are respectively a large amount of spores of traditional water agar method rape plasmodiophora brassicae
Son and monospore photo, water agar block background is impure and bubble is more, and be not easily distinguishable rape plasmodiophora brassicae monospore.
Specific embodiment
Method of the invention is illustrated below by specific embodiment, but the present invention is not limited thereto, it is all at this
Any modifications, equivalent replacements, and improvements etc. done within the spirit and principle of invention, should be included in protection model of the invention
Within enclosing.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
A kind of side based on methylene blue staining liquid combination agarose method separation rape plasmodiophora brassicae monospore provided by the invention
Method, the processes such as processing daetylorhiz, preparation spore suspension, acquisition monospore and monospore inoculation including rape plasmodiophora brassicae.Side of the invention
Method is that Cruciferae clubroot incidence tissue 1) is broken into homogenate, and eight layers of filtered through gauze abandon precipitating, and preparation concentration is 5 × 103
A spore m L-1Rape clubroot resting spores of bacteria suspension, 4 DEG C save backup；2) 0.01% methylene blue of 100 μ L is taken
The 30mL that sterilizing is added in dyeing liquor, which melts and is cooled in 50 DEG C or so of 1.3% agarose solution, to be mixed.3) by the load glass of sterilizing
Piece dips in the methylene blue staining liquid agarose solution prepared, and slide surface is made to form solidifying layer.4) 2 μ are then taken
The resting spore hanging drop that the μ of L~5 L has diluted coagulates layer surface in dyeing liquor agarose.It is observed under 400 power microscopes, really
Determine only one spore of the visual field, cuts about 1mm with scalpel2Dyeing liquor agarose containing monospore coagulates layer, and back-off is in susceptible big
At Chinese cabbage seedling root hair, cultivation obtains rape plasmodiophora brassicae monospore system.
This test with chrysanthemum heart Chinese cabbage (be purchased from middle vegetable kind industry scientific and technological (Beijing) Co., Ltd) for test material, using biography
Two methods of system agar block method and dyeing liquor agarose method are respectively inoculated with 600 plants of Seedling of Chinese Cabbage, amount to 3 repetitions.It is inoculated with 45d
It is investigated left and right, the results showed that after not only substantially increasing inoculation using dyeing liquor agarose method picking plasmodiophora brassicae monospore
The survival rate of plant, the monospore disease incidence after also improving inoculation, the disease incidence that compares improve 16.71%.
The screening of embodiment 1, agarose optium concentration
The glass slide of sterilizing is dipped in various concentration agarose solution, slide surface is made to form solidifying layer.Then
The resting spore hanging drop for taking 5 μ L to dilute coagulates layer surface in agarose.It observes, is cut about with scalpel under the microscope
The agarose that 1mm2 contains the dyeing liquor of monospore coagulates layer.Filter out best agar block concentration.Agarose solution concentration is set 1%,
1.2%, 1.3%, 1.5%, 1.8%, 2%, 2.5% and 3% totally 8 processing analyze agarose under various concentration and coagulate the saturating of layer
The complexity of lightness, quality hardness and monospore picking.
1 various concentration agarose analysis of results table of table
As the result is shown when concentration is 1.3%, agarose is solidifying, and layer transparency is preferable, quality is moderate, easy picking, is most suitable for
Rape plasmodiophora brassicae single spore separation is carried out, detailed results description is shown in Table 1.
Embodiment 2, optimum dyeing agent and the screening of stin of thickness
The configuration of dyeing liquor: weigh that 0.05g acid fuchsin, 0.01g are Congo red, 0.01g Eosin Y is received, in 0.05 g respectively
Red, the 0.01g sarranine of property, 0.005g methylene blue, 0.01g methylene blue, 0.02g methylene blue, 0.03g methylene blue,
100mL sterile water is added in 0.04g methylene blue, 0.02g bromophenol blue, 0.01g methyl green.It is configured to quality concentration expressed in percentage by volume
Respectively 0.05% acid fuchsin, 0.01% Congo red, 0.01% Eosin Y is received, 0.05% dimethyl diaminophenazine chloride, 0.01% sarranine,
0.005% methylene blue, 0.01% methylene blue, 0.02% methylene blue, 0.03% methylene blue, 0.04% methylene blue,
The dyeing liquor of 0.02% bromophenol blue and 0.01% methyl green is set 4 DEG C of brown bottle and is saved backup.
Dyeing liquor agarose coagulates layer preparation: taking above-mentioned 12 kinds each 50 μ L of different dyeing liquors, 15mL is added and melts and is cooled to 50
DEG C or so 1.3% agarose solution in, be placed in sterile petri dish and solidify, prepare dyeing liquor agarose coagulate layer.
Dyeing liquor agarose coagulates influence of the layer to rape plasmodiophora brassicae conidium vitality: using the bis- fluorescence of Hochest 33342-PI
Staining counter detects influence of the different dyeing liquors processing to rape clubroot resting spores of bacteria vigor.By concentration 1 × 108A spore
mL-1Rape plasmodiophora brassicae spore suspension 10mL be spread evenly across each dyeing liquor agarose coagulate layer surface, control take equivalent spore
Suspension is placed in the solidifying layer surface of agarose that dyeing liquor is not added, stewing process 1h.The rape clubroot resting spores of bacteria that takes that treated
Supernatant is abandoned in each 1mL of suspension, centrifugation, and spore precipitates first with 10 μ gmL-133342 dye liquor of Hoechst suspend, 37 DEG C incubation
10min, then with 5 μ gmL-14 DEG C of PI dye liquor be protected from light be incubated for 15min, be subsequently placed in fluorescence microscopy under the microscope.Through
After the bis- dyes of Hochest 33342-PI, great-hearted resting spore sends out blue-fluorescence, and dead resting spore sends out red fluorescence.Often
A processing observes and records 100 resting spores, screens the dyeing liquor on rape clubroot resting spores of bacteria vigor without influence.As a result
Such as table 2:
The different coloring agents of table 2 are to rape clubroot resting spores of bacteria effect of vigor
3 various concentration methylene blue staining liquid of table is to rape clubroot resting spores of bacteria effect of vigor
As shown in table 2, table 3: different dyeing liquors have a certain impact to rape clubroot resting spores of bacteria vigor.Its Central Asia
Methyl blue dyeing liquor is minimum to clubroot resting spores of bacteria effect of vigor, using sterile water as 0.01% methylene blue staining of solvent
It is 97% that liquid, which handles resting spore survival rate, handles suspend mode spore by 0.01% methylene blue staining liquid of solvent of PBS buffer solution
Sub- survival rate is 95%, not significant with 98% difference of processing Conidia persistence that coloring agent is not added is compareed.In a certain range,
As using sterile water as the increase of solvent methylene blue staining liquid concentration, the survival rate of resting spore declines after processing.It is acid
Magenta, bromophenol blue, methyl green etc. all have a certain impact to rape plasmodiophora brassicae conidium vitality, and resting spore survival rate is 61%
Between~87%.In addition, after rape plasmodiophora brassicae is placed in the solidifying layer processing of methylene blue staining liquid agarose, in optical microscopy
Lower observation, agarose background and rape daetylorhiz residual tissue bacterium are contaminated to be light blue, and great-hearted plasmodiophora brassicae spore is not contaminated
Color, therefore convenient for distinguishing rape plasmodiophora brassicae monospore.In conclusion methylene blue staining liquid to rape plasmodiophora brassicae conidium vitality without
It influences, can be used for single spore separation is the dyeing that agarose coagulates layer background (see Fig. 1).
Embodiment 3, methylene blue Agarose plug method are compared with traditional water agar method single spore separation effect
The methylene blue Agarose plug method and reported water agar method established using the present invention (are referred in background technique
Method one) carry out rape plasmodiophora brassicae monospore connect bacterium, compare plasmodiophora brassicae single spore separation result.To the chrysanthemum heart Chinese cabbage of vernalization for 24 hours
After (being purchased from scientific and technological (Beijing) Co., Ltd of middle vegetable kind industry) seedling is using 2 kinds of different method inoculation processing, it is transplanted to diameter
The sterile seedlings nursing plate of 5cm × 5cm, is placed in a greenhouse culture.Each 200 plants of processing, 3 repetitions.It is placed in greenhouse, is kept for day
Warm 20~25 DEG C, 11~16 DEG C of night temperature, illumination 16hd-1, the last fortnight humidity 100%, watering on demand later, greenhouse is unified to manage
Reason.
The inoculation of 4 water agar block method of table and methylene blue Agarose plug method inoculation survey data statistics
The results are shown in Table 4: under same cultivation condition, using identical bacterial strain and inoculation host material, more traditional water
The incidence of Chinese cabbage, i.e. acquisition rape plasmodiophora brassicae monospore after agar block method and methylene blue Agarose plug method inoculation monospore
Strain situation.By comparison of test results, traditional agarose method monospore disease incidence is 0.74%, obtains 3 plants of monospore strain altogether；
And the disease incidence of dyeing liquor Agarose plug method used in the present invention is 17.45%, obtains 85 plants of monospore strain altogether, significantly larger than
The host's disease incidence and monospore strain of traditional water agar block method obtain probability.The result shows that the present invention is based on methylene blue agar
The rape plasmodiophora brassicae single spore separation method of sugar method substantially increases disease incidence and the rape plasmodiophora brassicae of inoculation host Chinese cabbage
Monospore obtains probability, and this method is reliable, feasible.
Picking rape plasmodiophora brassicae monospore is suitable for using method of the invention, replaces water agar, free from admixture with agarose；
Be added dyeing liquor after, can more block, more accurately observe monospore；And dyeing liquor on conidium vitality without influence.It can be fundamentally
Chinese cabbage is not easy to survive after solving the problems, such as to be not readily separated the operational issue and inoculation of rape plasmodiophora brassicae monospore.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, appoints
The change or replacement why not expected by creative activity, should be covered by the protection scope of the present invention.Therefore, this hair
Bright protection scope should be determined by the scope of protection defined in the claims.
Claims (10)
1. a kind of rape plasmodiophora brassicae single spore separation method based on methylene blue staining liquid combination agarose method, including walk as follows
It is rapid:
1) Cruciferae clubroot incidence tissue is broken into homogenate, filtered, abandon precipitating, prepare rape plasmodiophora brassicae spore suspension；
2) it prepares quality concentration expressed in percentage by volume and is the agarose solution of 1.3%-1.5%, and sterilize；
Prepare the methylene blue staining liquid that quality concentration expressed in percentage by volume is 0.005%-0.01%；
The methylene blue staining liquid and the agarose solution after sterilizing are mixed according to the ratio of 100 μ L:30mL of volume ratio
It closes and mixes, obtain methylene blue staining liquid agarose solution；
3) clean glass slide is taken, is dipped in step 2) the methylene blue staining liquid agarose solution, makes glass slide table
Face forms solidifying layer；
4) rape clubroot resting spores of bacteria uniform suspension described in step 1) methylene blue staining liquid agarose is coated on to coagulate
Layer surface is observed under the microscope, when determining only one spore under the visual field, separates the methylene containing the rape plasmodiophora brassicae monospore
Base indigo plant dyeing liquor agarose coagulates layer, is buckled at the seedling root hair of crucifer, and cultivation obtains rape plasmodiophora brassicae monospore system.
2. separation method according to claim 1, it is characterised in that: in the step 1), the mode of the filtering is to adopt
With eight layers of filtered through gauze.
3. separation method according to claim 1 or 2, it is characterised in that: in the step 1), the rape plasmodiophora brassicae stops
The concentration of dormancy spore suspension miospore is 5 × 102A spore mL-1~5 × 104A spore mL-1。
4. separation method according to any one of claim 1-3, it is characterised in that: in the step 1), the rape
Plasmodiophora brassicae spore suspension is placed on 4 DEG C~8 DEG C preservations before use.
5. separation method described in -4 according to claim 1, it is characterised in that: in the step 2), the agarose solution and
Solvent in methylene blue staining liquid is sterile water；The condition of the sterilizing are as follows: 121 DEG C of sterilizing 20min.
6. separation method according to any one of claims 1-5, it is characterised in that: in the step 2), after sterilizing
The agarose solution melt and be cooled to 50 DEG C -60 DEG C, the methylene blue staining liquid is then added.
7. separation method described in any one of -4 according to claim 1, it is characterised in that: described micro- in the step 4)
The amplification factor of mirror is 400 times.
8. separation method described in any one of -7 according to claim 1, it is characterised in that: in the step 4), the separation
Methylene blue staining liquid agarose containing the rape plasmodiophora brassicae monospore coagulates the mode of layer are as follows: cuts about 1mm with scalpel2Contain
The methylene blue staining liquid agarose of monospore coagulates layer.
9. separation method according to claim 1 to 8, it is characterised in that: in the step 4), the cross
Flower section plant is Chinese cabbage.
10. separation method according to claim 9, it is characterised in that: in the step 4), the condition of the cultivation are as follows:
25 DEG C of temperature, humidity 80%, dark culture are for 24 hours.
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| CN112522107A (en) * | 2020-12-21 | 2021-03-19 | 浙江大学 | Stable and efficient monospore separation method for plasmodiophora brassicae, which is a strong parasitic protozoon |
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522107A (en) * | 2020-12-21 | 2021-03-19 | 浙江大学 | Stable and efficient monospore separation method for plasmodiophora brassicae, which is a strong parasitic protozoon |
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