CN109652496A - A kind of plasmodiophora brassicae floatation type bacterium solution inoculation method - Google Patents
A kind of plasmodiophora brassicae floatation type bacterium solution inoculation method Download PDFInfo
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Abstract
The invention belongs to plasmodiophora brassicae indoor inoculation technical fields, disclose a kind of plasmodiophora brassicae floatation type bacterium solution inoculation method, the wild cabbage daetylorhiz of freezen protective is taken out, thaw at RT is weighed after being cut into small pieces, and adds the sterile water of 5 times of weight, after stirring into homogenate with tissue mashing refiner, 6 layers of filtered through gauze adjust pH=5~6, utilize dormant spore concentration in blood counting chamber measurement homogenate；The seed after disinfection is broadcast in the floatation type seedlings nursing plate equipped with sterilization matrix, inoculation liquid is added in floating disc；Make in the matrix after mixing dormant spore concentration up to 108A/g matrix, the moisturizing 48h under 25 DEG C of dark conditions carry out normal management.Plasmodiophora brassicae floatation type bacterium solution inocalation method of the invention is designed for quick, uniform vaccination ways, with rapid onset after inoculation, kind growing way is uniform, and morbidity is uniform, the state of an illness differentiates high sensitivity, and can correctly mark off the true disease resistance of kind and simple to operate for objective.
Description
Technical field
The invention belongs to plasmodiophora brassicae indoor inoculation technical field more particularly to a kind of plasmodiophora brassicae floatation type bacterium solution inoculation sides
Method.
Background technique
Currently, the prior art commonly used in the trade is such that establishing a set of stable artificial disease inoculation method is screening
The basis of anti-clubroot Cruciferae material.Existing plasmodiophora brassicae indoor inoculation method mainly has rhizosphere soil note bacterium method, mixes soil
Method, root-pouring method, root dipping method etc..Pouring root inocalation method is to pour into plasmodiophora brassicae around host's root system when seedling grows to two leaves wholeheartedly
Resting spore suspension, but the disadvantage is that the daetylorhiz amount used is larger, waste inoculation source operates waste of manpower；Root dipping inocalation method is
Bacterial suspension inoculation is dipped in after cleaning root system extraction with clear water in one heart stage of two leaves, although bacterium source can be saved, is operated more multiple
It is miscellaneous；The characteristics of rhizosphere soil note bacterium method is after planting to carry out independent injection inoculation one by one in nursery cave, and workload is cumbersome, takes
When it is laborious；Mixing local method is the clubroot resting spores of bacteria suspension that will be prepared, and admixes in sterile seedling medium, be easy to cause matrix
Middle inoculation bacterial concentration is uneven, influences statistical result.
In conclusion problem of the existing technology is:
(1) pouring root inocalation method is to pour into clubroot resting spores of bacteria around host's root system when seedling grows to two leaves wholeheartedly
Suspension, but the disadvantage is that the daetylorhiz amount used is larger, waste inoculation source operates waste of manpower.
(2) root dipping inocalation method be in one heart stage of two leaves, by root system extraction with clear water clean after, dip in bacterial suspension inoculation, although
Bacterium source can be saved, but is operated more complex.
(3) the characteristics of rhizosphere soil note bacterium method is after planting to carry out independent injection inoculation one by one in nursery cave, work
Measure it is cumbersome, it is time-consuming and laborious.
(4) mixing local method is the clubroot resting spores of bacteria suspension that will be prepared, and admixes in sterile seedling medium, be easy to cause
It is uneven that it is inoculated with bacterial concentration in matrix, influences statistical result.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of plasmodiophora brassicae floatation type bacterium solution inoculation methods.
The invention is realized in this way a kind of plasmodiophora brassicae floatation type bacterium solution inoculation method, the plasmodiophora brassicae floatation type bacterium solution
Inoculation method includes:
The first step, using the disease plant daetylorhiz of Cruciferae clubroot morbidity field acquisition as trying pathogen, low temperature
It saves backup；To sterilize vermiculite, sterilizing turf, the mixing of sterile soil again, as test matrix, loaded on disinfection floatation type seedlings nursing plate
It is spare；
Second step, the wild cabbage daetylorhiz of freezen protective is taken out, and thaw at RT weighs after being cut into small pieces, adds sterile water, uses group
Knit and smash to pieces after refiner stirs into homogenate, 6 layers of filtered through gauze adjust pH=5~6, make spore cracking sufficiently, impurity screening, utilize blood
Dormant spore concentration in the measurement homogenate of ball count plate；
Third step, the seed after the first step is sterilized are broadcast in the floatation type seedlings nursing plate equipped with sterilization matrix, by inoculation liquid
It is added in floating disc；Make in the matrix after mixing dormant spore concentration up to 108A/g matrix, moisturizing carry out normal management；
4th step, the Resistance Identification time is susceptible variety serious susceptible period, after sowing 6 weeks, according to incidence
Investigation carries out disease index classification investigation.
It further, is vermiculite: turf: sterilized soil=1:1:2, pH=5~6 according to mass ratio in the first step.
Further, the sterile water of 5 times of weight is added in the second step.
Further, in the third step under 25 DEG C of dark conditions moisturizing 48h, carry out normal management.
In conclusion advantages of the present invention and good effect are as follows: plasmodiophora brassicae floatation type bacterium solution inocalation method overcomes other skills
The problem that the consumption of bacterium source amount is big, inoculation bacterial concentration is uneven, complicated for operation etc. is inoculated in art, reduce because be inoculated with it is uneven due to
The number for needing to be inoculated with repeatedly；In addition in shorter time symptom can occur for the inoculation method compared with other methods, fall ill after inoculation
Fastly, kind growing way is uniform, and uniformly, the state of an illness differentiates high sensitivity, and can correctly mark off the true disease resistance of kind for morbidity；Both it was applicable in
In the pathogen Race identification of small sample material, it is also applied for the variety resistance identification of high-volume material, it is simple and quick,
Inoculation efficiency is high, at low cost, environmentally friendly, strong operability in production application, and the light degree that simplifies is high, which is suitble to indoor anti-
Property identification and agent activity screening study work, more applicable crop field breeding material scale inoculation screening and evaluation.
Plasmodiophora brassicae floatation type bacterium solution inocalation method of the invention is designed for quick, uniform vaccination ways, to send out after inoculation
Sick fast, kind growing way is uniform, and uniformly, the state of an illness differentiates high sensitivity, and can correctly mark off the true disease resistance of kind and behaviour for morbidity
Make simple and convenient for objective；Plasmodiophora brassicae floatation type bacterium solution inocalation method is a kind of large batch of variety resistance mirror of suitable Cruciferae
Fixed best inoculation method is disease-resistant varieties breeding and disease-resistant variety rational deployment service.
Detailed description of the invention
Fig. 1 is plasmodiophora brassicae floatation type bacterium solution inoculation method flow chart provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The daetylorhiz amount used for the prior art is larger, operates waste of manpower；Workload is cumbersome, time-consuming and laborious；It is inoculated with bacterium
Liquid uneven concentration, the problem of influencing statistical result.Plasmodiophora brassicae floatation type bacterium solution inocalation method of the invention is for quick, uniform
Vaccination ways design, with rapid onset after inoculation, kind growing way is uniform, and uniformly, the state of an illness differentiates high sensitivity for morbidity.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
As shown in Figure 1, plasmodiophora brassicae floatation type bacterium solution inoculation method provided in an embodiment of the present invention the following steps are included:
S101: the selection of material to be tested: using the disease plant daetylorhiz of Cruciferae clubroot morbidity field acquisition as confession
It is spare to try pathogen, cryo-conservation；Sterilizing vermiculite, sterilizing turf, sterile soil are mixed that (mass ratio is leech according to the proportion again
Stone: turf: sterilized soil=1:1:2, pH=5~6), as test matrix, loaded on sterilizing, floatation type seedlings nursing plate is spare；Finally select
At least one germ plasm resource is taken to carry out subsequent qualification test；
S102: preparation inoculation liquid: the wild cabbage daetylorhiz of freezen protective is taken out, and thaw at RT weighs after being cut into small pieces, adds 5
The sterile water of times weight, after stirring into homogenate with tissue mashing refiner, 6 layers of filtered through gauze adjust pH=5~6, fill spore cracking
Divide, impurity screening, utilizes dormant spore concentration in blood counting chamber measurement homogenate；
S103: inoculation: the seed after the first step is sterilized is broadcast in the floatation type seedlings nursing plate equipped with sterilization matrix, will be connect
Kind liquid is added in floating disc；Make in the matrix after mixing dormant spore concentration up to 108A/g matrix reaches indoor identification morbidity mark
Standard, the moisturizing 48h under 25 DEG C of dark conditions carry out normal management；
S104: the Resistance Identification time is susceptible variety serious susceptible period, usually after sowing 6 weeks, according to incidence
Investigation carries out disease index classification investigation.
Application effect of the invention is explained in detail below with reference to experiment.
1 material and method
Strain: disease plant daetylorhiz, -20 DEG C of freezen protectives are acquired in club-root morbidity field.
For trying wild cabbage material: height susceptible variety: Qiu Shi 4；Disease-resistant variety: hundred admire Tian Shangpin.
2 methods
The preparation of 2.1 inoculation liquids
The wild cabbage daetylorhiz that partial freeze saves is taken out, thaw at RT weighs after being cut into small pieces, adds equivalent sterile water, use group
Knit smash to pieces refiner stir into homogenate after 4 layers of filtered through gauze, 4000r/min be centrifuged 15min, abandon supernatant；It is heavy with sterile aqueous suspension
Shallow lake 3500r/min is centrifuged 10min, abandons supernatant and repeats this step 3 times, finally abandons supernatant and precipitated again with sterile aqueous suspension, system
At clubroot resting spores of bacteria suspension, resting spore suspension concentration is measured using blood counting chamber, and be adjusted to 3.8 × 108A/
mL.4 DEG C save backup.
2.2 inoculation method
Rhizosphere soil infuse bacterium method: by the seed after disinfection, broadcast equipped with sterilization matrix (mass ratio is vermiculite: turf: sterilizing
Soil=1:1:2, pH=5~6) nutritive cube in, with liquid-transfering gun draw 2mL prepared 3.8 × 108A/mL resting spore is outstanding
Supernatant liquid is injected in rhizosphere matrix normal management.It is inoculated with to for examination wild cabbage material, every part 50 plants of material, setting control, 3 weights
Multiple culture, investigated incidence after 6 weeks.
Floatation type bacterium solution inocalation method: the wild cabbage daetylorhiz of freezen protective is taken out, and thaw at RT weighs after being cut into small pieces, adds 5
The sterile water of times weight, after stirring into homogenate with tissue mashing refiner, 6 layers of filtered through gauze adjust PH=5~6, utilize blood count
Dormant spore concentration in plate measurement homogenate.By the seed after disinfection, broadcast equipped with certain sterilization matrix (mass ratio is vermiculite: grass
Charcoal: sterilized soil=1:1:2, pH=5~6) floatation type seedlings nursing plate in, by inoculation liquid be added floating disc in, after finally making mixing
Matrix in dormant spore concentration up to 108A/g matrix, the moisturizing 48h under 25 DEG C of dark conditions, hereafter carries out normal management.For
The floating of holding seedlings nursing plate, should suitably plus water.The Resistance Identification time is susceptible variety serious susceptible period, usually in sowing 6 weeks
Afterwards.
3 data statistics and analysis
Disease grade standard is as follows for the inoculation of 3.1 club-root indoors artificials:
Club-root grade scale and symptom in table Room 1
3.2 disease index
Disease index (DI)=∑ (morbidity grade typical value × diseased plant number × 100 at different levels)/(investigation total strain number × superlative degree hair
Sick typical value)
Club-root population resistance grade scale is as follows:
Immune (I): DI=0；Highly resistance HR:0 < DI≤5；Disease-resistant R:5 < DI≤15；In anti-MR:15 < DI≤30；Susceptible S:30
<DI≤50；Height sense HS:DI > 50.
4 results
After being inoculated with six weeks, incidence is investigated, is inoculated with severity Scaling standard according to club-root indoors artificial
Docking plant is classified, and disease incidence and disease index are calculated.It can be seen that 2 kinds of inoculation methods can make from table 2, table 3
Plant morbidity, wherein floatation type bacterium solution inocalation method effect of inoculation is preferable, and the average attack rate that Qiu Shi 4 is 97.95%, average disease
Feelings index is 95.50；Tian Shangpin, average attack rate 7.14% are admired for highly resistance kind hundred, average disease index is 3.06.
Rhizosphere soil infuses bacterium method effect compared with slightly worse, and the average attack rate that Qiu Shi 4 is 89.58%, and average disease index is 84.22；
Tian Shangpin, average attack rate 5.95% are admired for highly resistance kind hundred, average disease index is 3.06.Therefore 2 kinds of inoculation sides
In method more preferably with the effect of inoculation of floatation type bacterium solution inocalation method.
" autumn is No. 4 real " incidence under 2 distinct methods of table
Significant difference (P=0.05) is indicated with * identical after column data, and * * indicates that difference is extremely significant (P=0.01).
" hundred admire Tian Shangpin " incidence under 3 distinct methods of table
Significant difference (P=0.05) is indicated with * identical after column data, and * * indicates that difference is extremely significant (P=0.01).
The present invention is directed to quick vaccination ways design experiment, the results showed that, used 2 kinds of inoculation methods, rhizosphere soil
Infuse in bacterium method and floatation type bacterium solution inocalation method, be with floatation type bacterium solution inocalation method effect of inoculation it is best, can in breeding for disease resistance
It is stable and consistent effectively there is morbidity at typical condition with simple, and can simultaneously accurately identify the disease-resistant of a large amount of plant
Property.The inoculum of 2 kinds of inoculation methods is all pathogen resting spore, and difference is that floatation type bacterium solution inocalation method is sterilization matrix master
The dynamic resting spore suspension that absorbs is inoculated with, without carrying out independent injection inoculation one by one；And rhizosphere soil note bacterium method feature is
Independent injection inoculation will be carried out one by one, and workload is cumbersome, time-consuming and laborious.According to the superiority and inferiority of effect of inoculation, the base that sterilizes can be passed through
The effect of inoculation of matter active absorption inoculation liquid is more preferable, makes the process of matrix absorption inoculation liquid that resting spore more be promoted to sprout, benefit
It is infected in it.During present invention test, the resting spore in root nodule sprouts zoospore out, can move about with water flow, due to educating
The water of seed plate base portion does not flow out, so most of zoospore remains in seedlings nursing plate, compares other inoculation methods, wastes bacterium
Measure less, and it is uniform to fall ill.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (4)
1. a kind of plasmodiophora brassicae floatation type bacterium solution inoculation method, which is characterized in that the plasmodiophora brassicae floatation type bacterium solution inoculation method packet
It includes:
The first step, using the disease plant daetylorhiz of Cruciferae clubroot morbidity field acquisition as trying pathogen, cryo-conservation
It is spare；Sterilizing vermiculite, sterilizing turf, sterile soil are mixed again, as test matrix, loaded on sterilizing, floatation type seedlings nursing plate is standby
With；
Second step, the wild cabbage daetylorhiz of freezen protective is taken out, and thaw at RT weighs after being cut into small pieces, adds sterile water, smash with tissue
After broken refiner stirs into homogenate, 6 layers of filtered through gauze adjust pH=5~6, so that spore is cracked abundant, impurity screening, utilize hemocytometer
Dormant spore concentration in number plate measurement homogenate；
Third step, the seed after the first step is sterilized are broadcast in the floatation type seedlings nursing plate equipped with sterilization matrix, inoculation liquid are added
In floating disc；Make in the matrix after mixing dormant spore concentration up to 108A/g matrix, moisturizing carry out normal management；
4th step, the Resistance Identification time is susceptible variety serious susceptible period, after sowing 6 weeks, according to incidence investigate
Carry out disease index classification investigation.
2. plasmodiophora brassicae floatation type bacterium solution inoculation method as described in claim 1, which is characterized in that according to matter in the first step
Amount is than being vermiculite: turf: sterilized soil=1:1:2, pH=5~6.
3. plasmodiophora brassicae floatation type bacterium solution inoculation method as described in claim 1, which is characterized in that add 5 times in the second step
The sterile water of weight.
4. plasmodiophora brassicae floatation type bacterium solution inoculation method as described in claim 1, which is characterized in that at 25 DEG C in the third step
Moisturizing 48h under dark condition carries out normal management；
S104: the Resistance Identification time is susceptible variety serious susceptible period, usually after sowing 6 weeks, according to the incidence of S3
Investigation carries out disease index classification investigation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114303516A (en) * | 2022-01-04 | 2022-04-12 | 沈阳农业大学 | Method for inoculating plasmodiophora brassicae and application of plasmodiophora brassicae in screening of plasmodiophora brassicae resistant breeding materials |
-
2019
- 2019-01-29 CN CN201910084609.4A patent/CN109652496B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 于晓坤等: "水介导十字花科作物根肿病的传播及其化学防治", 《华中农业大学学报》 * |
| 柴阿丽等: "十字花科根肿病接种技术及发病条件研究", 《华北农学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114303516A (en) * | 2022-01-04 | 2022-04-12 | 沈阳农业大学 | Method for inoculating plasmodiophora brassicae and application of plasmodiophora brassicae in screening of plasmodiophora brassicae resistant breeding materials |
| CN114303516B (en) * | 2022-01-04 | 2022-12-13 | 沈阳农业大学 | Method for inoculating plasmodiophora brassicae and application of plasmodiophora brassicae in screening of plasmodiophora brassicae resistant breeding materials |
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