CN107245452B - A kind of single spore separation method of Chinese cabbage plasmodiophora brassicae biological strain - Google Patents
A kind of single spore separation method of Chinese cabbage plasmodiophora brassicae biological strain Download PDFInfo
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Abstract
A kind of single spore separation method of Chinese cabbage plasmodiophora brassicae, it is characterised in that: freeze box filter paper culture: the agar block containing monospore is moved into freeze box, is set and is carried out connecing bacterium on the Seedling of Chinese Cabbage root of filter paper cultivation；Dark culture is first carried out in freeze box；Agar block is allowed to touch root hair, convenient for infecting for plasmodiophora brassicae；Transition culture: water planting culture is carried out after growth 6 days, seedling is transferred in the centrifuge tube of distilled water, it is placed in lighttight centrifuge tube box in illumination box at 23 DEG C of temperature, after being cultivated 2 days in the environment of humidity 60% and illumination 16h/d, it changes Gram nutrient solution suddenly and carries out water planting, seedling is gone to matrix culture when root fleece infects by daily Additional nutrient solution；It is long to progress sterile soil culture after 4 true leaves after seedling, knee is collected after 40 days is used for Race Identification.The present invention makes disease incidence be increased to 47.86%, and so that survival rate of plant is increased to 67% from 27% by connecing the selection of bacterium seedling period and training systern.
Description
Technical field
The present invention relates to the single spore separation methods of Chinese cabbage plasmodiophora brassicae, are plant pathogen biological strain isolation technics, belong to
In field of biotechnology.
Background technique
Crop in cruciferae clubroot is also referred to as crown gall, is invaded by rape plasmodiophora brassicae (Plasmodiophora brassicae)
Worldwide soil-borne disease caused by contaminating, seriously affects Chinese cabbage and the production of other crop in cruciferae.Since plasmodiophora brassicae is usual
It is infected with the mixed raw state of multiple biological strains, prevent its resistance is from lasting, therefore by plasmodiophora brassicae single spore separation technology,
Each plasmodiophora brassicae biological strain is separated with important application value.
Currently with plasmodiophora brassicae single spore separation technology carry out Race Identification research there are reports, such as 2008
Year, xue etc. has carried out single spore separation using the method that culture dish culture combination bacterium earth culture is supported, 3 lifes obtained using 5 bacterial strains
Manage microspecies infection rate mostly all 6% hereinafter, the maximal infection rate 16.95% [xue etc., 2008, Isolation and
Variation in Virulence of Single-Spore Isolates of Plasmodiophora brassicae
from Canada,Plant Disease,29(3):456-462]；Li Qian in 2012 etc. utilizes micro-pipe absorption method single spore separation skill
Art carries out plasmodiophora brassicae single spore separation, obtains 4 biological strains using 2 bacterial strains, infection rate is between 1.49-3.96%
[Li Qian etc., 2012, rape plasmodiophora brassicae (Plasmodiophora brassicae) single spore separation is inoculated with and the mirror of biological strain
It is fixed, plant protection, 2012,38 (3): 95-101].From these results of study as it can be seen that the physiology being separated to using existing technology
Microspecies are very limited, the main reason is that being limited to that isolation technics is incomplete, and especially infection rate is too low, planting percent is not also high
It is caused.Therefore this research main purpose is to develop a kind of infection rate, disease incidence and all higher single spore separation method of planting percent.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of quick separating plasmodiophora brassicae monospore, establish more perfect monospore point
From technology and subsequent identification biological strain system.The separation method is convenient and efficient, improves survival rate of plant and the morbidity of plant
Rate reduces the probability of mixed spore when identification biological strain, adapts to promote and apply.
In order to achieve the above objectives, the technical solution adopted in the present invention is as follows:
(1) freeze box filter paper culture
1% agar block containing monospore is moved into freeze box, the 2 age in days seedling root of Chinese cabbage for setting filter paper cultivation is enterprising
Row connects bacterium.With 23 DEG C of temperature first in freeze box, humidity 60% carries out dark culture.Pay attention to having to allow agar block with sporangium
Face contact is to root hair, in order to infecting for plasmodiophora brassicae.
(2) transition culture
Since freeze box growing space is limited, after growth 6 days, water planting culture is carried out, seedling is transferred to the dress with sponge
Have in the 2ml centrifuge tube of distilled water, is placed in lighttight centrifuge tube box in illumination box at 23 DEG C of temperature, humidity 60%
It after being cultivated 2 days in the environment of illumination 16h/d, changes Gram (Hongland ' s) nutrient solution suddenly and carries out water planting, pay attention to needing daily
With syringe Additional nutrient solution to 2ml.When microscope tabletting observes that root fleece infects, seedling is gone into sterilization matrix
Culture, matrix are made of turf, perlite, vermiculite, weight ratio 7:2:1, after seedling length to 4 true leaves, carry out sterile soil
Culture collects knee and is used for Race Identification after 40 days.
Method of the invention has the advantage that compared with prior art
The present invention establishes more perfect single spore separation technical system for the first time, and single spore separation infection rate is low in forefathers' research
(only 1.5-4%), the present invention make disease incidence be increased to 47.86% by connecing the improvement of bacterium method, and by connecing the choosing of bacterium seedling period
It selects and training systern makes survival rate of plant be increased to 67% from 27%, while shortening experiment process.
Detailed description of the invention
Fig. 1 is bacterium solution water planting schematic diagram.
Fig. 2 is agar block filter paper method schematic diagram.
Fig. 3 is that 1 age in days seedling connects 32 days schematic diagrames of bacterium.
Fig. 4 is that 2 age in days seedling connect 12 days schematic diagrames of bacterium.
Fig. 5 is that 3 age in days seedling connect 12 days schematic diagrames of bacterium.
Fig. 6 is that 4 age in days seedling connect 24 days schematic diagrames of bacterium.
Fig. 7 is one plant of scheme culture process.
Fig. 8 is two plant of scheme culture process.
Fig. 9 is one plant growth condition of scheme.
Figure 10 is two plant growth condition of scheme.
Figure 11 is water planting and matrix culture plant maximum blade width (cm) average value column diagram.
Figure 12 is the monospore plant disease plant that scheme one obtains.
Figure 13 is the monospore plant disease plant that scheme two obtains.
Figure 14 is host's onset state.
Compared with the prior art, the present invention has the following advantages:
This project establishes more perfect single spore separation technical system for the first time, and method of the invention is compared with prior art
Good effect is shown:
(1) increased freeze box filter paper connects bacterium incubation step in this technique, is increased to infection rate from 1.5-16.95%
47.86%.
(2) the excessive step of matrix culture is increased in this technology, so that survival rate of plant is increased to 67% from 27%, and shorten
Experiment process.
Specific embodiment
(1) prepared by bacterium solution
1g morbidity old complaint is taken to be ground with aseptic water washing 3~5 times using the mortar of sterilizing, until root tissue is broken
It is broken, 10mL sterile water is added, mixed liquor passes through 8 layers of filtered through gauze, and filtrate 3100r/min is centrifuged 15min, abandons supernatant, will sink
Shallow lake is redissolved in 50mL aqua sterilisa, and 3100rpm is centrifuged 15min, this step operation is 2~3 times repeatable, supernatant is abandoned, in precipitating
Add 4mL50% sucrose solution, shake up, 3100rpm is centrifuged 10min, supernatant is moved into clean centrifuge tube with liquid-transfering gun, added
30mL aqua sterilisa, 3100rpm are centrifuged 15min, discard supernatant liquid, and precipitating is dissolved in 30mL aqua sterilisa again, and 4000rpm is centrifuged 5min,
This step operation is repeated 2 times, and finally precipitating is dissolved in spare in 5mL aqua sterilisa, is detected by blood counting chamber by diluted rue
The concentration of a kind of sedge plasmodiophora brassicae spore suspension.Microscopy is carried out before carrying out single spore separation, guarantees only have 1 in every 0.5 μ L bacterium solution
Monospore.
(2) freeze box agar method single spore separation
It is put into two layers of filter paper on the small lattice of each of freeze box, a small amount of distilled water is added, keeps filter paper wet, Chinese cabbage is normal
Rule seed is uniformly layered on filter paper, is placed under 25 DEG C of dark surrounds vernalization for 24 hours.
It is 2000/mL that the rape plasmodiophora brassicae spore suspension finally obtained, which is diluted to spore concentration, is inhaled with liquid-transfering gun
It takes 1.0% agarose solution of 80 μ L to drip on sterilized glass slide, slowly jiggles glass slide, 15min is left
After the solidification of right agarose, on the agarose that takes the glass slide of the hanging drop of 0.5 μ L after solidification, it is placed in 16 × 40 power microscopes
Under observed and gradually adjust the visual field, until it is central to find that single plasmodiophora brassicae zoospore is located at the visual field, then be gradually increased micro-
Mirror multiple is observed, and determines that microscope without other spores, is transferred under 16 × 40 times of object lens by visual field center again.Then
It is sheared on agar plate with special transfer needle, not needing mobile glass slide and microscope, transfer needle can directly carry
It is operated on object platform.Notice that in order to further verify only include 1 spore in field range, the agar plate after observation cutting
On scratch and scratch outside and nearby either with or without other spores, then can guarantee that cutting is the agar block containing monospore.
During separating monospore, then transfer needle alcolhol burner flame calcination 1min is placed in sterile water and is cooled down.To avoid
Transfer needle touches other zoospores, and needle falling point should be as far as possible on agar plate within the vision.
1% agar block containing monospore is moved into the 2 age in days seedlings roots cultivated in freeze box and carries out connecing bacterium, in temperature
Degree is 23 DEG C, and humidity is dark culture in the environment of 60%.Pay attention to having to allow agar block band sporangium face contact to arrive when this step
Root hair, in order to infecting for plasmodiophora brassicae.
(3) transition culture
Since freeze box growing space is limited, water planting culture is carried out after growth 6 days.Seedling is transferred to filling with sponge
In the 2ml centrifuge tube of distilled water, sets in centrifuge tube box, be put into illumination box, at 23 DEG C of temperature, humidity 60% and illumination
After being cultivated 2 days in the environment of 16h/d, changes Gram (Hongland ' s) nutrient solution suddenly and carry out water planting, need to use syringe daily
Additional nutrient solution is to 2ml.When microscope tabletting observes that root fleece infects, seedling is gone into sterilization matrix culture, matrix
It is made of turf, perlite, vermiculite, weight ratio 7:2:1, after seedling length to 4 true leaves, carries out in next step.
(4) sterile soil culture
The sterile soil (with the nursery soil of 121 DEG C of 30min that sterilize in autoclave) that volume 2/3 is first filled in pot for growing seedlings, will
Seedling after matrix culture moves into pot for growing seedlings, and a little fine sand is spread on upper layer, prevents " jumping root ", and every plant of plant is furnished with an independence
Pallet, at 23 DEG C~25 DEG C of temperature, soil moisture is maintained at 70%~90% and is cultivated.
(5) fungus block is collected
After plant carries out sterile soil culture 40 days, carries out washing root observation, to disease plant root nodule Subsampling and number, protect
In the presence of in -20 DEG C of refrigerators so that the later period prepares monospore bacterium solution.Waste water and bacterium soil after washing root, which will be collected, to be uniformly processed, in order to avoid
Contaminated soil.
(6) WILLIAMS-DARLING Ton (Williams) Race Identification
The seed disinfection-of WILLIAMS-DARLING Ton (Williams) knee dientification of bacteria host is hoted water treatment of seeds (by 4 layers of yarn of seed
Cloth is wrapped, and is tightened with rubber band, is placed in 50 DEG C of warm water and is impregnated 30min, regular pendulum vernalization in culture dish) vernalization afterwards
It 1 day, is sowed in hole tray of 50 holes equipped with sterile soil, every cave 2-3.After planting 6 days with 2ml syringe by monospore bacterium solution
Squeeze into hole tray (method that sporangium liquid and preparation method thereof is prepared with bacterium solution, by blood counting chamber calculating reach spore concentration
1x107A/mL), hole tray is placed in sterile environment, paying attention between each hole tray will there are certain gap, each caves
Disk has an independent pallet, in case the mutual pollution between causing plant, watering at regular time and quantity, holding soil moisture are
75%~90%, while planting lower susceptible variety and being compareed the incidence for investigating plant after the onset to susceptible variety.
Disease investigation standard: 0 grade, without tumour, root system development is normal for root；1 grade, lateral root raw a little tumour or main root slightly
Have swollen thick, there is nodositas lump small but that naked eyes are high-visible；3 grades, main root raw tumour, have apparent larger nodositas or ball
Shape lump；5 grades, occurring big tumour on main root, enlarges into spindle, big knee extends to hypocotyl, and plant is short and small,
Growth stops.
Disease index < 10 be it is disease-resistant, disease index >=10 be susceptible (Strandberg&Williams, 1967)
Biological strain is determined according to table 1 and Disease investigation result.
1 Williams biological strain identification system of table
Table 1 Possible host reactions to Plasmodiophora brassicae races in
Williams system
Note :+represent susceptible reaction；Represent disease resistance response
Note:+represents susceptible reaction ,-represents resistant reaction
Embodiment 1: infection rate investigation
Agar method replaces bacterium solution water planting to carry out single spore separation (Fig. 1), and agar method single spore separation is in the agar containing 1.0%
The bacterium solution of suitable concentration is added dropwise on the glass slide of sugar, observes under the microscope.It will be observed that the agar block of single spore is cut,
Continue to observe, until only one spore in agar block.Agar block containing monospore is moved into the children cultivated in freeze box
It carries out connecing bacterium on seedling, carries out water planting after a few days, carry out matrix culture (Fig. 2) after root system is infected.Change in this step
Into original technology, ordinary culture medium is replaced with freeze box, save the cost guarantees that single spore has sufficient time contact root
Hair improves infection rate, and replaces pervious concave surface glass slide with common glass slide, every time can be in microscope with mode with this
Lower 2-3 monospore of observation, save the cost and time.Infection rate is greatly improved by this method.
It is compared by experimental result, the infection rate of bacterium solution water culture is 1.5-4% (Li Qian etc., 2012), used in this experiment
The infection rate of agar block filter paper method be increased to 47.86%.
Embodiment 2: the determination in vaccine age is connect
It selects 1 age in days, 2 ages in days, 3 ages in days and the seedling in 4 age in days periods to carry out monospore respectively and connects bacterium, as a result, it has been found that 1 age in days
Seedling meets 32 talentes after bacterium and is infected, and the time that 2 ages in days and 3 age in days seedling are infected is more early, enters within 8 days and infects initial stage, and 12 days
Into infecting peak period.And 4 age in days seedling connect bacterium 24 days and are infected.So 2 ages in days and 3 ages in days are most preferably to connect bacterium period (see Fig. 3-
6).Analyzing 1 age in days seedling and infecting may be caused by causing agar block to shift due to its just germination continued growth slowly.
Embodiment 3: survival rate of plant investigation
Since the space of freeze box is limited, seedling can only grow 6 days in freeze box, and the later period will use water planting, tradition side
Monospore seedling is placed in centrifuge tube in method and is cultivated, pours nutrient solution per daily syringe to maintain plant strain growth, it is time-consuming to take
Power, and seedling percent is low in this process, and later period culture is also relatively difficult.Experiment it was found that, after seedling is infected
The problem that one of can substantially improve is transferred in the matrix of sterilizing.Specific experimental method is as follows:
It is tested based on the seedling of 2 ages in days, 400 plant, divides two batches and cultivated, connect bacterium after a few days, a part
First water planting switchs to earth culture (scheme one) (see Fig. 7) after infecting；Another part elder generation water planting carries out matrix culture (side after infecting
Case two) (see Fig. 8), compare plant growing way and survival rate.By the comparison of the two schemes, we are the following results were obtained: side
The survival rate of case one is 27%, the survival rate 67% of scheme two.The upgrowth situation of water planting and matrix culture plant (see Fig. 9,10).
In order to make result more it is credible we suitable growth environment compared by the measurement to maximum blade width.Experiments have shown that matrix
Than water planting culture, n plant survival rate is higher for culture, and growth is more preferable, also saves the time (see Figure 11) of half, carries out matrix training
It supports after a week, the growing state difference of Rooted Cuttings and matrix culture is little, starts significant difference occur after growth 3 weeks.
Embodiment 4: plant morbidity survey
Reach certain growth degree about in 35 days or so progress Disease investigations to plant, disease plant is sampled.
Wherein one disease incidence of scheme is 54.1%, and two disease incidence of scheme is 63.01%.The plant the most apparent that wherein falls ill is chosen to carry out
Compare, the monospore plant that right side plant scheme one obtains, left side is that the monospore plant that scheme two obtains can be found that scheme two more
Add suitable, not only disease incidence has greatly improved, and the more original method of occurring degree has also greatly reinforced (see Figure 12,13).
Embodiment 5: Race Identification
Due to the particularity of sporangium, the probability that prevent mixed spore from occurring in this step, and in previous studies
Bacterium local method has been generallyd use to carry out connecing bacterium, but the probability that this method mixes spore is larger, in order to avoid this case, we are used
The method of injection bacterium solution carries out connecing bacterium, not only ensure that connecing mycoplasma amount decreases the possibility of mixed spore.
The host of disinfection and vernalization 1 day is sowed in the hole tray containing different sporangiums, every cave 2-3 seeds,
40 sporangiums are identified altogether, and each each host of monospore investigates 30 plants respectively, behind hot-house culture 60 days of 5-25 DEG C, carries out disease
Sentiment is looked into.Qualification result shows, removes outside original No. 4 biological strains in this area, we also identify new No. 8, No. 9,
No. 11 biological strains.Wherein No. 4 biological strains are dominant races, and proportion reaches 57.5%, and No. 11 biological strains take second place, and account for
32.5%, No. 9 biological strains only there are three, account for 7.5%, No. 8 microspecies only one, account for 2.5%.4 hosts are carried out
Williams identification and analysis finds that the identification of two wild cabbages host JQ, BS are stronger to the knee disease resistance of areas of Shenyang etc., 40 lists
There is WB in spore, LT almost 100% morbidity, occurring degree is most heavy, and JQ disease incidence 57.5%, occurring degree is most light, BS disease incidence
90%, occurring degree is heavier (see Figure 14).
Claims (1)
1. a kind of single spore separation method of Chinese cabbage plasmodiophora brassicae, it is characterised in that following key step:
(1) freeze box filter paper culture
1% agar block containing monospore is moved into freeze box, sets and is connect on the 2 age in days seedling root of Chinese cabbage of filter paper cultivation
Bacterium；With 23 DEG C of temperature first in freeze box, humidity 60% carries out dark culture；Pay attention to having to allow agar block band sporangium face to connect
Root hair is contacted, in order to infecting for plasmodiophora brassicae；
(2) transition culture
Since freeze box growing space is limited, after growth 6 days, water planting culture is carried out, seedling is transferred to being equipped with sponge and is steamed
It in the 2ml centrifuge tube of distilled water, is placed in lighttight centrifuge tube box in illumination box at 23 DEG C of temperature, humidity 60% and light
It after being cultivated 2 days in the environment of 16h/d, changes Gram nutrient solution suddenly and carries out water planting, pay attention to being needed daily with syringe Replacement Battalion
Nutrient solution is to 2ml, and when microscope tabletting observes that root fleece infects, seedling is gone to sterilization matrix culture, matrix by turf,
Perlite, vermiculite composition, three's weight ratio are 7:2:1；After seedling length to 4 true leaves, sterile soil culture is carried out, is received after 40 days
Collect knee and is used for Race Identification.
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| CN115581196A (en) * | 2022-10-28 | 2023-01-10 | 云南省农业科学院经济作物研究所 | Inoculation method for rapidly screening clubroot disease resistant varieties in seedling stage |
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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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Non-Patent Citations (1)
| Title |
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| 芸薹根肿菌(Plasmodiophora brassicae)单孢分离接种及生理小种的鉴定;李茜等;《植物保护》;20121231;第38卷(第3期);摘要和材料与方法 * |
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