CN104651240A - Method for separating monospores of plasmodiophoromycetes and method for establishing a monospore line by host propagation - Google Patents
Method for separating monospores of plasmodiophoromycetes and method for establishing a monospore line by host propagation Download PDFInfo
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Abstract
The invention provides a method for separating monospores of plasmodiophoromycetes. The method comprises the following steps: carrying out decomposition, water-adding grinding and filtration and separation on a root nodule with clubroot of a cruciferae crop to obtain a spore suspension; diluting the solution obtained in the step by distilled water to obtain a diluted spore suspension; and picking spores in the diluted spore suspension by using an acupuncture needle and pressing the spores on an agar block, and observing one spore on the agar block by using an optical microscope and marking and retaining. The invention further provides a method for establishing a monospore line by host propagation. The method comprises the following steps: putting the agar block on a seedling root hair of a sterile cruciferae crop and culturing to obtain the root nodule with clubroot; and pouring the ground root nodule into sterilized soil to form culture soil, seedling the sterile cruciferae crop seeds on the culture soil, cultivating to obtain the root nodule with clubroot, and pouring the part of root nodule ground into the culture soil. The success rate of picking monospores by using the separating method is high, and soil for propagation by virtue of the propagation method can be used for a long time, so that the utilization ratio of the root nodule is improved.
Description
Technical field
The present invention relates to single spore separation inoculation method field, expand numerous method setting up monospore system in particular to a kind of plasmodiophora brassicae single spore separation, host.
Background technology
Club root is by the worldwide important disease on the microbial crop in cruciferae of biogenous rape knee.Anti-club root breeding and Appropriate application disease-resistant variety are important channel and the method for this disease of control.Owing to there is physiological strain under knee bacterial classification, and tradition utilize Williams differential host to identify the inoculation bacterium source of physiological strain is root nodule, instead of adopt monospore, therefore be considered to identify result should be " pathotype " instead of physiological strain.Adopt single spore separation, host to expand numerous, set up plasmodiophora brassicae monospore system qualification plasmodiophora brassicae physiological strain, antagonism club root breeding and Appropriate application disease-resistant variety significant.
It is by dilution spore suspension that now popular plasmodiophora brassicae single spore separation method mainly contains two kinds: one, the concentration making suspension reach 0.5 μ L to contain a spore, then draw suspension with microtubule repeatedly to observe on slide glass, inoculate after determining to only have a spore.Two is use the punch tool method of frame on microscope to be separated, the plasmodiophora brassicae spore be distributed on agar block is first observed under the microscope, punch with the micro-drill device be erected on Nosepiece, the agar after picking punching is inoculated on previously prepd host plant root again.Incidence is observed after cultivating for some time.
But, first method in aforesaid method is repeatedly drawn when suspension is observed due to microtubule and is easily occurred that spore sticks on microtubule wall, even there is situation about not observing, efficiency is lower, second method is owing to Nosepiece being settled micro-drill device very difficult, cannot determine whether that statospore sticks on punch tool in operation, and the real-time sterilization also more difficult realization to punch tool.And consuming time relatively more of a specified duration when using above method picking monospore, success ratio is not high.
In view of this, special proposition the present invention.
Summary of the invention
The advantages such as the first object of the present invention is to provide a kind of plasmodiophora brassicae single spore separation method, and this method improves the efficiency of picking spore, and the success ratio of choosing monospore is high, and acupuncture needle used is easily sterilizing also, easy to operate.
The second object of the present invention be to provide a kind of adopt plasmodiophora brassicae single spore separation after host expand numerous method setting up monospore system, the advantages such as this expanding propagation method carries out expanding numerous soil can life-time service, improves root nodule utilization ratio, easy to operate.
Embodiments provide a kind of plasmodiophora brassicae single spore separation method, comprise the steps:
(A) root nodule of crop in cruciferae being suffered from club root obtains spore suspension after become thoroughly decomposed, add water grinding and filtering separation step;
(B) strength of solution that above-mentioned steps obtains is controlled 1 × 10
7-3 × 10
7individual spore/ml, and then doubly obtain dilution spore suspension with distilled water diluting 30-50;
(C) with acupuncture needle in described dilution spore suspension after picking spore by being pressed on agar block, and on described agar block, only have the laggard row labels of spore to retain with observation by light microscope.
The plasmodiophora brassicae single spore separation method that the embodiment of the present invention provides, in picking process, monospore only can by fractographic place at agar surface, and observation and comparison is convenient, uses 10 times of object lens can meet observation requirement, and the easy sterilization of acupuncture needle.Wherein strength of solution is also an important Con trolling index, should first control 1 × 10
7-3 × 10
7individual spore/ml, and then it is further diluted, the multiple of dilution 30-50 doubly between, 33,34,35,36 times etc. can be chosen.If the excessive concentration of solution, the success ratio of picking can be affected, also be not easy to use microscopic examination, therefore need to control in suitable scope.
In the present invention, to the root nodule of club root be suffered from the step of becoming thoroughly decomposed, adding water grinding and filtering separation, first tentatively filter with 8 layers of gauze, the mode of centrifugation is adopted to carry out when being then separated, generally need to carry out 4-5 centrifugation, first time centrifugation stays supernatant liquor to abandon precipitation, because the soil block of some bulks can be contained in precipitation, for removing the impurity such as host cell in liquid, after centrifugation be several times then stay precipitation to abandon supernatant liquor, isolate the component lighter than spore, as some bacteriums, the sporangiocyst etc. after sprouting.
In step (C), preferably select by being pressed on the edge of agar block after picking spore, and the speed of picking as quickly as possible, prevent from carrying out easily agar being dried up owing to offering ventilation system in the process operated, affect microscopic examination, be pressed on agar and also do not exert oneself very much, easily too dark bad observation in the middle of pressure, the edge being therefore pressed in agar block facilitates control dynamics also as too excessively dark with what defeat.
In addition, preferably select to operate in an aseptic environment in whole operating process, prevent plasmodiophora brassicae from polluteing whole Experimental Area, affect experimental result.
The embodiment of the present invention additionally provides a kind of host's expanding propagation method after plasmodiophora brassicae single spore separation, comprises the steps:
(D) the described agar block containing single spore is placed on the crop in cruciferae seedling root hair of aseptically process, cultivates the root nodule that for some time obtains suffering from club root;
(E) soil is cultivated by pouring in sterilized soil to be formed after described root nodule grinding, crop in cruciferae seed through aseptically process is sowed on described cultivation soil, cultivation for some time obtains the root nodule of suffering from club root, pours in described cultivation soil to ensure the plasmodiophora brassicae content that described cultivation is native and life-time service after taking out the grinding of part root nodule.
The expanding propagation method that the embodiment of the present invention provides by cultivate the root nodule extraction portion cultivated of for some time divide grind after be reentered in soil, to ensure the plasmodiophora brassicae content in soil, soil can be made to expand numerous permanently effective matrix as carrying out, and the utilization ratio that improve as expanding numerous initial plasmodiophora brassicae statospore, unlike soil in prior art carried out a generation expand numerous after then directly abandon, not only wasted raw material but also can not ensure to expand numerous continuity.
In the present invention, the culturing process of crop in cruciferae seedling is: get a certain amount of Cruciferae seed, sterilize in the alcohol of 75% after 5min with aseptic water washing 3 times, a grain is put into and is completed in the enamel tray of filter paper, water spray moisturizing, with transparent preservative film plastic paper by after the sealing of enamel tray parcel, put into constant temperature illumination growth cabinet and cultivate 72h.Use this method can to ensure in culturing process not pollute by other bacterial classifications, use the sealing of plastic paper parcel effectively can prevent moisture evaporation.By repeatedly testing, find that crop in cruciferae used preferably selects precocious No. five these kinds of Chinese cabbage, because this kind is more easily infected, sickness rate is higher, can significantly improve the infection rate of plasmodiophora brassicae.
After cultivating seedling, agar block containing single spore is placed on the crop in cruciferae seedling root hair of aseptically process, continue to cultivate, its concrete steps of cultivating are preferably: first in constant temperature growth cabinet, cultivate 40-50h under the condition of dark, then on seedling pan, 60-65d is cultivated, and preferably hot-house culture, owing to producing zoospore after the sprouting of plasmodiophora brassicae statospore, illumination can make the germination rate of statospore reduce and have impact to the vigor of zoospore.Cultivating for some time is in dark conditions to improve statospore germination rate, improves and infects probability.
Finally expand in numerous process, normal cultivation 60-65d, be typically chosen in common flowerpot and carry out, the control of density will be noted in the process of sowing, not overstocked too unloose yet, overstocked normal growth space affect crop, excessively thin waste energy uneconomical, general control is in the density of 25-35 strain/basin, and the diameter of flowerpot is 12-15cm.
In the present invention, also prepare root sepage, in order to improve the germination rate of statospore thus to improve further plasmodiophora brassicae infect success ratio on root hair, between described step (A) and described step (B), get root sepage after also comprising the steps: that plant seed is carried out cultivation for some time after disinfection, the volume ratio of described sepage and described spore suspension is controlled to obtain solution between the ratio of 1:8-10.The concrete volume ratio of its root sepage and spore suspension needs in a suitable scope, because the on the one hand concentration of solution in its concrete scale effect follow-up (B) step, if the excessive concentration of solution can affect microscopical observation difficulty, control on the one hand in addition the germination rate of the statospore in spore suspension can be made to reach best in this scope, its ratio can also get 1:8.5,1:9,1:9.5 etc.
Solution preferably preserves 48-50h under the dark condition of 25-30 DEG C, and the pH value of solution controls at 6-7, because under this operating parameters condition, and the sprouting best results of its statospore.
The concrete grammar preparing root sepage is: by plant seed with 1% clorox or use aseptic water washing again 3 times with after 75% alcohol disinfecting 3min, seed is placed in the culture dish that moist aseptic filter paper is housed.Culture dish is placed in illumination growth cabinet and cultivates growth cabinet.Sterilize root Vetstrep (50ppm) when seedling grows to 2-10cm 5min, then use aseptic water washing 3 times.In disposal plastic cup, add a certain amount of Hoagland nutritive medium, rim of a cup gauze wraps up, and is inserted by plant seedlings in gauze fixing, is positioned over growth cabinet and cultivates, and carry out humidification with humidifier, get root sepage, save backup after 12-14d.Use Hoagland nutritive medium to have the effect improving statospore germination rate further, then adopt gauze parcel because seedling growth is higher, by the volatile humidity therefore needing to adopt humidifier to ensure needed for its growth of the nutrient solution in gauze parcel cup.
It is wherein a kind of that plant seed can be chosen in the seed of rye grass, leek, capsicum, white turnip and Chinese cabbage.Wherein, the sprouting of root sepage to statospore that ryegrass seed is prepared is the most effective, and in order to improve effect further, can drip described sepage, play the effect of double insurance on described crop in cruciferae seedling root hair.
Compared with prior art, beneficial effect of the present invention is:
(1) plasmodiophora brassicae single spore separation method improves the efficiency of picking spore, and the success ratio of choosing monospore is high, easily observes under the microscope, and acupuncture needle used is easily sterilizing also, easy to operate;
(2) adopting host's expanding propagation method of the present invention to make to carry out to expand numerous soil can life-time service, and it is higher to expand plasmodiophora brassicae infection rate in numerous process, and the morbidity situation risen has appearred in plant;
(3) the root sepage by using Hoagland nutritive medium to prepare processes statospore, improves statospore germination rate, increases and infects success ratio.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is that the plasmodiophora brassicae single spore separation of the embodiment of the present invention and host expand numerous method steps schema;
Fig. 2 is that the graphic representation affected is sprouted in root sepage prepared by the different plant seed of experimental example 1 of the present invention on statospore.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
Embodiment 1
Spore suspension processed: get 10g root nodule after being become thoroughly decomposed by crop in cruciferae club root root nodule and add 50mL water in grinding machine for grinding.Move into 50mL centrifuge tube with after 8 layers of filtered through gauze, the centrifugal 10min of 500r/min, abandons precipitation; Supernatant liquor is continued the centrifugal 15min of 3100r/min, abandon supernatant liquor and stay precipitation; 50mL aqua sterilisa, shakes up precipitation, and the centrifugal 10min of 3100r/min, abandons supernatant liquor, and precipitation is heavily dissolved in 50mL aqua sterilisa, the centrifugal 10min of 3100r/min, and the operation of this step can repeat 2-3 time.Adjustment spore suspension concentration is to 1 × 10
8individual spore/about ml, saves backup.
Embodiment 2
Root sepage processed: by plant seed with 1% clorox or use aseptic water washing again 3 times with after 75% alcohol disinfecting 3min, be placed on by seed in the culture dish that moist aseptic filter paper is housed, culture dish is placed in illumination growth cabinet and cultivates.Sterilize root Vetstrep (50ppm) when seedling grows to 2-10cm 5min, then use aseptic water washing 3 times.Add a certain amount of Hoagland nutritive medium in disposal plastic cup, rim of a cup gauze wraps up, and is inserted by plant seedlings in gauze fixing, is positioned over growth cabinet and cultivates, and carry out humidification with humidifier, get root sepage, save backup after 12-14d.
Embodiment 3
Spore suspension mixes with root sepage: spore suspension obtains solution after mixing according to the volume ratio of 1:8-10 with root sepage.Regulator solution pH value is control its concentration 1 × 10 after preserving 48-50h under putting into the dark condition of 25-30 DEG C after 6-7
7-3 × 10
7between individual spore/ml, and then doubly obtain dilution spore suspension with distilled water diluting 30-50.
Embodiment 4
Host plant seedling is cultivated: use precocious No. five Chinese cabbages to be host, get a certain amount of seed, sterilize in the alcohol of 75% after 5min with aseptic water washing 3 times, a grain is put into and is completed in the enamel tray of filter paper, water spray moisturizing, with transparent preservative film plastic paper by after the sealing of enamel tray parcel, put into growth cabinet and cultivate 72 hours.
Embodiment 5
Plasmodiophora brassicae single spore separation: cut good clear water agar on slide glass, is cut into of uniform size square, holds dilution spore suspension in culture dish.Use acupuncture needle to carry out picking in dilution spore suspension, needle point is gently stained with in culture dish, then presses at the edge of agar block.Observe needle point impression under an optical microscope and around with or without single spore, if any and only have a spore to exist, then carry out marking rear reservation, whole process is carried out in an aseptic environment.
Embodiment 6
Infect host Gen Mao: drip the root sepage of embodiment 2 on Chinese cabbage seedling root hair after, to mark above the agar block with single spore tips upside down on, 48-50h is cultivated with being placed in the dark growth cabinet of constant temperature after preservative film sealing, proceed to afterwards in seedling pan and continue to cultivate 60-65d, by the root nodule Collection and conservation of morbidity.
Embodiment 7
Host expands numerous: fill in the flowerpot of sterilized soil by pouring into after the root nodule mortar grinder of morbidity, Chinese cabbage seeds is sowed according to every basin 25-35 strain density with after 75% alcohol disinfecting, and the diameter of flowerpot is 12-15cm, 60-65d " Invest, Then Investigate " incidence.One strain of getting in disease plant is poured in original flowerpot after grinding equally, and ensure the bacterial content in flowerpot, and reduce the situation that subculture expands the pathogenic decline in numerous process, the soil in this flowerpot will be preserved separately, life-time service.
The concrete grammar flow process of above embodiment 1-7 is shown in the step 1-7 in accompanying drawing 1.
Experimental example 1
Plant seed is selected rye grass respectively, leek, capsicum, white turnip and Chinese cabbage, root sepage is prepared according to the preparation method of the embodiment of the present invention 2, the temperature of preserving controls at 4 DEG C, the root sepage of above-mentioned five kind of plant is adopted to mix with the spore suspension that embodiment 1 is prepared according to the method for embodiment 3 again, the volume ratio of mixing is 1:10 according to the ratio of spore suspension and root sepage, pH value is 6.3, what wherein select rye grass is first group, what select leek is second group, what select capsicum is the 3rd group, what select white turnip is the 4th group, what select Chinese cabbage is the 5th group, and get respectively nutritive medium solution and distilled water in contrast one group with contrast two groups and mix with spore suspension, the volume ratio of mixing is 10:1 according to the ratio with spore suspension, pH value is 6.3, often organizing 2mL adds in 2mL centrifuge tube, often group does 3 Parallel testings, assemble for avoiding statospore precipitation in transfer pipet put procedure, place it in shaking table and preserve (light culture, temperature 25 DEG C), the interval 24h sprouting situation of spore under biology microscope sem observation different treatment, each Duplicate Samples observes 3 times, each counting 100 spores, observe 7 days altogether, concrete outcome sees the following form 1.
As can be seen from following table 1, the statospore that front 48h respectively organizes is sprouted and is not especially significantly changed, from the 3rd day, use the statospore germination rate after rye grass, capsicum and white turnip root sepage process to have significant raising, leek and Chinese cabbage started there has been considerable change at the 4th day.Statospore germination rate after rye grass process was up to 93.00% at the 7th day, and it sprouts speed is also the highest; The effect of leek slightly lower than rye grass, but more a lot of than Chinese cabbage; The result of capsicum is similar with the effect of nutritive medium; Statospore after white turnip process reached 84.00% of climax by the 6th day; Use the statospore germination rate of nutritive medium process also to have a higher growth at the 3rd day, the statospore germination rate effect of distilled water process of comparing still clearly.Can find out that from accompanying drawing 2 in 7 days, the sepage of several plant root affects the sprouting of statospore more intuitively.Visible use of the present invention sepage can effectively promote that plasmodiophora brassicae sprouts, and then success ratio and sickness rate are infected in raising.
The impact that table 1 different roots of plants sepage process is sprouted statospore
Different small English alphabet is the significant difference in P < 0.05 level
Experimental example 2
Operate according to embodiment 1-6, the plant seed wherein selected in embodiment 2 is rye grass, detects the success ratio of inoculation of embodiment 6, and carry out 5 groups of experiments altogether, concrete outcome sees the following form 2.
Table 2 success ratio of inoculation cartogram
| Group | Inoculation monospore quantity | Morbidity number | Success ratio (%) |
| 1 | 66 | 4 | 6.06 |
| 2 | 66 | 3 | 4.55 |
| 3 | 66 | 5 | 7.58 |
| 4 | 66 | 4 | 6.06 |
| 5 | 66 | 6 | 9.09 |
As can be seen from Table 2, the success ratio of inoculation of the embodiment of the present invention is generally more than 4.5%, reach as high as 9.09%, but in prior art, as Li Qian, Shen Xiangqun, Li Lin, in this section of document of the qualification of the inoculation of rape plasmodiophora brassicae single spore separation and physiological strain, the highest success ratio of inoculation only has 3.96%.
Experimental example 3
Operate according to embodiment 1-7, the plant seed wherein selected in embodiment 2 is rye grass, to embodiment 7 host expand numerous after sickness rate detect, carried out three generations altogether and expanded numerous, concrete outcome sees the following form 3.
Table 3 morbidity statistics table
| Group | Survive strain number (individual) | Morbidity strain number (individual) | Sickness rate (%) |
| The first-generation | 25 | 17 | 68.00 |
| The s-generation | 33 | 21 | 63.64 |
| The third generation | 31 | 27 | 87.10 |
As can be seen from upper table 3, each sickness rate is 68.0%, 63.6% and 87.10%, has occurred the morbidity situation risen.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. a method for plasmodiophora brassicae single spore separation, is characterized in that, comprises the steps:
(A) root nodule of crop in cruciferae being suffered from club root obtains spore suspension after become thoroughly decomposed, add water grinding and filtering separation step;
(B) strength of solution that above-mentioned steps obtains is controlled 1 × 10
7-3 × 10
7individual spore/ml, and then doubly obtain dilution spore suspension with distilled water diluting 30-50;
(C) with acupuncture needle in described dilution spore suspension after picking spore by being pressed on agar block, and on described agar block, only have the laggard row labels of spore to retain with observation by light microscope.
2. the method for plasmodiophora brassicae single spore separation according to claim 1, is characterized in that, in described step (C), by being pressed on the edge of agar block after picking spore.
3. the method for the plasmodiophora brassicae single spore separation according to any one of claim 1-2, is characterized in that, described step (C) is carried out in gnotobasis.
4. adopt the host of the plasmodiophora brassicae single spore separation method described in any one of claim 1-3 to expand numerous method setting up monospore system, it is characterized in that, comprise the steps:
(D) the described agar block containing single spore is placed on the crop in cruciferae seedling root hair of aseptically process, cultivates the root nodule that for some time obtains suffering from club root;
(E) soil is cultivated by pouring in sterilized soil to be formed after described root nodule grinding, crop in cruciferae seed through aseptically process is sowed on described cultivation soil, cultivation for some time obtains the root nodule of suffering from club root, pours in described cultivation soil to ensure the plasmodiophora brassicae content that described cultivation is native and life-time service after taking out the grinding of part root nodule.
5. host according to claim 4 expands numerous method setting up monospore system, it is characterized in that, between described step (A) and described step (B), get root sepage after also comprising the steps: that plant seed is carried out cultivation for some time after disinfection, the volume ratio of described sepage and described spore suspension is controlled to obtain solution between the ratio of 1:8-10.
6. host according to claim 5 expands numerous method setting up monospore system, it is characterized in that, described plant seed is wherein a kind of in the seed of rye grass, leek, capsicum, white turnip and Chinese cabbage.
7. host according to claim 5 expands numerous method setting up monospore system, it is characterized in that, described solution preserves 48-50h under the dark condition of 25-30 DEG C; Preferably, the pH value of described solution controls at 6-7.
8. host according to claim 5 expands numerous method setting up monospore system, it is characterized in that, in described step (D), before agar block containing single spore being placed in the step on the crop in cruciferae seedling root hair of aseptically process, described method also comprises: on described crop in cruciferae seedling root hair, drip described sepage.
9. host according to claim 4 expands numerous method setting up monospore system, it is characterized in that, in described step (D), the concrete steps of cultivation are: in constant temperature growth cabinet, cultivate 40-50h under the condition of dark, then on seedling pan, cultivate 60-65d;
Preferably, in described step (E), the time of cultivation is 60-65d.
10. the host according to any one of claim 4-9 expands numerous method setting up monospore system, it is characterized in that, described crop in cruciferae selects precocious No. five Chinese cabbages.
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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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| CN107245452A (en) * | 2017-04-28 | 2017-10-13 | 沈阳农业大学 | A kind of single spore separation method of Chinese cabbage plasmodiophora brassicae biological strain |
| CN107245452B (en) * | 2017-04-28 | 2019-10-01 | 沈阳农业大学 | A kind of single spore separation method of Chinese cabbage plasmodiophora brassicae biological strain |
| CN110029083A (en) * | 2019-04-01 | 2019-07-19 | 中国农业科学院蔬菜花卉研究所 | A method of rape plasmodiophora brassicae monospore is separated based on methylene blue agarose method |
| CN110029083B (en) * | 2019-04-01 | 2020-08-18 | 中国农业科学院蔬菜花卉研究所 | Method for separating plasmodiophora brassicae monospores based on methylene blue agarose method |
| 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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