CN105165604B - A kind of application for identifying soybean to the method for soybean blight resistance and its in breeding - Google Patents
A kind of application for identifying soybean to the method for soybean blight resistance and its in breeding Download PDFInfo
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- CN105165604B CN105165604B CN201510671821.2A CN201510671821A CN105165604B CN 105165604 B CN105165604 B CN 105165604B CN 201510671821 A CN201510671821 A CN 201510671821A CN 105165604 B CN105165604 B CN 105165604B
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Abstract
The present invention provides a kind of application for identifying soybean to the method for soybean blight resistance and its in breeding.Methods described includes step：Soybean phytophthora bacterial strain is inoculated in CA culture mediums, V8 culture mediums or butter bean culture medium, light culture makes bacterial strain rejuvenation, and the medium treatment that thalline is will be enriched in afterwards is broken, produces inoculum；(2) it is inoculated with soybean plant strain material：Inoculum obtained by step (1) is contained in detection container, the just open and flat green band handle blade of soybean plant strain to be checked is chosen, petiole is inserted in the inoculum in the detection container；(3) light culture：Step (2) is vaccinated with the detection container of soybean material and is placed in the confined space of moistening judged result after light culture.The authentication method of the present invention is easy to operate, with low cost, saves the time, and may be repeated experiment, while experiment material to be identified will not be lost, it is to avoid conventional method can cause the drawbacks of experiment material group is lost.
Description
Technical field
The invention belongs to breeding for disease resistance technical field, and in particular to a kind of identification soybean to the method for soybean blight resistance and
Its application in soybean breeder.
Background technology
The soybean blight as caused by soybean phytophthora (Phytophthora sojae Kaufmann＆Gerdemann) is soybean
A kind of crushing soil-borne disease in production.The disease has generation in the multiple provinces of China.Soybean phytophthora is in all lifes of soybean
Soybean can be infected by educating the phase, cause root-rot, stem rot, plant to downgrade, wither and dead, general field underproduction 10%-30%, sternly
Important place block is up to 60%-90%, or even causes total crop failure.The annual economic loss caused by soybean blight in the whole world is about at present
1000000000 dollars (Tyler, 2007).
At present, researcher typically identifies resistance of the soybean to soybean blight by hypocotyl wound inoculation method.Under
Plumular axis inocalation method, can only be carried out in Seedling Stage, and susceptible material is dead after inoculation, causes subsequent experimental not carry out.This lacks
Point is particularly evident in the identification of heterozygosis Soybean Resistance epidemic disease plant F2 colonies, can cause the loss of colony, and can not be repeated
Identification.In consideration of it, being necessary the new method of developmental research to identify resistance of the soybean to soybean blight.
The content of the invention
The invention aims to improve determination rates of the soybean to soybean blight resistance, and avoid conventional identification method
The defect that heterozygosis disease-resistant plant F2 colonies can be caused to lose, and a kind of method of new identification soybean to soybean blight resistance is provided
And its application in soybean breeder.The authentication method of the present invention is easy to operate, with low cost, saves the time, and can carry out
Repeat to test, while experiment material to be identified will not be lost, it is to avoid conventional method can cause experiment material group to lose
Drawback.
One of technical scheme that the present invention is provided is：A kind of identification soybean is to the method for soybean blight resistance, and it is included such as
Lower step：
(1) preparation of inoculum：By soybean phytophthora (Phytophthora sojae Kaufmann＆Gerdemann) bacterial strain
CA culture mediums, V8 culture mediums or butter bean culture medium are inoculated in, light culture makes bacterial strain rejuvenation, and the culture medium of thalline is will be enriched in afterwards
Processing is broken, produces inoculum；
(2) it is inoculated with soybean plant strain material：Inoculum obtained by step (1) is contained in detection container, chooses to be checked big
The just open and flat green band handle blade of beans plant, petiole is inserted in the inoculum in the detection container；
(3) light culture：The detection container of soybean material will be vaccinated with step (2) and be placed in the confined space of moistening and secretly trained
Judged result after supporting, the temperature of the confined space is 22~27 DEG C, and the relative humidity of the confined space is 90~100%；
The criterion of the judged result is, after cultivating 3~5 days, if blade and petiole keep the length of scab on green or petiole
Degree is less than or equal to 1.5cm, then soybean plant strain to be checked is disease-resistant plant, to be checked big if the length of scab is more than 1.5cm on petiole
Beans plant is disease plant.
In the present invention, step (1) is the preparation of inoculum.
Wherein, the soybean phytophthora bacterial strain is the soybean phytophthora bacterial strain described in the routine of this area, including various different virulences
The bacterial strain of genotype, such as PsMC1, Ps41-1 and PsUSAR2.
Described CA culture mediums are that this area is conventional described, and its specific formula is：With 1L stereometers, including 200g Hus
Radish and 20g agar.
Described V8 culture mediums are that this area is conventional described, and its specific formula is：With 1L stereometers, wherein containing fruit juice
200g, CaCO33g, agar 20g, remaining is distilled water, pH5.8.
Described butter bean culture medium is that this area is conventional described, and its compound method is：With 1L stereometers, lima bean flour is taken
60g, add water 1000ml, in 60 DEG C of water-bath 1h, double gauze filter and remove residue, and supernatant complements to 1000ml, adds agar 20g,
Boil rear constantly boiling several minutes, cooling is produced.
Before soybean phytophthora bacterial strain is inoculated in into CA culture mediums, preferably also include carrying out the soybean phytophthora bacterial strain
The step of separation and purifying (S)：By soybean phytophthora bacterial strain on CA culture mediums 7~10d of light culture, cultivation temperature be 22~27 DEG C
(preferably 25 DEG C), be inoculated with afterwards susceptible soybean varieties seedling recover virulence, plant fall ill after carry out pathogen separation and
Purifying, is used for subsequent inoculations by obtained soybean phytophthora bacterial strain.
The temperature of light culture described in step (1) is preferably 22~27 DEG C (preferably 25 DEG C)；The time of the light culture
Preferably 7~10 days.
The broken finger of the medium treatment that will be enriched in thalline described in step (1) is using the feasible method of any routine in this area
The CA medium treatments that will be enriched in soyabean phytophthora thalline are broken (preferably to uniform), can such as stir in small, broken bits uniform,
It can smash to pieces.It is preferred that the medium treatment for being processed as will be enriched in thalline using soy bean milk making machine is broken.
In the present invention, step (2) is inoculation soybean plant strain material.
Wherein, described detection container can be any conventional container in this area, such as EP pipes, small test tube, centrifuge tube
, preferred 2~5mL centrifuge tube.
The just open and flat green band handle blade of described soybean plant strain to be checked can be any blade of plant proper site,
As long as its healthy, children is tender and in green, the green compound leaf of 4cm petiole is preferably more than with length.Such as this area is conventional,
The green band handle blade is before inoculation, and petiole position generally requires progress explant sterilization, is infected with antiforeign bacteria.It is preferred that
The green compound leaf removes partial leaflet (preferably removing side life leaflet, retain middle leaflet) before inoculation.
After in the inoculum that petiole inserts in the detection container, it is preferred that using wet absorbent cotton in vessel port to leaf
Handle is fixed, and the wound for removing leaflet formation is completely cut off air by the fixation simultaneously.
In the present invention, step (3) is light culture.
Wherein, the confined space can be the conventional sterilized space in this area, such as can be from the larger plastics of specification
Box.In concrete operations, then the detection container (such as centrifuge tube) that step (2) first can be inoculated with into soybean plant strain material is placed in cold
Freeze box, then freeze box be placed in big plastic casing, finally by big plastic casing be placed in hot and humid environment (preferably 25 DEG C, 90~
100%RH) carry out light culture.
One preferred embodiments of the authentication method comprise the following steps：
(1) preparation of inoculum：By soybean phytophthora bacterial strain on CA culture mediums 7~10d of light culture, cultivation temperature is 25
DEG C, the seedling that susceptible soybean varieties are inoculated with afterwards recovers virulence, and the separation and purifying of pathogen are carried out after plant falls ill, will be pure
Change obtained soybean phytophthora bacterial strain and be inoculated in CA culture mediums, 7~10d of light culture will be enriched in the culture medium soya-bean milk of thalline afterwards
Machine is smashed to uniform, produces inoculum；
(2) it is inoculated with soybean plant strain material：Inoculum obtained by step (1) is contained in 2mL uncapping centrifuge tubes, selection is treated
Inspection soybean plant strain has the just open and flat green compound leaf of petiole of the length more than 4cm, and the leaflet of the compound leaf both sides is removed,
Petiole is inserted in the inoculum in the centrifuge tube, petiole is fixed in the centrifugation mouth of pipe with the absorbent cotton of moistening and simultaneously will
Remove the wound isolation air of leaflet formation；
(3) light culture：The centrifuge tube that soybean material is vaccinated with step (2) is put into freeze box, by the freeze box
It is placed on bottom to be covered with the sealed plastic box of moistening sponge, the sealed plastic box is placed in into 25 DEG C of light cultures judges to tie for 4 days
Really, the criterion of the judged result is, after cultivating 4 days, if blade and petiole keep the length of scab on green or petiole
Degree is less than or equal to 1.5cm, then soybean plant strain to be checked is disease-resistant plant, to be checked big if the length of scab is more than 1.5cm on petiole
Beans plant is disease plant.
The two of the technical scheme that the present invention is provided are：Aforementioned identification soybean is to the method for soybean blight resistance in soybean breeder
In application.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and produce each preferable reality of the present invention
Example.
Agents useful for same and raw material of the present invention are commercially available.
The positive effect of the present invention is：
The authentication method of the present invention has following advantages：1) growth of soybean plant strain is not influenceed；2) easy to operate, cost is low
It is honest and clean；3) experiment condition controllability is high, tests the very little that takes up space, and takes short；4) it can be carried out in soybean most breeding times
Identification experiment, can carry out repeating experiment；5) identification to multiple soybean phytophthora bacterial strains can be carried out simultaneously；6) it will not lose and treat
The experiment material of identification, it is to avoid conventional method can cause the drawbacks of experiment material group is lost.Therefore, identification side of the invention
Method has broad application prospects in soybean breeder.
Brief description of the drawings
Fig. 1 is the schematic diagram after the green band handle compound leaf chosen removes both sides leaflet in embodiment 1, and will be eliminated
The schematic diagram after petiole insertion inoculum after leaflet.
Fig. 2 is the schematic diagram that embodiment 1 observes susceptible or disease-resistant situation after petiole light culture is inoculated with 4 days, wherein, the left side
Blade be have determined that be disease-resistant plant state, the right blade be have determined that be susceptible plant state.
Embodiment
Following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.In the present invention, except special theory
Outside bright, all devices, reagent and material are that conventional commercial can be obtained.
A kind of method for identifying soybean to soybean blight resistance of embodiment 1
(1) preparation of soybean phytophthora inoculum
Soybean phytophthora bacterial strain (52696TM) be stored on CA culture mediums, bacterial strain is transferred to containing 15ml during experiment
In the culture dish of the CA culture mediums of 1.5% agar, 25 DEG C of dark culturings 7-10 days, culture environment relative humidity is 90~100%.
Being inoculated with susceptible variety Williams, (kind is in " Genetic Analysis of Soybean Plant Introductions
with Resistance to Phytophthora sojae,Gordon SG,et al.Phytopathology,2007Jan；
97(1):Have disclosure in 106-12 ", be conventional soy susceptible variety) seedling recovery virulence, carry out pathogen after plant morbidity
Separation and purifying, for being inoculated with.The last week is inoculated with, bacterial strain is transferred to the CA culture mediums containing 1.5% agar containing 15ml
In culture dish, 25 DEG C of dark culturings 7-10 days, culture environment relative humidity is 90~100%., will with nine positive soy bean milk making machines during inoculation
Culture medium containing mycelia smashes preparation inoculum, is dispensed into 5ml syringe, is inoculated with standby.
(2) it is inoculated with soybean plant strain material：The young tender compound leaf of one, clip soybean plant strain top health, stays 4cm petioles, by both sides
Leaflet cut off (as shown in Figure 1).
(3) light culture
0.5ml inoculum is injected in the 2ml centrifugation bottom of the tube of uncapping with syringe, the petiole insertion of soybean leaves is connect
Plant in body, at the centrifugation mouth of pipe, wrap up petiole (as shown in Figure 1) with the cotton of moistening, play fixed and moisture-keeping function.Then will be from
Heart pipe is put into the freeze box of 100 lattice.Freeze box is put into bottom to be covered with the big plastic casing of moistening sponge, box top is with one layer
Plastic cloth sealing is tight, and Disease investigation (as shown in Figure 2) is carried out after being put into 25 DEG C of culturing room's moisturizing cultures 4 days.Record blade and leaf
Handle state, if petiole has infected, the length of scab on record petiole.
Choose the above-mentioned experiment of progress (as shown in table 1) of 13 soybean lines, the hypocotyl wound inoculation method of early stage
(Schmitthenner,A.F.,&Bhat,R.G.(1994).Useful methods for studying Phytophthora
in the laboratory.Special Circular-Ohio Agricultural Research and Development
Center, (143)) show, wherein 7 strains show resistance to 3 soybean phytophthora bacterial strains, Henan beans 25 to PsUSAR2 and
PsMC1 shows resistance, and new soya bean only shows resistance to PsMC1, and also 4 strains are equal to 3 soybean phytophthora bacterial strains
Show susceptible, the qualification result of petiole inocalation method is consistent therewith.After statistical analysis, draw and judge susceptible and disease-resistant as follows
Standard is：After step (3) light culture 4 days, if blade and petiole keep green, or the of length no more than 1.5cm of petiole scab
It is then disease-resistant plant (R)；If petiole browning softens, and it is then disease plant (S) that scab length, which is more than 1.5cm,.
The result that each soybean line of table 1 is identified with hypocotyl wound inoculation method and petiole inocalation method
A kind of method for identifying soybean to soybean blight resistance of embodiment 2
(1) preparation of soybean phytophthora inoculum
Soybean phytophthora bacterial strain is stored on carrot juice agar (CA) culture medium, and bacterial strain is transferred to containing 15ml's during experiment
In the CA medium culture wares of 1.5% agar, 22 DEG C of dark culturings 7-10 days, inoculation susceptible variety Williams seedling recovers poison
Power, carries out pathogenicbacteria separation and purifying, for being inoculated with after plant morbidity.The last week is inoculated with, bacterial strain is transferred to containing containing 15ml
Have in the culture dish of CA culture mediums of 1.5% agar, 27 DEG C of dark culturings 7-10 days.During inoculation, it will be contained with nine positive soy bean milk making machines
The culture medium of mycelia smashes preparation inoculum, is dispensed into 5ml syringe, is inoculated with standby.
(2) it is inoculated with soybean plant strain material：The young tender compound leaf of one, clip soybean plant strain top health, stays 4cm petioles, by both sides
Leaflet cut off.
(3) light culture
0.5ml inoculum is injected in the 2ml centrifugation bottom of the tube of uncapping with syringe, the petiole insertion of soybean leaves is connect
Plant in body, at the centrifugation mouth of pipe, wrap up petiole with the cotton of moistening, play fixed and moisture-keeping function.Then centrifuge tube is put into 100
In the freeze box of lattice.Freeze box is put into bottom to be covered with the big plastic casing of moistening sponge, box top is sealed with one layer of plastic cloth
Tightly, Disease investigation is carried out after being put into 25 DEG C of culturing room's moisturizing cultures 4 days.Blade and petiole state are recorded, if petiole has infected, note
Record the length of scab on petiole.
Judge susceptible and disease-resistant standard judged result according to what embodiment 1 was drawn, in these plant strain growths for a period of time
Post-sampling carries out repeating experiment, as a result shows that the result for repeating experiment is consistent with the testing result of early stage, same in be the same as Example 1
Ground, result that these tested plant are measured in early stage with hypocotyl wound inoculation method and in the present embodiment with petiole inocalation method institute
Obtain result consistent.This shows the authentication method and its criterion of the present invention accurately and reliably.
Although above having made to retouch in detail to the present invention with general explanation, embodiment and experiment
State, but on the basis of the present invention, it can be made some modifications or improvements, this is aobvious and easy to those skilled in the art
See.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed
Scope.
Claims (8)
1. a kind of method for identifying soybean to soybean blight resistance, it is characterised in that it comprises the following steps：
(1) preparation of inoculum：Soybean phytophthora (Phytophthora sojae Kaufmann ＆Gerdemann) bacterial strain is connect
Plant in CA culture mediums, V8 culture mediums or butter bean culture medium, light culture makes bacterial strain rejuvenation, at the culture medium that thalline is will be enriched in afterwards
Reason is broken, produces inoculum；
(2) it is inoculated with soybean plant strain material：Inoculum obtained by step (1) is contained in detection container, soybean to be checked is chosen and plants
The just open and flat green band handle blade of strain, petiole is inserted in the inoculum in the detection container；
Wherein, described green band handle blade is the green compound leaf for the petiole for being more than 4cm with length；The green compound leaf is connecing
Before kind, remove partial leaflet;
(3) light culture：It is placed in the detection container of soybean material is vaccinated with step (2) in the confined space of moistening after light culture
Judged result, the temperature of the confined space is 22~27 DEG C, and the relative humidity of the confined space is 90~100%；It is described
The criterion of judged result is, after cultivating 3~5 days, if blade and petiole keep the length of scab on green or petiole small
In equal to 1.5cm, then soybean plant strain to be checked is disease-resistant plant, if the length of scab is more than 1.5cm on petiole, soybean to be checked is planted
Strain is disease plant.
2. according to the method described in claim 1, it is characterised in that trained soybean phytophthora bacterial strain is inoculated in into CA culture mediums, V8
Before supporting base or butter bean culture medium, in addition to the step of the soybean phytophthora bacterial strain is separated and purified (S)：By soybean
Mtr mutant 7~10d of light culture on CA culture mediums, cultivation temperature is 22~27 DEG C, and the children of susceptible soybean varieties is inoculated with afterwards
Seedling recovers virulence, and the separation and purifying of pathogen are carried out after plant falls ill, obtained soybean phytophthora bacterial strain is used for into step (1)
Inoculation.
3. according to the method described in claim 1, it is characterised in that the temperature of light culture described in step (1) is 22~27 DEG C；
The time of the light culture is 7~10 days.
4. according to the method described in claim 1, it is characterised in that will be enriched at the culture medium of thalline described in step (1)
Broken reason is that the medium treatment that will be enriched in thalline using soy bean milk making machine is broken.
5. according to the method described in claim 1, it is characterised in that described detection container is 2~5mL centrifuge tube.
6. according to the method described in claim 1, it is characterised in that in step (2), inserted in petiole in the detection container
After in inoculum, petiole is fixed in vessel port using wet absorbent cotton.
7. according to the method described in claim 1, it is characterised in that it comprises the following steps：
(S) separation and purifying of soybean phytophthora bacterial strain：By soybean phytophthora bacterial strain on CA culture mediums 7~10d of light culture, culture temperature
Spend for 25 DEG C, the seedling that susceptible soybean varieties are inoculated with afterwards recovers virulence, the separation of pathogen is carried out after plant falls ill and pure
Change, the soybean phytophthora bacterial strain purified；
(1) preparation of inoculum：Obtained soybean phytophthora bacterial strain will be purified and be inoculated in CA culture mediums, 7~10d of light culture, afterwards
The culture medium that will be enriched in thalline is broken into homogenate with soy bean milk making machine, produces inoculum；
(2) it is inoculated with soybean plant strain material：Inoculum obtained by step (1) is contained in 2mL uncapping centrifuge tubes, chooses to be checked big
The green compound leaf of the just open and flat petiole that there is length to be more than 4cm, the leaflet of the compound leaf both sides is removed on beans plant, will
Petiole is inserted in the inoculum in the centrifuge tube, petiole is fixed in the centrifugation mouth of pipe while will go with the absorbent cotton of moistening
Except the wound of leaflet formation completely cuts off air；
(3) light culture：The centrifuge tube that soybean material is vaccinated with step (2) is put into freeze box, the freeze box is placed
In the sealed plastic box that moistening sponge is covered with bottom, the sealed plastic box is placed in 25 DEG C of light cultures, 4 days judged results, institute
The criterion for stating judged result is, after cultivating 4 days, if blade and petiole keep the length of scab on green or petiole to be less than
Equal to 1.5cm, then soybean plant strain to be checked is disease-resistant plant, if the length of scab is more than 1.5cm, soybean plant strain to be checked on petiole
For disease plant.
8. application of any one of the claim 1~7 identification soybean to the method for soybean blight resistance in soybean breeder.
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| CN108103140B (en) * | 2017-11-09 | 2021-04-06 | 广东省农业科学院蔬菜研究所 | Method for rapidly identifying resistance of cucumber to epidemic disease |
| CN110199711B (en) * | 2019-06-20 | 2021-11-05 | 黑龙江省农业科学院耕作栽培研究所 | Accurate identification method for soybean pythium root rot resistance resources |
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