CN110501295A - A kind of method of indoor quickly screening prevention and treatment Ustilago maydis medicament - Google Patents
A kind of method of indoor quickly screening prevention and treatment Ustilago maydis medicament Download PDFInfo
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Abstract
The present invention provides a kind of methods of indoor quickly screening Ustilago maydis pesticide control, belong to maize diseases Prevention Technique field, it the described method comprises the following steps: preparing a series of potato dextrose broths containing different effective component concentration medicaments to be measured, the Ustilago maydis bacterial strain of activation is moved into above-mentioned pastille culture medium, with initial light absorption value OD of the microplate reader measurement bacterium solution at 590nm1With the final light absorption value OD after culture 16-20h2；The increasing value Δ OD for calculating each processing bacterium solution OD, calculates inhibiting rate；Using the logarithm of fungicide effective component concentration as independent variable, inhibiting rate probability value are dependent variable, calculate virulence regression equation, and find out concentration EC in inhibition50.Method time-consuming of the invention is short, easy to operate, data are reliable, repeatability is strong, is highly suitable for the screening to Ustilago maydis fungicide, has very strong practicability.
Description
Technical field
The present invention relates to maize diseases Prevention Technique fields, and in particular to a kind of indoor quickly screening prevention and treatment black powder of corn tumor
The method of sick medicament.
Background technique
Ustilago maydis (or corn smut) is by Ustilago maydis (Ustilago maydis), Huo Chengwei
Maize diseases caused by ustilago zeae, corn tumor smut etc., take place mostly in warm Arid Area, and spring sowing area compares summer sowing
Area's morbidity is serious, and general disease incidence is 5~10%, it is serious cause 30~80% production loss, be China the Yellow River and Huai He River sea area,
NORTHEAST REGION IN and Major Diseases in the maize production of northwest and area's corn variety carry out the disease of evaluation of resistance when authorizing
Evil.
It is main for the prevention and treatment of the disease at present due to lacking the specificity fungicide for Ustilago maydis in the market
It aims at prevention.But the pesticide control that the prevention and treatment for Ustilago maydis, exploitation and screening are suitable for is still largely effective
And a kind of essential means.With the increase of environment supervision and pesticide market competitive pressure, market is for effectively anti-
The demand for controlling the new pesticide of Ustilago maydis is also more more and more urgent.Due to the black powder of pathogenic bacteria maize of Ustilago maydis
Bacterium is yeast shape fungi, and bacterium colony is not in the radial growth of rule, and slow growth, using traditional on plating medium
Pastille culture medium right-angled intersection mensuration carries out toxicity test to it, and not only minute is long, and the accuracy of measurement is also difficult to protect
Card.The slide used in other researchs sprouts method, and not only cumbersome and observation spore sample is small, and evaluating drug result is persuaded
Power is limited.Therefore, a set of accurately and reliably indoor medicament rapid screening method for the disease is researched and developed, is conducive to improve
For the field reagent screening efficiency of the disease, the successful screening rate of new pesticide is improved, there is weight for the prevention and treatment of the disease
The practical study value wanted.
Summary of the invention
The purpose of the present invention is to provide a kind of method of indoor quickly screening Ustilago maydis pesticide control, this method
Have the characteristics that fast and easy, result are accurate and reliable, effectively can provide reference for field reagent screening.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of methods of indoor quickly screening Ustilago maydis pesticide control, comprising the following steps:
1) a series of potato dextrose broths containing different effective component concentration medicaments to be measured are prepared, will be lived
The Ustilago maydis bacterial strain of change moves into above-mentioned pastille culture medium, so that the concentration of pastille culture medium miospore is 1 × 105-1
×106A/ml, with initial light absorption value OD of the microplate reader measurement bacterium solution at 590nm1；
2) by the above-mentioned fluid nutrient medium containing Ustilago maydis bacterial strain under dark condition shaken cultivation 16-20h, use
Microplate reader measures final OD2；
3) the increasing value Δ OD for calculating each processing bacterium solution OD, calculates inhibiting rate；With the logarithm of fungicide effective component concentration
Value is independent variable, and inhibiting rate probability value are dependent variable, calculates virulence regression equation, and find out concentration EC in inhibition50。
Preferably, the activation step of Ustilago maydis bacterial strain described in step 1) of the present invention are as follows: by Ustilago maydis
Strain inoculated vibrates activation culture under dark condition and obtains activated strains in potato dextrose broth.
It is further preferred that the temperature for vibrating activation culture described in step 1) of the present invention is 25 DEG C -28 DEG C, frequency is
150-250r/min, time 16-20h.
Preferably, OD described in step 2) of the present invention2The range of value is between 0.8-1.
Preferably, the temperature of shaken cultivation described in step 2) of the present invention is 25 DEG C -28 DEG C, frequency 150-250r/
min。
Preferably, in step 3) of the present invention, the inhibiting rate calculates according to the following formula: inhibiting rate (%)=[(control bacterium solution Δ
OD- handles bacterium solution Δ OD)/control bacterium solution Δ OD] * 100%.
Preferably, the constituent and its weight ratio of potato dextrose broth described in step 1) of the present invention
Are as follows: potato 200g, glucose 20g, distilled water 1000ml.
It is further preferred that the preparation method of potato dextrose broth of the present invention includes: by potato
It is cut into small pieces after peeling, is put into distilled water and boils, after filtered through gauze, glucose is proportionally added, it is fixed boils rear distilled water
Hold to 1000ml, sterilizes after packing.
Still more preferably, the sterilising conditions are as follows: 121 DEG C of sterilizing 20min.
Preferably, microplate reader measurement OD value of the present invention repeats 2~4 times.
Beneficial effects of the present invention:
(1) detection and the poison of a kind of effect of chemical control can be completed in method fast and easy of the invention in 20 hours
The measurement of force curve, and 10-15d is needed using traditional right-angled intersection rule, it is extremely easy to pollute since incubation time is long
Situations such as lead to the failure of an experiment.
(2) present invention prevention and treatment Ustilago maydis Chemicals method testing result is accurate and reliable, and repeatability is strong, Ke Yiyou
Effect ground provides reference for field reagent screening.
(3) present invention measures strain cultured solution OD value using microplate reader to judge that fungicide imitates the inhibition of strain growth
Fruit, not only easy to operate but also more objective and accurate compared with slide sprouts method, repeatability is strong, and data are reliable.
Method time-consuming of the invention is short, easy to operate, data are reliable, repeatability is strong, is highly suitable for the black powder of corn tumor
The screening of sick fungicide has very strong practicability.
Specific embodiment
The present invention provides a kind of methods of indoor quickly screening Ustilago maydis pesticide control, comprising the following steps:
1) a series of potato dextrose broths containing different effective component concentration medicaments to be measured are prepared, will be lived
The Ustilago maydis bacterial strain of change moves into above-mentioned pastille culture medium, so that the concentration of pastille culture medium miospore is 1 × 105-1
×106A/ml, with initial light absorption value OD of the microplate reader measurement bacterium solution at 590nm1；
2) by the above-mentioned fluid nutrient medium containing Ustilago maydis bacterial strain under dark condition shaken cultivation 16-20h, use
Microplate reader measures final OD2；
3) the increasing value Δ OD for calculating each processing bacterium solution OD, calculates inhibiting rate；With the logarithm of fungicide effective component concentration
Value is independent variable, and inhibiting rate probability value are dependent variable, calculates virulence regression equation, and find out concentration EC in inhibition50。
The present invention is not particularly limited the source of Ustilago maydis, the black powder of maize of taken outdoors or indoor preservation
Bacterium.Ustilago maydis used in the specific embodiment of the invention is the Ustilago maydis bacterial strain that indoor low temperature saves.
The present invention preferably activates Ustilago maydis.Preferably, the present invention is by the maize of cryo-conservation
Smut strain inoculated vibrates activation culture in potato dextrose broth.The present invention preferably vibrates activation culture
It is carried out under dark condition.The temperature of activation culture is preferably 25 DEG C -28 DEG C, and further preferably 26 DEG C -27 DEG C.The oscillation
Frequency be preferably 150-250r/min, further preferably 200r/min.The present invention preferred activation culture time is 16-20h,
It is further 17-19h.Activation Ustilago maydis bacterial strain is obtained after activation culture.
In the present invention, the constituent and its weight ratio of potato dextrose broth are preferred are as follows: potato 200g,
Glucose 20g, distilled water 1000ml.Those skilled in the art can prepare potato grape according to the conventional method in this field
Sugar liquors culture medium.In the present invention, it is preferred to be prepared in accordance with the following methods: will be cut into small pieces after peeling potatoes, be put into
It is boiled in distilled water, after filtered through gauze, glucose is proportionally added, boiled rear distilled water and be settled to 1000ml, go out after packing
Bacterium.The preferred potato boiling time of the present invention is 20-40min.The present invention is preferably filtered with 3-4 layers of gauze.This field skill
Art personnel can select packing size according to the actual situation.It is preferably packed as in the specific embodiment of the invention in every 50ml triangular flask
Dispense 15ml culture medium.In the present invention, the condition of the sterilizing is preferred are as follows: 121 DEG C of sterilizing 20min.
The present invention prepares a series of potato dextrose broths containing different effective component concentration medicaments to be measured,
Preparation method is prepared according to the conventional method in this field.The present invention is not particularly limited the type of medicament to be measured, adopts
Known in this field or unknown fungicide.The effective component concentration of medicament to be measured is according to medicament to Ustilago maydis
Inhibitory effect selectively adjusts.In the specific embodiment of the invention select this field in conventional sterilization agent thiram, Tebuconazole,
Pyraclostrobin, Fluoxastrobin, propiconazole and fluorine azoles ring bacterium amine, above-mentioned medicament no strongly absorbing region at wavelength 590nm will not be right
The measurement of bacterium solution OD value has an impact.
The present invention moves into the Ustilago maydis bacterial strain of activation in above-mentioned pastille culture medium, so that spore in pastille culture medium
The concentration of son is 1 × 105-1×106A/ml, with initial light absorption value OD of the microplate reader measurement bacterium solution at 590nm1.The present invention will
Fluid nutrient medium containing Ustilago maydis bacterial strain shaken cultivation 16-20h under dark condition is measured final with microplate reader
OD2.The temperature of the preferred shaken cultivation of the present invention is 25 DEG C -28 DEG C, further preferably 26 DEG C -27 DEG C.The present invention is preferably described
The frequency of shaken cultivation is 150-250r/min, further preferably 200r/min.The present invention is 16- in the shaken cultivation time
In 20h, the OD2The range of value improves the accuracy of microplate reader measurement result between 0.8-1.The present invention is surveyed with microplate reader
It measures OD value to carry out according to the routine operation in this field, the present invention preferred microplate reader measurement OD value repetition 2~4 times, preferably 3
It is secondary.
The present invention calculates increasing value the Δ OD, Δ OD=OD of each processing bacterium solution OD according to above-mentioned measurement result2-OD1。
The present invention calculates inhibiting rate according to the following formula:
Inhibiting rate (%)=[(control bacterium solution Δ OD- handles bacterium solution Δ OD)/control bacterium solution Δ OD] * 100%.
Using the logarithm of fungicide effective component concentration as independent variable, inhibiting rate probability value are dependent variable, calculate virulence and return
Return equation, and finds out concentration EC in inhibition50.The calculation of concentration is according to this field in above-mentioned virulence regression equation and inhibition
In conventional method carry out.
The method fast and easy of lab screening Ustilago maydis pesticide control of the present invention, can be completed one in 20 hours
The detection of kind effect of chemical control and the measurement of virulence curve, testing result is accurate and reliable, and repeatability is strong, is highly suitable for jade
The screening of rice gall smut fungicide, has very strong practicability.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Ustilago maydis bacterial strain used is that Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie saves in following embodiments
Existing bacterial strain, but should not be understood as the restriction to technical solution of the present invention.Technical solution of the present invention can measure medicament to appoint
The control efficiency of the Ustilago maydis bacterial strain in meaning source.
1 Ustilago maydis pesticide control screening process of embodiment
(1) it prepares potato dextrose broth: will be cut into small pieces after potato 200g peeling, be put into 800ml steaming
It boils 20min in distilled water, after 4 layers of filtered through gauze, glucose 20g is added, 1000ml, 121 DEG C of sterilizings are settled to after boiling
20min。
By the Ustilago maydis strain inoculated of cryo-conservation in potato dextrose broth, in dark condition
Under, 25 DEG C -28 DEG C, 200r/min culture 16-20h obtain activated strains.
(2) a series of potato glucose liquid containing different effective component concentration medicaments to be measured are prepared according to table 1 to train
Base is supported, is respectively charged into triangular flask, activated strains obtained in a certain amount of step (1) is drawn with pipettor and moves into above-mentioned triangular flask
In, make the concentration 1 × 10 of spore in the medium5-1×106A/ml is added and medicament equivalent aqua sterilisa in control group.With
Microplate reader measures initial light absorption value OD of the bacterium solution at 590nm1.Triangular flask is placed in shaking table, under dark condition, 25 DEG C-
28 DEG C, 200r/min culture 16-20h, measure final OD with microplate reader2, 3 repetitions of each sample are averaged.
(3) the increasing value Δ OD for calculating each processing bacterium solution OD, calculates inhibiting rate.With fungicide effective component concentration (mg/L)
Logarithm be independent variable, inhibiting rate probability value be dependent variable, calculate virulence regression equation, and find out inhibition in concentration EC50。
Δ OD=OD2-OD1
Inhibiting rate (%)=[(control bacterium solution Δ OD- handles bacterium solution Δ OD)/control bacterium solution Δ OD] * 100%
1 Select to use medicament of table and concentration
The selection result is as shown in table 2.It can be seen that can effectively distinguish different agents to maize using method of the invention
The virulence effect of smut, EC stronger to the virulence of Ustilago maydis by test Tebuconazole and fluorine azoles ring bacterium amine50Value is only
0.03, and Fluoxastrobin is most weak to Ustilago maydis virulence, EC50Value is 0.34, should preferentially be made in further field trial
It is carried out with stronger fungicide of virulence such as Tebuconazole, fluorine azoles ring bacterium amine.
Table 2 screens measurement of the medicament to Ustilago maydis virulence curve
| Medicament title | Virulence curve | R2 | EC50(mg/L) |
| Thiram | Y=2.1767x+6.4308 | 0.8269 | 0.22 |
| Tebuconazole | Y=1.5971x+7.6519 | 0.9623 | 0.03 |
| Pyraclostrobin | Y=1.6387x+6.4767 | 0.9875 | 0.13 |
| Fluoxastrobin | Y=1.7727x+5.8245 | 0.9367 | 0.34 |
| Propiconazole | Y=0.6987x+6.2802 | 0.9725 | 0.16 |
| Fluorine azoles ring bacterium amine | Y=3.2061x+11.556 | 0.9693 | 0.03 |
The most suitable initial inoculation concentration of embodiment 2 determines test
(1) it prepares potato dextrose broth: will be cut into small pieces after potato 200g peeling, be put into 800ml steaming
It boils 20min in distilled water, after 4 layers of filtered through gauze, glucose 20g is added, 1000ml, 121 DEG C of sterilizings are settled to after boiling
20min。
By the Ustilago maydis strain inoculated of cryo-conservation in potato dextrose broth, in dark condition
Under, 25 DEG C -28 DEG C, 200r/min culture 16-20h obtain activated strains.
(2) maize activated in a certain amount of step (1) is added in the triangular flask that packing has equivalent fluid nutrient medium
Smut culture solution is detected by blood counting chamber and is counted, and making the concentration of Ustilago maydis spore in every bottle is respectively 108、
107、106、105、104With 103A/ml, 3 repetitions of each concentration.28 DEG C, 200r/min are placed in shaking table, 16h is cultivated, is used
Microplate reader measures light absorption value OD of the bacterium solution at 590nm590, it is averaged for each sample duplicate measurements 3 times.It is dense due to solution
The too high or too low accuracy that can influence microplate reader measurement result is spent, therefore we select light absorption value after culture to exist
Processing between 0.8-1 is as optimal initial inoculum density.
Test result: after 16h is cultivated, culture solution OD under different initial concentrations590Value is as shown in table 3 below, as shown in Table 3,
It is 10 in initial inoculation concentration6When, OD590Value is 0.941, the maximum OD value that can accurately measure close to microplate reader, is most suitable
Initial inoculation concentration.
The most suitable initial inoculation concentration of table 3
3 different agents concentration of embodiment influences to test on measurement system precision
(1) it prepares potato dextrose broth: will be cut into small pieces after potato 200g peeling, be put into 800ml steaming
It boils 20min in distilled water, after 4 layers of filtered through gauze, glucose 20g is added, 1000ml, 121 DEG C of sterilizings are settled to after boiling
20min。
By the Ustilago maydis strain inoculated of cryo-conservation in potato dextrose broth, in dark condition
Under, 25 DEG C -28 DEG C, 200r/min culture 16-20h obtain activated strains.
(2) it is added in the fluid nutrient medium that pyraclostrobin concentration is 1mg/l, 0.025mg/l and 0mg/l respectively certain
The bacterium solution activated in step (1) is measured, Ustilago maydis spore count in culture medium is made to reach 1 × 105-1×106A/ml is used
Microplate reader measures and records initial light absorption value OD of each processing at 590 μm1(measure 3 times and be averaged), is placed in 28 in shaking table
DEG C, 200r/min is cultivated 16h, is measured the light absorption value OD of bacterium solution again using microplate reader2(measure 3 times and be averaged) calculates every
The incrementss Δ OD of a sample light absorption value, each processing are repeated 4 times, and calculate the relative standard deviation of each Δ OD under same concentrations
(RSD%).
Δ OD=OD2-OD1
Test result: as shown in Table 4, after various concentration pastille culture medium culture 16h, OD value variation delta between each repetition
OD is as shown in the table, and pastille culture medium group relative standard deviation is not smaller, and pastille culture medium group relative deviation is larger.But each deviation
Value all in allowed limits, can satisfy the needs of Chemicals test.
4 Precision test result of table
4 virulence curve of embodiment and EC50Value measurement repetitive test
(1) it prepares potato dextrose broth: will be cut into small pieces after potato 200g peeling, be put into 800ml steaming
It boils 20min in distilled water, after 4 layers of filtered through gauze, glucose 20g is added, 1000ml, 121 DEG C of sterilizings are settled to after boiling
20min。
By the Ustilago maydis strain inoculated of cryo-conservation in potato dextrose broth, in dark condition
Under, 25 DEG C -28 DEG C, 200r/min culture 16-20h obtain activated strains.
(2) this test is subject medicament using pyraclostrobin.It in superclean bench first will subject medicament mother liquor dilution
For 5 different quality concentration, the 15 μ l of medical fluid for drawing each concentration respectively is added to the triangle that packing has 15ml sterilising medium
It in bottle, sufficiently shakes up, the band medicine fluid nutrient medium of various concentration is made, while setting the culture medium that equivalent amount of water is added as blank pair
According to every processing sets 3 repetitions.The Ustilago maydis training activated in equivalent step (1) is added in different pastille culture mediums
Nutrient solution makes culture medium miospore number reach optimal initial concentration 1 × 105-1×106A/ml measures each processing at 590nm
Initial light absorption value OD1(measure 3 times and be averaged).
(3) triangular flask for pastille culture medium of carrying disease germs is placed in shaking table 28 DEG C, 200r/min, cultivates 16h, uses microplate reader
The light absorption value OD of bacterium solution is measured again2(measure 3 times and be averaged).
(4) increasing value for calculating each processing bacterium solution OD, calculates inhibiting rate.With pair of fungicide effective component concentration (mg/L)
Numerical value is independent variable, and inhibiting rate probability value are dependent variable, calculates virulence regression equation, and find out concentration EC in inhibition50.This test
It is repeated 3 times altogether, carries out 3 groups of repetitions every time, finally more each EC acquired50The difference of value.
Δ OD=OD2-OD1
Inhibiting rate (%)=[(control bacterium solution Δ OD- handles bacterium solution Δ OD)/control bacterium solution Δ OD] * 100%
Test result: test result is as shown in table 5 below, computes repeatedly to obtain EC by 3 times50Average value is 0.13, standard
Deviation is 0.015, illustrates that this method has preferable repeatability, test data is reliable.
5 repetitive test result of table
| It repeats | Virulence equation | R2 | EC50(mg/L) |
| 1 | Y=2.0977x+6.7354 | 0.9786 | 0.15 |
| 2 | Y=1.6387x+6.4767 | 0.9875 | 0.13 |
| 3 | Y=1.4846x+6.3751 | 0.9089 | 0.12 |
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method of indoor quickly screening Ustilago maydis pesticide control, comprising the following steps:
1) a series of potato dextrose broths containing different effective component concentration medicaments to be measured are prepared, by activation
Ustilago maydis bacterial strain moves into above-mentioned pastille culture medium, so that the concentration of pastille culture medium miospore is 1 × 105-1×106
A/ml, with initial light absorption value OD of the microplate reader measurement bacterium solution at 590nm1；
2) by the above-mentioned fluid nutrient medium containing Ustilago maydis bacterial strain under dark condition shaken cultivation 16-20h, with enzyme mark
Instrument measures final OD2；
3) the increasing value Δ OD for calculating each processing bacterium solution OD, calculates inhibiting rate；Logarithm with fungicide effective component concentration is
Independent variable, inhibiting rate probability value are dependent variable, calculate virulence regression equation, and find out concentration EC in inhibition50。
2. the method according to claim 1, wherein the activation of Ustilago maydis bacterial strain described in step 1) walks
Suddenly are as follows: by Ustilago maydis strain inoculated in potato dextrose broth, the oscillation activation training under dark condition
It supports and obtains activated strains.
3. according to the method described in claim 2, it is characterized in that, the temperature for vibrating activation culture described in step 1) is 25
DEG C -28 DEG C, frequency 150-250r/min, time 16-20h.
4. the method according to claim 1, wherein OD described in step 2)2The range of value is between 0.8-1.
5. the method according to claim 1, wherein the temperature of shaken cultivation described in step 2) is 25 DEG C -28
DEG C, frequency 150-250r/min.
6. the method according to claim 1, wherein inhibiting rate described in step 3) calculates according to the following formula: inhibiting
Rate (%)=[(control bacterium solution Δ OD- handles bacterium solution Δ OD)/control bacterium solution Δ OD] * 100%.
7. the method according to claim 1, wherein potato dextrose broth described in step 1)
Constituent and its weight ratio are as follows: potato 200g, glucose 20g, distilled water 1000ml.
8. the method according to the description of claim 7 is characterized in that the preparation method of the potato dextrose broth
Include: to be put into being cut into small pieces after peeling potatoes in distilled water and boil, after filtered through gauze, glucose is proportionally added, boils
Distilled water is settled to 1000ml after boiling, sterilizes after packing.
9. according to the method described in claim 8, it is characterized in that, the sterilising conditions are as follows: 121 DEG C of sterilizing 20min.
10. the method according to claim 1, wherein microplate reader measurement OD value repeats 2~4 times.
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