CN103555811A - Method for detecting sensitivity of phytophthora parasitica var nicotianae to bactericide - Google Patents
Method for detecting sensitivity of phytophthora parasitica var nicotianae to bactericide Download PDFInfo
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- CN103555811A CN103555811A CN201310580090.1A CN201310580090A CN103555811A CN 103555811 A CN103555811 A CN 103555811A CN 201310580090 A CN201310580090 A CN 201310580090A CN 103555811 A CN103555811 A CN 103555811A
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Abstract
The invention discloses a method for detecting the sensitivity of phytophthora parasitica var nicotianae to a bactericide. The method comprises the following steps of diluting dimethomorph by using a lima bean liquid culture medium or an oat liquid culture medium to prepare agents with various mass concentrations; culturing the phytophthora parasitica var nicotianae on a lima bean solid culture medium plate, inducing by using a V8 liquid culture medium to obtain a zoospore suspension liquid, regulating the concentration of the zoospore suspension liquid to be 1+10<4>/mL; placing a 96-hole micropore plate in a monitoring culture box, culturing in the dark at a temperature of 28 DEG C for 72h, reading data of a phytophthora parasitica var nicotianae growth curve of 0-72h by adopting growth curve monitoring software; and when culturing for 0 and 72h, determining an OD value of the phytophthora parasitica var nicotianae in each pore of 590nm by adopting turbidity determination software, calculating an inhibition rate of phytophthora parasitica var nicotianae spore germination and mycelial growth of the bactericide according to a formula (1), and calculating an EC50 value of the bactericide to a strain. According to the method, the sensitivity of the phytophthora parasitica var nicotianae to the bactericide can be detected conveniently, simply, rapidly, efficiently and accurately.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method of tobacco black shank bacterium to fungicide sensitivity that detect.
Background technology
By tobacco black shank bacterium (
phytophthora parasiticavar.
nicotianae) black shank that causes is one of the most serious disease of tobacco epidemic in world wide, tobacco, in whole breeding time, all a plurality of tissues such as tobacco root, cane and blade be can infect, root system necrosis, blade yellow, plant wither, the symptom such as even withered conventionally caused.In the situation that not taking prophylactico-therapeutic measures, loss can reach 100%.At present, the control of black shank mainly relies on chemical pesticide, and control pharmacy variety is comparatively single.Phenylamide fungicide metalaxyl is used for many years continuously on tobacco, and resistance problem becomes increasingly conspicuous, and prevention effect reduces gradually, and the sensitivity Detection of carrying out in time is rapidly and efficiently the prerequisite of balck shank resistance management.In addition, the exploitation of new chemical sterilant need to adopt the method for indoor bioassay to carry out high flux screening to the biological activity of pathogenic bacteria.The traditional indoor bioassay method of pathogenic fungi has spore germination method and mycelial growth rate method.The former adopts " slide glass method " and " nutrient agar medium method " conventionally, but all relates to micro-Microscopic observation, and counting is trouble; The latter adopts " culture dish culture method " conventionally, but the test duration is large compared with length, workload.Two kinds of methods are difficult to the method as pathogenic bacteria sensitivity Detection in enormous quantities and high flux screening Fungicidal compounds.Find easy, quick, efficiently and accurately bioassay method becomes the important goal of home and abroad pathogenic bacteria sensitivity testing and sterilant high flux screening.
Microdilution plate method (MicropLate method) is to utilize monitoring incubator (BioLog OminLog) to cultivate the growth curve that pathogenic bacteria to be measured, growth curve monitoring software (OminLog-PM) obtain pathogenic bacteria, the OD value of bacteria suspension in turbidity measurement software (Microstation) rapid detection microwell plate (96 hole), using OD increased value as evaluation index, by the difference of blank group and chemicals treatment group OD increased value, calculate the method for medicament inhibiting rate.This method can either obtain the growth curve that medicament is selected lower pathogenic bacteria, can record again the susceptibility of pathogenic bacteria to chemical bactericide.Gerd had once measured the comprehensive inhibition of boscalid amine to ash arrhizus bacteria spore germination and mycelial growth with YBA nutrient solution, but the method only can obtain the turbidity value of pathogenic bacteria growth, can not reflect the concrete behavioral characteristics of pathogenic bacteria growth.
Summary of the invention
The object of the invention is to overcome the problems referred to above and provide a kind of easy, quick, detect the method for tobacco black shank bacterium to fungicide sensitivity efficiently, accurately.
A kind of method of tobacco black shank bacterium to fungicide sensitivity that detect of the present invention, comprises the following steps:
(1), by lima bean liquid nutrient medium or oat medium dilution dimethomorph, configure the medicament of various mass concentrations;
(2) tobacco black shank bacterium is cultivated to 4d on lima bean solid medium flat board, get 10 of the mycelia pieces that bacterium colony 1/3 place's diameter is 5mm, put into and fill 20mL10%V8 nutrient solution culture dish, after illumination cultivation 4d, mycelia surface forms a large amount of sporocysts; The sporangial balck shank flat board of formation is placed in to 4 ℃ of refrigerator subzero treatment 30min, and then is placed in room temperature 2h, from solution, collect zoospore suspension, its concentration is adjusted into 1 * 104/mL, storage, stand-by;
(3) 8 passage electric pipettors join the every hole 140 μ L of the medicament of 8 kinds of different mass concentration in 96 hole microwell plates, and blank group is set; Under aseptic condition, in 96 hole microwell plates, every hole adds 1 * 10
4the zoospore suspension 10 μ L of individual/mL; Build 96 orifice plate plate lids, 96 hole microwell plates are placed in to monitoring incubator (BioLog OminLog), under 28 ℃ of dark conditions, cultivate 72h, adopt growth curve monitoring software (OminLog-PM) to read the data of 0-72h hour black shank bacterium growth curve; Cultivate 0 and during 72h, adopt turbidity measurement software (Microstation) measure the OD value of the every hole of 590nm black shank bacterium (process 0 and the absorbance of 72h be designated as respectively OD
0and OD
72), according to formula (1), calculate the inhibiting rate of sterilant to black shank bacterium spore germination, and calculate the EC of medicament to bacterial strain spore germination
50value.
Or under aseptic condition, in 96 hole microwell plates, every hole adds 1 * 10
4the zoospore suspension 10 μ L of individual/mL, be placed in monitoring incubator (BioLog OminLog), under 28 ℃ of dark conditions, cultivate after 24h, add respectively again 140 μ L through lima bean substratum, to dilute that mass concentration is 2,1,0.5,0.25,0.13,0.063, the dimethomorph medicament of 0.031mg/L, arrange that to add same volume sterilized water be blank group; (absorbance of processing 0h is OD to adopt turbidity measurement software (Microstation) to measure the OD value of 96 hole microwell plate 590nm
0), microwell plate is continued to be placed under monitoring incubator (BioLog OminLog) dark condition and cultivate, adopt growth curve monitoring software (OminLog-PM) to read the data of 0-72h hour black shank bacterium growth curve; (absorbance of processing 72h is OD after 72h, again to measure OD value
72).According to formula (1), calculate the inhibiting rate of medicament to mycelial growth, and calculate the EC of medicament to bacterial strain mycelial growth
50value.
Described lima bean liquid nutrient medium, its collocation method is: take 60g lima bean and boil 1h in 800mL sterilized water, filter, filtrate is settled to 1L, sterilizing, stand-by;
Described 10%V8 liquid nutrient medium, its collocation method is: centrifugal 5 minutes of 100mLV8 nutritive medium 1500rpm, collects supernatant liquor, and adds 0.2gCaCO
3, add deionized water to 1L, pH6.5～7.5, after sterilizing, packing is stand-by.
The present invention compared with prior art, has obvious beneficial effect, as can be known from the above technical solutions: sterilant is different to the susceptibility of balck shank different steps.Metaxanin does not have activity to the sprouting of spore, EC
50value >200mg/L, and very strong to mycelial growth susceptibility, EC
50value <0.25mg/L. and dimethomorph is all very sensitive to mycelial growth and spore germination, so sterilant of the present invention is selected dimethomorph.Then by the optimization to processing condition, improved spore suspension and mycelium OD value gathers way, met the requirement of Fast Measurement.By " thalline turbidity measurement " and " growth curve monitoring " combined, obtained the OD value for sensitivity testing, obtained again " the growth kinetics curve " of thalline.Thereby easy, quick, detect tobacco black shank bacterium in germ spore germination, the susceptibility of vegetative stage to sterilant efficiently, accurately, thereby realize the effective control to black shank.
Embodiment
embodiment 1:
Utilize present method to measure dimethomorph to picking up from the inhibition of 3 strain tobacco black shank bacterium spore germinations on the sick stem of Tobacco in Guizhou, comprise the following steps:
(1) preparation lima bean liquid nutrient medium: take 60g lima bean and boil 1h in 800mL sterilized water, filter, filtrate is settled to 1L, sterilizing, stand-by; By lima bean liquid nutrient medium dilution dimethomorph, configuration quality concentration is 100,10,1,0.1,0.05,0.025,0.013, the medicament of 0.006mg/L;
(2) tobacco black shank bacterium is cultivated to 4d on lima bean solid medium flat board, get 10 of the mycelia pieces that bacterium colony 1/3 place's diameter is 5mm, put into and fill 20mL10%V8 nutrient solution culture dish, after illumination cultivation 4d, mycelia surface forms a large amount of sporocysts; The sporangial balck shank flat board of formation is placed in to 4 ℃ of refrigerator subzero treatment 30min, and then is placed in room temperature 2h, from solution, collect zoospore suspension, its concentration is adjusted into 1 * 10
4individual/mL, storage, stand-by;
(3) 8 passage electric pipettors join the every hole 140 μ L of the medicament of above-mentioned 8 kinds of different mass concentration in 96 hole microwell plates, and blank group is set; Under aseptic condition, in 96 hole microwell plates, every hole adds 1 * 10
4the zoospore suspension 10 μ L of individual/mL; Build 96 orifice plate plate lids, 96 hole microwell plates are placed in to monitoring incubator (BioLog OminLog), under 28 ℃ of dark conditions, cultivate 72h, adopt growth curve monitoring software (OminLog-PM) to read the data of 0-72h hour black shank bacterium growth curve; While cultivating 0-72h, adopt turbidity measurement software (Microstation) measure the OD value of the every hole of 590nm black shank bacterium (process 0 and the absorbance of 72h be designated as respectively OD
0and OD
72), according to formula (1), calculate the inhibiting rate of sterilant to black shank bacterium spore germination, and calculate the EC of medicament to bacterial strain spore germination
50value.
The inhibiting rate % list that 3 strains are surveyed goes out as follows:
The susceptibility of table 1 dimethomorph to 3 strain tobacco black shank bacterium spore germinations
| Bacterial strain | EC 50Value (mg/L) |
| 1 | 0.14 |
| 2 | 0.13 |
| 3 | 0.12 |
from table, result shows, to tobacco black shank bacterium, spore germination has very strong inhibition activity to dimethomorph, and 3 bacterial strains that strain is surveyed are all more responsive to dimethomorph, its EC
50
be worth equal <0.15 mg/L.
embodiment 2
Utilize present method to measure dimethomorph to picking up from the inhibition of 3 strain tobacco black shank bacterium mycelial growths on Tobacco in Guizhou, comprise the following steps:
(1) preparation lima bean liquid nutrient medium: take 60g lima bean and boil 1h in 800mL sterilized water, filter, filtrate is settled to 1L, sterilizing, stand-by; By lima bean liquid nutrient medium dilution dimethomorph, configuration quality concentration is 2,1,0.5,0.25,0.13,0.063, the medicament of 0.031mg/L;
(2) tobacco black shank bacterium is cultivated to 4d on lima bean solid medium flat board, get 10 of the mycelia pieces that bacterium colony 1/3 place's diameter is 5mm, put into and fill 20mL10%V8 nutrient solution culture dish, after illumination cultivation 4d, mycelia surface forms a large amount of sporocysts; The sporangial balck shank flat board of formation is placed in to 4 ℃ of refrigerator subzero treatment 30min, and then is placed in room temperature 2h, from solution, collect zoospore suspension, its concentration is adjusted into 1 * 104/mL, storage, stand-by;
(3), under aseptic condition, in 96 hole microwell plates, every hole adds 1 * 10
4the zoospore suspension 10 μ L of individual/mL, be placed in monitoring incubator (BioLog OminLog), under 28 ℃ of dark conditions, cultivate after 24h, add respectively again 140 μ L through lima bean substratum, to dilute that mass concentration is 2,1,0.5,0.25,0.13,0.063, the dimethomorph medicament of 0.031mg/L, arrange that to add same volume sterilized water be blank group; (absorbance of processing 0h is OD to adopt turbidity measurement software (Microstation) to measure the OD value of 96 hole microwell plate 590nm
0), microwell plate is continued to be placed under monitoring incubator (BioLog OminLog) dark condition and cultivate, adopt growth curve monitoring software (OminLog-PM) to read the data of 0-72h hour black shank bacterium growth curve; (absorbance of processing 72h is OD after 72h, again to measure OD value
72).According to formula (1), calculate the inhibiting rate of medicament to mycelial growth, and calculate the EC of medicament to bacterial strain mycelial growth
50value,
The inhibiting rate % list that 3 strains are surveyed goes out as follows:
The susceptibility of table 2 dimethomorph to 3 strain tobacco black shank bacterium mycelial growths
| Bacterial strain | EC 50Value (mg/L) |
| 1 | 0.17 |
| 2 | 0.16 |
| 3 | 0.17 |
from table, result shows, it is active that dimethomorph also has very strong inhibition to tobacco black shank bacterium mycelial growth, and 3 bacterial strains that strain is surveyed are dimethomorph sensitive strain, its EC
50
be worth equal <0.2mg/L.
Claims (4)
1. detect the method for tobacco black shank bacterium to fungicide sensitivity, comprise the following steps:
(1), by lima bean liquid nutrient medium or oat medium dilution dimethomorph, configure the medicament of various mass concentrations;
(2) tobacco black shank bacterium is cultivated to 4d on lima bean solid medium flat board, get 10 of the mycelia pieces that bacterium colony 1/3 place's diameter is 5mm, put into and fill 20mL10%V8 nutrient solution culture dish, after illumination cultivation 4d, mycelia surface forms a large amount of sporocysts; The sporangial balck shank flat board of formation is placed in to 4 ℃ of refrigerator subzero treatment 30min, and then is placed in room temperature 2h, from solution, collect zoospore suspension, its concentration is adjusted into 1 * 10
4individual/mL, storage, stand-by;
(3) 8 passage electric pipettors join the every hole 140 μ L of the medicament of 8 kinds of different mass concentration in 96 hole microwell plates, and blank group is set; Under aseptic condition, in 96 hole microwell plates, every hole adds 1 * 10
4the zoospore suspension 10 μ L of individual/mL; Build 96 orifice plate plate lids, 96 hole microwell plates are placed in to monitoring incubator, under 28 ℃ of dark conditions, cultivate 72h, adopt growth curve monitoring software to read the data of 0-72h hour black shank bacterium growth curve; Cultivate 0 and during 72h, adopt turbidity measurement software measure the OD value of the every hole of 590nm black shank bacterium (process 0 and the absorbance of 72h be designated as respectively OD
0and OD
72), according to formula (1), calculate the inhibiting rate of sterilant to black shank bacterium spore germination, and calculate the EC of medicament to bacterial strain spore germination
50value;
2. a kind of method of tobacco black shank bacterium to fungicide sensitivity that detect as claimed in claim 1, wherein (3) step can be following method: under aseptic condition, in 96 hole microwell plates, every hole adds 1 * 10
4the zoospore suspension 10 μ L of individual/mL, be placed in monitoring incubator (BioLog OminLog), under 28 ℃ of dark conditions, cultivate after 24h, add respectively again 140 μ L through lima bean substratum, to dilute that mass concentration is 2,1,0.5,0.25,0.13,0.063, the dimethomorph medicament of 0.031mg/L, arrange that to add same volume sterilized water be blank group; (absorbance of processing 0h is OD to adopt turbidity measurement software (Microstation) to measure the OD value of 96 hole microwell plate 590nm
0), microwell plate is continued to be placed under monitoring incubator (BioLog OminLog) dark condition and cultivate, adopt growth curve monitoring software (OminLog-PM) to read the data of 0-72h hour black shank bacterium growth curve; (absorbance of processing 72h is OD after 72h, again to measure OD value
72), according to formula (1), calculate the inhibiting rate of medicament to mycelial growth, and calculate the EC of medicament to bacterial strain mycelial growth
50value.
3. a kind of method of tobacco black shank bacterium to fungicide sensitivity that detect as claimed in claim 1 or 2, wherein the collocation method of lima bean liquid nutrient medium is: take 60g lima bean and boil 1h in 800mL sterilized water, filter, filtrate is settled to 1L, sterilizing, stand-by; The collocation method of lima bean solid medium is: take 60g lima bean and boil 1h in 800mL sterilized water, filter, add 16g agar powder in filtrate, boil and be settled to 1L, sterilizing, stand-by.
4. a kind of method of tobacco black shank bacterium to fungicide sensitivity that detect as claimed in claim 3, wherein the collocation method of 10%V8 liquid nutrient medium is: centrifugal 5 minutes of 100mLV8 nutritive medium 1500rpm, collects supernatant liquor, and adds 0.2gCaCO
3, add deionized water to 1L, pH6.5～7.5, after sterilizing, packing is stand-by.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396597A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Real-time observation method for phytophthora nicotianae infection process |
| CN104396599A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Method for phytophthora nicotianae inoculation of tobacco |
Citations (1)
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| CN102759513A (en) * | 2012-07-25 | 2012-10-31 | 山东农业大学 | Method for quickly testing sensitivity of Botrytis cinerea to bactericide |
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2013
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102759513A (en) * | 2012-07-25 | 2012-10-31 | 山东农业大学 | Method for quickly testing sensitivity of Botrytis cinerea to bactericide |
Non-Patent Citations (5)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396597A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Real-time observation method for phytophthora nicotianae infection process |
| CN104396599A (en) * | 2014-12-04 | 2015-03-11 | 云南省烟草农业科学研究院 | Method for phytophthora nicotianae inoculation of tobacco |
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Application publication date: 20140205 |