CN104634768A - Plant pathogen activity evaluation and bactericide high throughput screening method and kit - Google Patents
Plant pathogen activity evaluation and bactericide high throughput screening method and kit Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a plant pathogen activity evaluation and bactericide high throughput screening method and kit on the basis of dual fluorescence dyeing. In the provided method, a micro-titer plate is taken as the tool carrier, two different fluorescent dyes are adopted, the active/inactive spores or mycelia are labeled by fluorescence respectively, the color of the active spores or mycelia is different from the color of the inactive spores or mycelia, then the survival rate of the plant pathogen is detected by a fluorescent microscope or flow cytometry, and a kit is prepared according to the method. The method and kit can be used to fields using plant pathogen survival rate as the index such as bactericide screening, food safety evaluation, water quality evaluation, and the like. The provided plant pathogen activity evaluation method and kit has the advantages of rapidness, objectiveness, simpleness, practicality, and low cost, can be used to research the mechanism of bactericide, and can also be applied to the fields of bactericide high throughput screening and toxicity evaluation.
Description
Technical field
The present invention relates to a kind of phytopathogen activity rating based on double fluorescent dyeing and germifuge high-throughput screening method and kit; the method and kit can be applied in pathogen activity rating, Screening of Fungicide, food safety assessment, water quality assessment etc., belong to plant protection and biological technical field.
Background technology
Phytopathogen is that plant disease occurs and a popular important factor, and the loss number that the whole world causes because of disease is every year in hundred million yuan.Detecting phytopathogen and activity thereof quickly and accurately, is the basis of the state of an illness being carried out to effectively prediction.
At present, the detection of phytopathogen mainly adopts conventional pathogenicbacteria separation culture technique, and the method is time-consuming, effort, and can not detect obligate parasite.The immunological technique grown up afterwards and molecular biology method, as PCR etc., this class methods can accurate, sensitive, carry out qualitative to phytopathogen rapidly and quantitatively detect, but can not detect that it is active.But the activity rating of phytopathogen is the basis of disease being carried out to Accurate Prediction forecast.Less about the research of phytopathogen activity detection analysis both at home and abroad, the spore germination counting method (Colony Forming Units, CFU) that main employing is traditional, although the method is accurate to the detection of activity, reliable, detection time is longer.
Fluorescence colour medically detects the important means of Apoptosis and meronecrosis.After Apoptosis or necrosis, cell volume change can be there is, the change such as cell membrane penetration sexually revises, core DNA degradation, according to these features, select the fluorescent dye be applicable to, accurately can detect cell state.The apoptotic phenotype of phytopathogen is similar to zooblast with physiological characteristic, and eucaryotic cell structure and metabolic process simple.At present, fluorescence colour has minority to report in pathogen research, utilize the specific fluorescence dye propidium iodide of non-viable non-apoptotic cell to detect the activity of soybean sudden death comprehensive germ, SYTOX Green is used for the activity of human disease bacterium Escherichia coli in testing environment and water and salmonella.
High flux screening (High throughput screening, HTS) technology is one of important technical finding original new drug, has become the important tool in pharmacy and biotechnology research.Modern agriculture is more and more stronger for the dependence of germifuge, and along with the cry of environment and consumption safety improves day by day, and the drug-fast generation of harmful organism makes germifuge shortening in serviceable life.The initiative of novel pesticide is more and more urgent, also more and more difficult with exploitation.According to statistics, the successful exploitation of efficient, low toxicity, an environmentally friendly novel pesticide, needs to synthesize screening 80,000 compounds, the more than one hundred million unit of cost.This is proposed new challenge to the synthesis of new type bactericide and screening.At present, China's many employings spore germination method and mycelial growth rate method carry out the screening of germifuge, and along with new type bactericide initiative technology fast development, require set up a kind of method of screening noval chemical compound simply, fast.
By finding out the prior art analysis of phytopathogen activity rating and Screening of Fungicide, prior art is time-consuming, effort and cost is higher or need expensive instrument and equipment, be badly in need of a kind of simple, with low cost, do not need expensive instrument and equipment just can carry out the new method of fast quantification phytopathogen activity rating.
Summary of the invention
The object of the invention is to take a lot of work for current phytopathogen Activity determination and Screening of Fungicide, time-consuming, the problems such as testing result is unstable, expense is high, a kind of phytopathogen activity rating based on double fluorescent dyeing and germifuge high-throughput screening method and kit are provided, the fluorescence signal of vigor and debility spore or hyphal cell is had by detecting pathogen, determine that pathogen is active, realize evaluating drug effect and the high flux screening of germifuge.The method and kit have that operation is simple, detection time is short, detection limit is large, with low cost, the features such as the drug effect of various sterilization agent can be evaluated simultaneously, the fields such as Screening of Fungicide, toxicity assessment, food safety assessment, water quality assessment can be widely used in.Particular content is as follows:
1, a kind of phytopathogen activity rating based on double fluorescent dyeing and germifuge high-throughput screening method
The present invention discloses a kind of phytopathogen activity rating and germifuge high-throughput screening method, using microtiter plate as tool carrier, adopt the fluorescent dye that two kinds different, vigor and unvital spore or hyphal cell is had to carry out fluorescence labeling to phytopathogen respectively, due to have vigor and debility spore or hyphal cell painted different, utilize fluorescent microscope or flow cytomery fluorescence signal, and add up pathogen survival rate, directly can reflect the activity of phytopathogen, realize germifuge evaluating drug effect and high flux screening.
The first fluorescent dye of the method application can make vigor spore or the painted generation specific fluorescent of hyphal cell, be selected from acridine orange (AO), fluorescein two acetic acid vinegar (FDA), Hoechst 33258,4', one in 6-diamidino-2-phenylindone (DAPI), Annexin V-FITC etc., but be not limited thereto; The second fluorescent dye can make dead spore or the painted generation specific fluorescent of hyphal cell, is selected from the one in propidium iodide (PI), Ethidum Eremide (EB), SYTOX etc., but is not limited thereto.
Wherein, the working concentration of the first fluorescent dye AO is 10-1000 μ g/mL, and 488 nm blue lights excite down, makes living cells core in green or the even fluorescence of yellow green; The working concentration of FDA is 100-1000 μ g/mL, and 488 nm blue lights excite down, in living cells, produce green fluorescence; The working concentration of Hoechst 33258 is 1-100 μ g/mL, and under 350 nm ultraviolet excitations, living cells sends blue-fluorescence; The working concentration of DAPI is 5-500 μ g/mL, and during 358 nm UV-irradiation, living cells sends bright blue fluorescence; The working concentration of Annexin V-FITC is 5-500 μ g/mL, and 480nm blue light excites down, and living cells sends green fluorescence; The working concentration of the second fluorescent dye PI is 5-1000 μ g/mL, under 358 nm ultraviolet excitations, makes dead cell produce red fluorescence; EB working concentration is 10-1000 μ g/mL, makes dead cell produce orange fluorescent; The working concentration of SYTOX is 0.1-1 μ g/mL, makes non-viable non-apoptotic cell fluoresced green.
Wherein, the dyeing time of the first fluorescent dye AO is lucifuge process 10-25 min, the dyeing time of FDA is 5-40 min, the dyeing time of Hoechst 33258 is 15-50 min, the dyeing time of DAPI is the dyeing time of 3-15 min, Annexin V-FITC is lucifuge process 15-40 min; The dyeing time of the dyeing time of the second fluorescent dye PI to be the dyeing time of lucifuge process 4-30 min, EB be 20-40 min, SYTOX is 5-20 min.
In the method, phytopathogen is that artificial pure culture obtains, or enrichment obtains from disease tissue.The detected object of phytopathogen activity rating comprises, the hyphal cell of plant pathogenic fungi or spore, the spore of plant pathogenetic bacteria, the resting spore of rape plasmodiophora brassicae.
By phytopathogen spore or hyphal suspension, equivalent adds in the microtiter plate containing multiple hole, and every hole spore quantity is 10
2-10
5individual.Add two kinds of fluorescent dyes in order respectively, dyeing temperature is 4-35 DEG C.Finally, adopt flow cytometer, fluorescent microscope or fluorescence spectrophotometer to detect fluorescence signal, quantitative evaluation is carried out to phytopathogen activity.
The method can be widely used in the fields such as new compound screening, Screening of Fungicide, food safety assessment and water quality assessment.
2, a kind of phytopathogen activity rating based on double fluorescent dyeing and germifuge high flux screening kit
Utilize the present invention can be prepared into a kind of fast, the kit of the active and germifuge high flux screening of quantitative evaluation phytopathogen, this kit contains one or more microtiter plate containing multiple hole, two kinds of fluorescent dyes, the first fluorescent dye made vigor phytopathogen spore or hyphal cell painted, the second fluorescent dye make debility phytopathogen spore or hyphal cell painted.
Wherein, one or more microtiter plate containing multiple hole, selects from 12 holes, 24 holes, 96 holes, 384 holes, 1536 hole microtiter plates, wherein with 96 orifice plates and 384 orifice plates the best.The every hole of microtiter plate comprises one or more phytopathogen spore or mycelia, wherein for 96 orifice plates, and every hole 10
3-10
5individual spore is best; For 384 orifice plates, every hole 10
2-10
3individual spore is best.
The first fluorescent dye that this kit contains is selected from acridine orange (AO), fluorescein two acetic acid vinegar (FDA), Hoechst 33258,4', one in 6-diamidino-2-phenylindone (DAPI) etc., but be not limited thereto, vigor spore or hyphal cell can have been made to produce specific fluorescent; The second fluorescent dye is selected from the one in propidium iodide (PI), Ethidum Eremide (EB), SYTOX etc., but is not limited thereto, and dead spore or hyphal cell can be made to produce specific fluorescent.
This kit can also comprise one or more phytopathogens, and the culture medium of this pathogen and cleansing medium, and this medium can be lyophilized form or liquid form.
This kit can also comprise germifuge high flux screening instructions, or the instructions of compound or additive method process phytopathogen.
This kit can also comprise the contrast medicament of certain specific pathogen bacterium of control.
3, in the art, many effective biological stains are also had to use.Different coloring agents in the different parts reaction or concentrated of pathogen cell or tissue, and can use these characteristics to disclose specific part region.Available biological stain comprises, Hoest 33342, carmine (Carmine), Coomassie brilliant blue (Coomassie blue), crystal violet, DAPI, Yihong, ethidium bromide, magenta, HAEMATOXYLIN (Haematoxulin), iodine, malachite green, methyl chloride, methylene blue, dimethyl diaminophenazine chloride, Nile red, osmium tetroxide, rhodamine and sand Huang (Safranin) etc.
the beneficial effect that the present invention has compared with background technology is:
(1) the present invention establishes the fluorescent staining method and working environment that are suitable for phytopathogen activity rating, comprising: selection, colouring method, the dyeing time of fluorescent dye classification, hatch environment, fluorescence detection method etc.;
(2) method provided and kit can direct-detection phytopathogen survival rates, and simple to operate, equipment, cost requirements are low, just can realize in common lab;
(3) 30 minutes are only needed from sample preparation to completing detection, can directly judge the activity of single spore or mycelia, there is the features such as quick, accurate, sensitive, reproducible, be expected to alternative traditional spore germination method and mycelial growth rate method, be suitable for the department such as agri-scientific research, plant protection and use;
(4) phytopathogen survival rate detection method provided by the invention and kit have operation fast, the features such as multiple compounds can be screened simultaneously, save human and material resources and time, the aspects such as germifuge high flux screening, food security and water quality assessment can be widely used in.
Accompanying drawing explanation
Fig. 1 be FDA and PI fluorescence colour differentiate the pathogen of Botrytis cinerea (
botrytis cinerea) conidial activity.In figure: A. fluorescent microscope green polarized light is observed, after FDA dyeing, spore fluoresced green of living; B. under fluorescent microscope, blue polarized light is observed, and after PI dyeing, dead spore sends out red fluorescence; C. spore alive and the mixing of dead spore are simultaneously with FDA and PI dyeing, observe under fluorescent microscope under green polarized light, and spore fluoresced green of living, dead spore sends out red fluorescence.
Fig. 2 be FDA and PI fluorescence colour differentiate Fusarinm solani (
fusarium solani) activity of conidium and mycelia.In figure: A. fluorescent microscope green polarized light is observed, after FDA dyeing, live spore and viable bacteria silk fluoresced green; B. under fluorescent microscope, blue polarized light is observed, and after PI dyeing, dead spore and dead mycelia send out red fluorescence; C. have vigor and debility spore, mycelia mixing with FDA and PI dyeing, to observe under green polarized light, live spore and viable bacteria silk fluoresced green, dead spore and dead mycelia send out red fluorescence simultaneously.
Fig. 3 be FDA and PI fluorescence colour differentiate pseudomonas syringae shed tears pvs oryzae and oryzicola (
pseudomonas syringaepv.
lachrymans) activity of spore.In figure: A. fluorescent microscope green polarized light is observed, after FDA dyeing, spore fluoresced green of living; B. under fluorescent microscope, blue polarized light is observed, and after PI dyeing, dead spore sends out red fluorescence.
Fig. 4 be FDA and PI fluorescence colour differentiate rape plasmodiophora brassicae (
plasmodiophora brassicae) activity of spore.In figure: A. fluorescent microscope green polarized light is observed, after FDA dyeing, spore fluoresced green of living; B. under fluorescent microscope, blue polarized light is observed, and after PI dyeing, dead spore sends out red fluorescence; C. have vigor and debility spore, mycelia mixing with FDA and PI dyeing, to observe under fluorescent microscope under green polarized light, spore fluoresced green of living, dead spore sends out red fluorescence simultaneously.
Fig. 5 be Hoest 33258 and PI to after the pathogen of Botrytis cinerea spore staining, different time fluorescence intensity.In figure: different time after A. dyeing, 96 hole microtiter plates collect PI fluorescence intensity change; B. different time after dyeing, 384 hole microtiter plates collect PI fluorescence intensity change; C. different time after dyeing, 96 hole microtiter plates collect Hoest 33258 fluorescence intensity change; D. different time after dyeing, 384 hole microtiter plates collect Hoest 33258 fluorescence intensity change.
Fig. 6 be the melon anthrax-bacilus of flow cytomery (
colletotrichumorbiculare) analysis of spore active level.In figure: A. negative control pipe; B.Annexin V-FITC dyes Dan Yangguan; C.PI dyes single negative tube; D. testing sample pipe.
The melon anthrax-bacilus of Fig. 7 flow cytomery has vigor spore ratio and spore germination rate linear relationship chart.
Fig. 8 is that germifuge is to rape plasmodiophora brassicae resting spore effect of vigor.In figure: A. variable concentrations thiophanate-methyl process different time is to the insect killing effect of rape plasmodiophora brassicae; B. variable concentrations fluazinam process different time is to the insect killing effect of rape plasmodiophora brassicae.
Fig. 9 be 2 mg/mL fluazinam process to rape plasmodiophora brassicae (
plasmodiophora brassicae) impact of resting spore vigor.In figure: A. 2 mg/mL fluazinam process 10 min rape plasmodiophora brassicae resting spore viability state; B. 2 mg/mL fluazinam process 30 min rape plasmodiophora brassicae resting spore viability states; C. 2 mg/mL fluazinam process 1 h rape plasmodiophora brassicae resting spore viability states; D. 2 mg/mL fluazinam process 2 h rape plasmodiophora brassicae resting spore viability states.
embodiment
Flesh and blood of the present invention and beneficial effect is described in detail below in conjunction with drawings and Examples.Following examples only for being further detailed the present invention, should not be construed as limitation of the present invention.
Reagent:
Fluorescein two acetic acid vinegar (Fluorescein diacetate, FDA): Sigma;
Propidium iodide (Propidine iodide, PI): Sigma;
Annexin V-FITC：Sigma；
Hoechst 33258：Sigma；
It is pure that other reagent is domestic analysis.
Instrument:
Olympus fluorescent microscope (OLYMPUS BX51);
Flow cytometer (BD FACS Calibur);
Electric-heated thermostatic water bath (XMTD-700).
embodiment 1 is active and evaluating drug target sieving based on the phytopathogen of fluorescent dual decoration method
Adopt FDA(fluorescein two acetic acid vinegar) and PI(propidium iodide) as dyestuff, to spore, the mycelia of plant pathogenic fungi, and the conidium vitality of plant pathogenetic bacteria is identified, the appraisable phytopathogen target of screening double fluorescent staining method.
1. experimental technique
With plant pathogenic fungi spore, mycelia and plant pathogenetic bacteria spore for target sieving object (table 1), adopt the two dye method plant identification pathogen of FDA/PI active.
The configuration of fluorescent dye liquid: take 0.05 g FDA, be dissolved in 1mL acetone, adds 9 mL PBS 7.4, is mixed with the FDA storing solution that concentration is 5 mg/mL, puts brown bottle-20 DEG C and save backup, and using front PBS to be diluted to final concentration is that 100 μ g/mL are stand-by.Take 0.005 g PI, add 10 mL PBS 7.4 damping fluids, be made into the storage liquid of 500 μ g/mL, put brown bottle 4 DEG C and keep in Dark Place, use front PBS to be diluted to dyeing liquor that final concentration is 5 μ g/mL.
Colouring method: get freshly prepared spore or hyphal suspension 2 mL, be packed as two groups, one group is spore alive without water bath processing or mycelia, and another group is for through the dead spore of 95 DEG C of water bath processing 10 min or mycelia.Getting initial concentration is that FDA dyeing liquor (being dissolved in PBS) the 10 μ L of 100 μ g/mL adds 90 μ L spore or hyphal suspension, mixing, and final concentration is 10 μ g/mL, and room temperature dyes after 15 min, centrifugally discards dye liquor; Then add the PI dyeing liquor that 100 μ L concentration are 5 μ g/mL, room temperature lucifuge dyes 15 min.Adopt fluorescent microscope to observe the spore of stained preparation or mycelia, gather pathogen coloration result image, acquisition parameters is: green channel (exciting light 450 ~ 490 nm, utilizing emitted light >510 nm), object lens are 40 times.
2. experimental result
Utilize FDA/PI double fluorescent decoration method, dye to phytopathogen spore or mycelia, at fluorescence microscopy Microscopic observation, have vigor spore or mycelia after FDA/PI dyeing, fluoresced green, debility spore or mycelia then send out red fluorescence.According to this fluorescent characteristics, determining fluorescent dual decoration method can carry out activity rating and qualification (table 1) to the spore of various plants disease fungus and bacterium or mycelia.Mainly comprise: the monadian conidium of (1) plant pathogenic fungi: Erysiphe (
erysiphe), downy mildew Pseudomonas (
pseudoperonospora), Staphlosporonites (
botrytis), Penicillium (
penicillium), Phyllosticta (
phyllosticta), Verticillium (
verticillium), colletotrichum (
colletotrichum), Phytophthora (
phytophthora) etc. the monadian conidium (Fig. 1) of plant pathogenic fungi; (2) two born of the same parents' type conidiums of plant pathogenic fungi: Ascochyta (
ascochyta) etc. two born of the same parents' type conidiums of plant pathogenic fungi; (3) many born of the same parents type conidium of plant pathogenic fungi: nail spore belongs to (
passalora), Blastocladia (
cladosporium), Fusarium (
fusarium), Stagonospora (
stagonospora) etc. many born of the same parents type conidium (Fig. 2) of plant pathogenic fungi; (4) brick of plant pathogenic fungi is every shape conidium: Corynespora (
corynespora), Alternaria (
alternaria), Stemphylium (
stemphylium) etc. the brick of plant pathogenic fungi every shape conidium; (5) the wire conidium of plant pathogenic fungi: Cercospora (
cercospora), Septoria (
septoria) etc. the wire conidium of plant pathogenic fungi; (6) mycelia of plant pathogenic fungi: Rhizoctonia (
rhizoctonia), Sclerotinia (
sclerotinia) etc. the mycelia of plant pathogenic fungi; (7) Dan Sheng, the twin or raw spore of chain of plant pathogenetic bacteria: Pseudomonas alba (
pseudomonas), clavibacter belong to (
clavibacter), Raul Salmonella (
ralstonia), xanthomonas (
xanthomonas), Erwinia (
erwinia) etc. the Dan Sheng of plant pathogenetic bacteria, the raw spore (Fig. 3) of twin or chain; (8) protozoan circle monospore type resting spore: knee Pseudomonas (
plasmodiophora), powder scab Pseudomonas (
spongospora) etc. the monospore type spore (Fig. 4) of phytopathogen.
the determination of embodiment 2 Hoechst 33258/PI decoration method Chemicals best fluoroscopic examination time
Using fluorescent dual decoration method to evaluate a phytopathogen key condition that is active or that carry out germifuge high flux screening is determine the best fluoroscopic examination time, have maximum repeatability to make the method result.Here with the pathogen of Botrytis cinerea (
b. cinerea) spore activity identification is example, adopts 96 hole microtiter plates and 384 hole microtiter plates to test respectively.
1. experimental technique
The pathogen of Botrytis cinerea is cultivated 10 days in PDA nutrient culture media, brush spore method is adopted to collect spore suspension, be divided into three groups, first group of spore is lethal through 95 DEG C of water bath processing 10 min, second group is have vigor spore without any process, 3rd group is the mixed in equal amounts of the lethal and new fresh and alive spore of thermal treatment, often organizes sample and establishes three repetitions.Added respectively by three groups of samples in 96 holes and 384 hole microtiter plates and test, it is 3 × 10 that the 96 every holes of orifice plate add concentration
6the spore suspension 200 μ L of individual spore/mL, it is 3 × 10 that the 384 every holes of orifice plate add concentration
6the spore suspension 50 μ L of individual spore/mL.Often organize sample and all adopt PI(5 μ g/mL) and Hoechst 33258(5 μ g/mL) dyeing, detect first order fluorescence intensity, until 120 min terminate at interval of 10 min after dyeing.Fluoroscopic examination intensity is three mean values repeated.
2. experimental result
The two dye method of Hoechst 33258/PI is adopted to evaluate the pathogen of Botrytis cinerea conidium vitality, the 96 every holes 6 × 10 of orifice plate
5individual spore, the 384 every holes 1.5 × 10 of orifice plate
5individual spore.When adopting 96 holes and 384 microtiter plate, along with time lengthening, PI and Hoest 33258 fluorescence intensity strengthens gradually, and finally reach a maximal value, relative intensity of fluorescence no longer increases and keeps stable.Detect the Best Times of PI for 4-30 min after dyeing, detect the Best Times of Hoest 33258 for 15-50 min(Fig. 5 after dyeing).
Table 1 is active and evaluating drug target sieving based on the phytopathogen of fluorescent dual decoration method
Table 1 is active and evaluating drug target sieving (Continued) based on the phytopathogen of fluorescent dual decoration method
embodiment 3 flow cytomery phytopathogen active level is analyzed
with melon anthrax-bacilus (
c. orbiculare) spore is target, utilizes flow cytometer, detect the melon anthrax spores vigor through the two dye of Annexin V – FITC/PI, carry out Conidia persistence quantitative test.Meanwhile, adopt spore germination method in contrast, compare the correlativity of fluorescent dual decoration method flow cytomery result and traditional spore germination method testing result.
1. experimental technique
Using melon anthrax-bacilus 5 bacterial strains as experiment material.Freshly prepared spore suspension, 3 kinds of time-triggered protocol are all carried out respectively at 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, i.e. 5 min, 10 min, 20 min, all samples is divided into two groups, one group with after the two dye of Annexin V – FITC/PI, flow cytometer loading, analyzes germ spore active; Another group carries out spore germination experiment, uses SAS software, carries out statistical study to the relation of each process flow cytomery result and spore germination rate.
2. experimental result
Melon anthrax spores sample is after different temperatures and time-triggered protocol, and the detection method (Fig. 6) simultaneously adopting Annexin V – FITC/PI double fluorescent staining to be combined with flow cytometer and spore germination method detect Conidia persistence.Flow cytomery have vigor spore ratio consistent with spore germination rate testing result, the two becomes positive correlation.Flow cytomery have vigor spore ratio y and spore germination rate x linear relationship remarkable, related coefficient is R
2=0.9357, regression equation is respectively y=0.9380x+1.3192(Fig. 7).After fluorescent dual dyeing is described, adopt flow cytomery phytopathogen Activity Results accurate, detection speed is fast, can be used for the quantitative test of phytopathogen activity.
embodiment 4 based onhoechst 33258/PI
the rape plasmodiophora brassicae pesticide control screening of double fluorescent dyeing
With rape plasmodiophora brassicae resting spore for target, employing Hoechst 33258/PI double fluorescent decoration method qualification rape plasmodiophora brassicae (
p. brassica) resting spore vigor, evaluate the different agents such as fluazinam, thiophanate-methyl to the insect killing effect of rape plasmodiophora brassicae, the high-efficiency prevention and control medicament of screening rape plasmodiophora brassicae.
1. experimental technique
Get freshly prepared rape plasmodiophora brassicae resting spore suspending liquid, with 3 × 10
4the density in spore/hole is inoculated on 96 well culture plates, every hole 100 μ L spore suspension.Add in hand-hole by the fluazinam of variable concentrations, thiophanate-methyl (0.1,0.5,1,2 mg/mL) respectively, each drug concentration establishes 4 parallel holes, and every hole adds 100 μ L medicaments.Process 10 min respectively, 30 min, 1 h, after 2 h, abandoning supernatant.Every hole adds spore staining damping fluid 50 μ L, Hoechst 33258 dyeing liquor 50 μ L, PI dyeing liquor 50 μ L, 4 DEG C hatch 30 min after, adopt fluorescent microscope to observe process spore, gather pathogen coloration result image, acquisition parameters is: ultraviolet excitation, and object lens are 40 times.
2. experimental result
Adopt the two dye method (40 times of mirrors) under ultraviolet excitation of Hoechst 33258/PI to observe spore anyway, have vigor spore to send out blue-fluorescence, debility spore sends out red fluorescence.Along with drug concentration increases, plasmodiophora brassicae killing effect is strengthened; Processing time is longer, killing effect better (Fig. 8).Wherein, fluazinam is by concentration 0.1,0.5,1,2 mg/mL process 1 h are respectively 60.8%, 68.7%, 93.3%, 95.8% to rape plasmodiophora brassicae killing effect, process 2 h and are respectively 72.9%, 78.4%, 95.4% and 99.5% to rape plasmodiophora brassicae killing effect, can find out, fluazinam 1 mg/mL process 1 h, rape plasmodiophora brassicae resting spore fatal rate reaches more than 90% (Fig. 9).Thiophanate-methyl, by concentration 1 mg/mL process 2 h or 2 mg/mL process 1 h, reaches more than 80% (table 2) to rape plasmodiophora brassicae killing effect.The killing effect of fluazinam to rape plasmodiophora brassicae is better than thiophanate-methyl, can be used for the alternate medications of the crucifer club root of field control.
The different germifuge of table 2 is to rape plasmodiophora brassicae resting spore effect of vigor
Claims (17)
1., based on a phytopathogen activity rating method for double fluorescent dyeing, comprising (1) provides a sample comprising phytopathogen spore or hyphal cell; (2) make in sample, have vigor spore or hyphal cell to produce detectable fluorescence with the first fluorescent dye of suitable concn, make dead spore or hyphal cell in sample produce detectable fluorescence with the second fluorescent dye of suitable concn; (3) two kinds of fluorescence of phytopathogen spore or hyphal cell are detected.
2., based on a germifuge high-throughput screening method for double fluorescent dyeing, comprising (1) provides a sample comprising phytopathogen spore or hyphal cell; (2) with one or more compounds or additive method process phytopathogen; (3) make in sample, have vigor spore or hyphal cell to produce detectable fluorescence with the first fluorescent dye of suitable concn, make dead spore or hyphal cell in sample produce detectable fluorescence with the second fluorescent dye of suitable concn; (4) two kinds of fluorescence of phytopathogen spore or hyphal cell are detected.
3. the method according to claim 1-2, it is characterized in that: the first fluorescent dye is selected from acridine orange (AO), fluorescein two acetic acid vinegar (FDA), Hoechst 33258,4', one in 6-diamidino-2-phenylindone (DAPI), Annexin V-FITC etc., but be not limited thereto, vigor spore or hyphal cell can have been made to produce specific fluorescent; The second fluorescent dye is selected from the one in propidium iodide (PI), Ethidum Eremide (EB), SYTOX etc., but is not limited thereto, and dead spore or hyphal cell can be made to produce specific fluorescent.
4. the method according to claim 1-3, is characterized in that: the working concentration of the first fluorescent dye AO is 10-1000 μ g/mL, makes living cells core in green or the even fluorescence of yellow green; The working concentration of FDA is 100-1000 μ g/mL, in living cells, produce green fluorescence; The working concentration of Hoechst 33258 is 1-100 μ g/mL, makes living cells send blue-fluorescence; The working concentration of DAPI is 5-500 μ g/mL, makes living cells send bright blue fluorescence; The working concentration of Annexin V-FITC is 5-500 μ g/mL, and 480nm blue light excites down, and living cells sends green fluorescence; The working concentration of the second fluorescent dye PI is 5-1000 μ g/mL, makes dead cell produce red fluorescence; The working concentration of EB is 10-1000 μ g/mL, makes dead cell produce orange fluorescent; The working concentration of SYTOX is 0.1-1 μ g/mL, makes non-viable non-apoptotic cell fluoresced green.
5. the method according to claim 1-3, it is characterized in that: the dyeing time of the first fluorescent dye AO is lucifuge process 10-25 min, the dyeing time of FDA is 5-40 min, the dyeing time of Hoechst 33258 is 15-50 min, the dyeing time of DAPI is the dyeing time of 3-15 min, Annexin V-FITC is lucifuge process 15-40 min; The dyeing time of the dyeing time of the second fluorescent dye PI to be the dyeing time of lucifuge process 4-30 min, EB be 20-40 min, SYTOX is 5-20 min.
6. the method according to claim 1-2, is characterized in that: phytopathogen manually cultivates acquisition, or enrichment obtains from plant disease tissue.
7. the method according to claim 1-2, is characterized in that: phytopathogen activity rating detected object comprises, the spore of plant pathogenic fungi or hyphal cell, the spore of plant pathogenetic bacteria, the resting spore of rape plasmodiophora brassicae.
8. the method according to claim 1-2, is characterized in that: fluoroscopic examination can adopt any instruments detecting fluorescence signal such as flow cytometer, fluorescent microscope or fluorescence spectrophotometer.
9. the phytopathogen activity rating based on double fluorescent dyeing or germifuge high flux screening kit, comprise (1) one or more microtiter plate containing multiple hole, (2) two kinds of fluorescent dyes, the first fluorescent dye made vigor phytopathogen spore or hyphal cell painted, the second fluorescent dye make debility phytopathogen spore or hyphal cell painted.
10. kit according to claim 9, is characterized in that: described one or more microtiter plates containing multiple hole, selects from 12 holes, 24 holes, 96 holes, 384 holes, 1536 hole microtiter plates, wherein with 96 orifice plates and 384 orifice plates the best.
11. kits according to claim 9, it is characterized in that: the first fluorescent dye is selected from acridine orange (AO), fluorescein two acetic acid vinegar (FDA), Hoechst 33258,4', one in 6-diamidino-2-phenylindone (DAPI) etc., but be not limited thereto, vigor spore or hyphal cell can have been made to produce specific fluorescent; The second fluorescent dye is selected from the one in propidium iodide (PI), Ethidum Eremide (EB), SYTOX etc., but is not limited thereto, and dead spore or hyphal cell can be made to produce specific fluorescent.
12. kits according to claim 9, is characterized in that: can also comprise one or more phytopathogens, and the artificial culture medium of this pathogen.
13. kits according to claim 9, is characterized in that: can also comprise cleansing medium, and this medium can be lyophilized form or liquid form.
14. kits according to claim 9, is characterized in that: can also comprise germifuge high flux screening instructions, or the instructions of compound or additive method process phytopathogen.
15. kits according to claim 9, is characterized in that: the contrast medicament that can also comprise certain specific pathogen bacterium of control.
16. kits according to claim 9, is characterized in that: the every hole of described microtiter plate comprises one or more phytopathogen spore or hyphal cells, wherein for 96 orifice plates, and every hole 10
3-10
5individual spore is best; For 384 orifice plates, every hole 10
2-10
3individual spore is best.
17. methods according to claim 1-16 and kit, is characterized in that: described phytopathogen comprise Erysiphe (
erysiphe), downy mildew Pseudomonas (
pseudoperonospora), Staphlosporonites (
botrytis), Penicillium (
penicillium), Phyllosticta (
phyllosticta), Verticillium (
verticillium), colletotrichum (
colletotrichum), Phytophthora (
phytophthora) etc. the monadian conidium of plant pathogenic fungi; Ascochyta (
ascochyta) etc. two born of the same parents' type conidiums of plant pathogenic fungi; Nail spore belongs to (
passalora), Blastocladia (
cladosporium), Fusarium (
fusarium), Stagonospora (
stagonospora) etc. many born of the same parents type conidium of plant pathogenic fungi; Corynespora (
corynespora), Alternaria (
alternaria), Stemphylium (
stemphylium) etc. the brick of plant pathogenic fungi every shape conidium; Cercospora (
cercospora), Septoria (
septoria) etc. the wire conidium of plant pathogenic fungi; Rhizoctonia (
rhizoctonia), Sclerotinia (
sclerotinia) etc. the hyphal cell of plant pathogenic fungi; Pseudomonas alba (
pseudomonas), clavibacter belong to (
clavibacter), Raul Salmonella (
ralstonia), xanthomonas (
xanthomonas), Erwinia (
erwinia) etc. Dan Sheng, the raw spore of twin or chain of plant pathogenetic bacteria; Knee Pseudomonas (
plasmodiophora), powder scab Pseudomonas (
spongospora) etc. the monospore type spore of phytopathogen.
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