CN101900730A - Quick detection method for cucumber fusarium axysporum - Google Patents
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Abstract
The invention discloses a quick detection method for cucumber fusarium axysporum, and belongs to the technical field of biology. The quick detection method for the cucumber fusarium axysporum is characterized in that: because an improved new Dot-ELISA technique of ELISA is applied in the method, the method has low reagent dosage and simple and quick operation, does not need special equipment condition, and is suitable for basic units; an antigen membrane has long preservation period and can be preserved for half a year under -20 percent, and the activity thereof is not affected; the method can be posted for on-site epidemiological survey; and the detection result can be stored for long term, and the method is convenient for review and suitable for on-site detection and quick prediction of epidemic occurrence of the disease. The invention also relates to an agent and an instrument for detection with detection program, working condition and detectable quantity.
Description
Technical field
The invention belongs to biological technical field, relate to cucumber and produce the method for quick of going up each a kind of important silborne fungal diseases.
Background technology
Cucumber is a kind of important vegetables of China, and is significant for the daily life demand that satisfies the people.Cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum) is a kind of important disease on the cucumber, can produce to cucumber and bring heavy losses, has hindered the development that cucumber produces.All there is generation in various degree in main green cucumber district in the whole nation, is the important method of control cucumber fusarium axysporum by grafting, but in Liaoning, the case that droop takes place the grafting cucumber successively appears in ground such as Beijing, Shandong.Cucumber fusarium axysporum is in case morbidity prevents and treats after serious lately again, and it is similar to add many root disease symptoms, is difficult in the production distinguish, bring difficulty to disease control, cause to be difficult to suit the remedy to the case, cause sing misdiagnosis and mistreatment easily, early stage diagnosis fast and accurately is most important.
The detection of China's plant pathogeny organism, identification system be imperfection also, and detection technique weakness, existing technology have suitable gap with comparing in the world.Fast development along with science and technology, biotechnology provides new way for the detection and the evaluation of plant pathogenic microorganisms, the molecular Biological Detection technology is owing to its sensitivity, the quick and versatile and flexible extensive attention that is subjected to each research field of life science, but owing to its required reagent cost an arm and a leg, to the experimenter have relatively high expectations and experimental drug to reasons such as operating personnel and the toxic effects of environment, thereby limited its widespread use on agricultural.And immunological technique, the detection to plant virus has obtained using widely on medically to harmful pathogenic microorganism and agricultural, and the method for detection and technical system are day by day ripe.The detection method of capsicum root-rot type eqpidemic disease and cucumber fusarium axysporum is studied less at home and abroad, China does not set up as yet to this disease detection method fast and effectively, thereby produce for China capsicum and stayed great hidden danger, it is extremely urgent, significant therefore to set up the accurate detection technical method.
Nineteen eighty-two Hawkes etc. copies the method for dot blot in the molecular biology (Dot Hybridzation) to grow up some immunity in conjunction with measuring technology (Dot immunobinding assay, DIBA), the same enzyme linked immunosorbent assay (ELISA) of this method (ELISA) reaction principle is similar, so also this method is called Dot-ELISA.This method compares with ELISA that to have required reagent few, does not need special instrument, saves time, and is suitable for on-the-spot the detection, has the advantages such as sensitivity identical with ELISA, and range of application is more and more wider.For adapting to cucumber fusarium axysporum requirement quick, that accurately detect, the present invention has set up the method for quick program of above-mentioned two kinds of diseases.
Summary of the invention
The present invention is intended to set up the method for quick of cucumber fusarium axysporum, and by preparing tire specific antisera and optimize spot-enzyme linked immunological absorption (dot-ELISA) method and realize the detection to target protein of height, concrete grammar and step are:
1. the anti-cucumber fusarium axysporum Antiserum Preparation of rabbit: will be kept at cucumber fusarium axysporum bacterial strain on the inclined-plane earlier and be transferred on the PDA flat board and cultivate a week, picking bacterium piece moves into and is equipped with in the triangular flask of 100mL PD fluid nutrient medium then, 25 ℃ of 120r/min shaken cultivation 15d, with culture with filter paper filtering of two-layer Whatman, collection contains the filtrate of exo-antigen, freeze drying, preserve down in-20 ℃, after getting the thawing of freeze-drying sample room temperature, centrifugal (10,000rpm 30min, 4 ℃) decon, the gained supernatant is packed in the bag filter, with sucrose volume is concentrated into about 20ml.Concentrate is carried out centrifugal (10,000rpm 30min, 4 ℃) decon, and supernatant is as the exo-antigen sample liquid.The supernatant that takes a morsel is measured protein content.Adjusting protein concentration with PBS is 1-2mg/ml, the every pipe packing of 1ml, and-20 ℃ of preservations are standby, are used for animal immune (immunogene) and ELISA and detect (antigen).
Totally 4 of healthy male rabbits about 4～6 monthly ages, body weight 2.5kg are selected in this experiment for use, are used for immunity.
Do the immunogen immune rabbit with the outer albumen of wilt mycelium, first immunisation dosage is fully emulsified for the 1ml immunogene adds equal-volume Fu Shi Freund's complete adjuvant, method is to connect 2 5mL syringes (4～No. 6 syringe needles) to taking out with proofed sleeve, become the Water-In-Oil state with adjuvant up to medicine, drip in water till the indiffusion, during injection, antigen 1 ml after every rabbit injection of first rabbit emulsification, the subcutaneous multi-point injection in back two rabbits (Cao Pengze, 1992; Shen Guanxin, 1998), near the preceding elder generation of the injection hair of edge in one's ear shaves off with blade, and 70% alcohol disinfecting, dimethylbenzene are rubbed the wiping earflap, make venous congestion.Slightly cut ear vein with blade, get the preceding ear edge vein exploitating blood 1mL of immunity, as negative serum; After this fully emulsified as booster immunization every 7d with immunizing dose 1.5mg and freund 's incomplete adjuvant, immunity is four times altogether, auricular vein injection bacterium liquid 1mL (table 2-1) behind the last immune 10d, ear vein is adopted a small amount of blood after two weeks, after spending the night, 4 ℃ of placements separate out serum, the centrifugal 15min of 5000rpm, getting the upper strata antiserum adopts indirect elisa method to survey it to tire to reach and get final product a large amount of bloodletting 10000 or more, separate out serum (spending the night), to separate out the centrifugal 15min of serum 5000rpm, packing ,-20 ℃ frozen standby down.
2. used damping fluid in reacting:
Bag is cushioned liquid: 0.05mol/L, pH9.6 carbonate buffer solution (CBS), Na2CO3,1.590g; NaHCO3,2.930g adds deionized water to 1000mL, adjust pH to 9.6,4 ℃ of storages.
Phosphate buffered solution (antigenic dilution) is (PBS): 0.01mol/L, pH7.4, NaCl, 8.000g; KH2PO4,0.200g; KCl, 0.200g; After Na2HPO412H2O, 2.900g are dissolved in the 900mL deionized water, regulate pH to 7.4, constant volume is to 1000mL, behind 121 ℃ of following autoclaving 20min, and room temperature storage.
Lavation buffer solution: 0.01mol/L, pH7.4 phosphate-tween damping fluid (PBST) adds Tween-200.5mL, Sodium Mercurothiolate sodium 0.1g, room temperature storage among the 1000mL PBS.
The sealing damping fluid: the skim milk of 5% (m/V) with the PBS preparation, adds 5g skimmed milk power and 0.01g Sodium Mercurothiolate sodium, 4 ℃ of storages among the 100mL 0.01mol/L PBS.
Antibody dilution buffer (cleansing solution): 0.01mol/L PBST (containing 2%PVP) adds PVP 4.0g, room temperature storage among the 200mL PBST.
AP mark goat anti-rabbit igg dilution: 10Mm HEPES, 0.15M NaCl, pH7.5,0.1% Tween-20,0.1% crystallization bovine serum albumin(BSA).
ELIAS secondary antibody solution: suitably dilute the alkali phosphatase enzyme mark goat anti-rabbit igg with the ELIAS secondary antibody dilution.
Substrate buffer solution (PNPP): the PNPP of 10mg is dissolved in the 10ml 0.05M pH9.8 carbonate buffer solution, and contains 0.5mM Mgcl2.
Stop buffer (2mol/LNaOH): get 8g NaOH adding distil water 100mL and be 100mL 2M NaOH.
Substrate solution: get NBT83mg, BCIP43mg is dissolved in the dimethyl formamide of 1mL respectively, respectively gets 20mL behind the mixing and adds in the 5mL substrate buffer solution, preserves about 20 days for frozen-20 ℃.
3. the preparation of the used solution of antibody purification
Saturated ammonium sulfate solution: remove ionized water 500.0mL, be heated to 80 ℃, take by weighing 400.0g (NH4) SO4, slowly add in the entry and constantly stir, up to (NH4) SO4 dissolve fully and the solution becomes clear after, put 4 ℃ and spend the night to crystallization and separate out, with preceding getting supernatant, pH is transferred to 7.0 with ammoniacal liquor.
The DE-52 cellulose: take by weighing 5g DE-52 and be dissolved in 25ml distilled water, boiling water is boiled three times repeatedly, removes the chromatographic column of packing into behind the impurity.
0.01mol/L phosphate buffered solution (PB): KH2PO4,0.200g; Na2HPO412H2O, 2.900g, be dissolved in the 900mL deionized water after, regulate pH to 7.4, constant volume is to 1000mL, room temperature storage.
20% sulfosalicylic acid solution: take by weighing the 2g sulfosalicylic acid and be dissolved in the 10mL distilled water room temperature preservation.
Barium chloride solution (2%): get 0.2g BaCl2 and be dissolved in the 10.0mL deionized water.
4. the processing of bag filter and use
Bag filter is cut into suitable length (10～15cm) segment, in 2% (W/V) NaHCO3 and EDTA (pH8.0), bag filter was boiled 10 minutes, thoroughly clean bag filter with distilled water, in the EDTA of 1mmol/L (pH8.0), boil 10min again, deposit in 4 ℃ after the cooling, must guarantee that bag filter is immersed in the solution all the time.With the preceding water of in bag filter, filling, discharge then and clean up.
Bag filter one end is had knotting, and whether the physiological saline of packing into tries with pressure slightly and leaks, if do not leak, then pour out physiological saline, extrude bubble, sample to be dialysed is packed in the bag, and fully extruding makes bag filter contact with solution, becomes a water column, then the other end is had knotting, put into dislysate, add lesser trochanter, place 4 ℃ of refrigerators slowly to stir, dialysed 72 hours, during change dislysate for several times.After dialysis finishes, the liquid in the bag filter is carefully injected the centrifuge tube of moist heat sterilization.
5. slightly propose the purifying and the concentration determination of IgG antibody
The antibody of slightly carrying is passed through the DE-52 cellulose chromatography, and with 0.01M pH7.2PB wash-out desalination, concrete grammar is as follows:
1) take out 1 chromatographic column, vertical fixing is on support.The water of the DE-52 (putting into distilled water immersion) that swelling is good is toppled over away, the PB damping fluid that adds the 0.01M pH7.4 of 2 volumes, and stir into suspending liquid, be filled into post then, open the lower end outlet of post, continue to add the DE-52 that stirs, make the gel natural subsidence, close outlet to about 4/5 of pillar height.After treating that gel column forms, adding PB damping fluid flows through gel column with the phosphate buffer of 3 times of column volumes in the wash-out bottle, with balanced gel.
2) after the gel balance, remove the solution of gel surface, the whole samples of gained of will saltouing be added to the gel column table and, open the post end opening, control flow velocity slowly enter in the gel by sample solution.Add one deck PB damping fluid on the gel cylinder, and use this buffer solution elution, about control flow velocity 0.5mL/min, collect eluent with test tube.
3) check when beginning to collect eluent whether protein has begun to flow out.By taking out 1 solution in every collection tube in the black colorimetric disc, add 1 20% sulfosalicylic acid, if present white flocculent deposit, then proof has protein, when white precipitate is can not check in inspection, stops to collect.The antibody of collecting is measured its concentration by Coomassie brilliant blue G-250 method (Bradford, 1976), and a small amount of packing postposition-20 ℃ preservation is standby.
Concrete detection method is:
1. get healthy plant and plant tissue to be measured, tissue to be detected is cut into small pieces, put into extraction tube
2. in extraction tube, add reagent No. 1, be advisable with the submergence piece of tissue
3. with the glass grinding device piece of tissue to be detected is ground
4. get tissue fluid to be detected with getting the liquid pin, put on immobilon-p
5. after treating that film is done, carry out point sample 5-7 time with getting the liquid pin repeatedly, treat that last film is dried
6. film is dipped in No. 2 reagent 1.5 hours (detection fusarium wilt);
7. sway with No. 3 reagent and give a baby a bath on the third day after its birth time each 5min;
8. film is dipped in the sickle-like bacteria immunity antiserum, room temperature was placed 2 hours;
9. sway with No. 3 reagent and give a baby a bath on the third day after its birth time each 5min;
10. film is dipped in No. 5 reagent, placed 2 hours in room temperature;
11. sway with No. 3 reagent and to give a baby a bath on the third day after its birth time each 5min;
12. film is dipped in No. 7 reagent, in room temperature lucifuge reaction 10min;
13. flowing water flushing cessation reaction, the visual method judged result: the test sample that shows of the black purple dot of all demonstrations is phytophthora root rot or droop, and the extremely shallow person of colourless or spot colors shows non-these the two kinds of diseases of test sample.
Description of drawings
The specific detection result of the indirect Dot-ELISA method of Fig. 1
A1-A8 is respectively Fusarium oxysporum 504281 body endoantigens, B1 body endoantigen, 06021601 body endoantigen, Fusarinm solani body endoantigen, Fusarium oxysporum 504281 exo-antigens, B1 exo-antigen, 06021601 exo-antigen, Fusarinm solani exo-antigen among Fig. 1.
B1-B8 is respectively Botrytis cinerea body endoantigen, black thorn dish spore body endoantigen, big beautiful Verticillium dahliae body endoantigen, melon and fruit pythium spp body endoantigen, miliary damping-off thalline endoantigen, Phytophthora capsici C2 body endoantigen, NJDP body endoantigen, NSGP body endoantigen.
C1-C8 is respectively Botrytis cinerea exo-antigen, black thorn dish spore exo-antigen, big beautiful Verticillium dahliae exo-antigen, melon and fruit pythium spp exo-antigen, Rhizoctonia solani Kuhn exo-antigen, Phytophthora capsici C2 exo-antigen, NJDP exo-antigen, NSGP exo-antigen.
Specific detection result shows, indirect Dot-ELISA method that this experiment is set up and Fusarium oxysporum can form tangible spot reaction and (see Figure of description, Fig. 1), except that can forming the more weak spot of color, infect with other that pathogen of cucumber root such as Botrytis cinerea, black thorn dish spore, downy mildew, big beautiful Verticillium dahliae, melon and fruit pythium spp, Rhizoctonia solani Kuhn, white powder, Phytophthora capsici all are negative reaction or the spot colors that forms is very shallow with Fusarinm solani.
Show that more than indirect ELISA detection method that this experiment sets up to not having specificity between the Fusarium oxysporum different strains, then has higher specificity to pathogen not of the same race.
The sensitivity testing result of the indirect Dot-ELISA method of Fig. 2
1-3 is three repetitions among Fig. 2, and last figure is the antibody that the mycelium soluble protein produces among Fig. 2; Figure below is the antibody that exo-antigen produces.To be respectively the antigen diluent multiple be 10 times, 100 times, 200 times, 400 times, 600 times, 800 times, 1000 times, 1200 times, 1500 times, 2000 times, 2500 times, 3000 times liquid for from left to right every row spot in last figure and figure below.
The indirect Dot-ELISA condition of work of setting up more than the employing is investigated the sensitivity of the detection method of setting up.The result shows, increase along with the antigen diluent degree, the formed spot colors of Dot-ELISA is more and more shallow (sees Figure of description, Fig. 2), when 1500 times of antigen diluents, still can present tangible spot, therefore the sensitivity of this experiment detection pure culture Fusarium oxysporum is 1500 times, i.e. 0.667 μ g/mL.
The effect assessment result is detected in the field of the indirect Dot-ELISA method of Fig. 3
A1-A4 is 1-4 number sick sample among Fig. 3, and B1-B4 is 5-8 number sick sample, and C1-C4 is the negative control of healthy plant; The antibody that left side figure produces for the mycelium soluble protein among Fig. 3, right figure is the antibody that exo-antigen produces.
Gather 16 strains of disease sample altogether, operate, detect effect with the field of estimating this method by indirect Dot-ELISA program.The result shows, the indirect Dot-ELISA method that adopts this experiment to set up detects the morbidity cucumber plant, 16 strain disease plants all can form tangible spot and (see Figure of description on the NC film, Fig. 3), show as positive reaction, healthy plant then shows as negative reaction, and this result is consistent with the indirect ELISA testing result.
Embodiment
By optimization to the Dot-ELISA fast detecting condition of cucumber fusarium axysporum, formulated the kit that the field is used for this kind disease quick diagnosis, the rate of accuracy reached to 95% of field prediction disease, sensitivity reaches 95%.
1. cucumber fusarium axysporum field Fast Detection Technique
1.1 the Dot-ELISA best operating condition determines indirectly
According to indirect Dot-ELISA method, determine that mycelium soluble protein and exo-antigen are respectively 1.0h and 1.5h best off-period; The working concentration of mycelium soluble protein and exo-antigen antibody is respectively 320 *; 160 *, i.e. 3.125 μ g/mL; 6.25 μ g/mL; One anti-best incubation time is 2.0h and 2.0～2.5h; The best effort concentration of mycelium soluble protein and exo-antigen ELIAS secondary antibody is respectively 800 *, 600 *; The best incubation time of ELIAS secondary antibody is 2.0h.
1.2 effect assessment is detected at the scene of cucumber fusarium axysporum cause of disease
Healthy plant, the their early stage plant, morbidity plant in mid-term, the color of morbidity plant in latter stage on the NC film deepened gradually.And the plant of a strain appearance health has in fact caught an illness, and we can draw the Dot-ELISA method and can carry out early diagnosis to cucumber fusarium axysporum thus.
2 brief summaries
With the nitrocellulose filter is solid phase carrier, is antigen with the Fusarium oxysporum, by optimizing the Dot-ELISA reaction conditions, has set up the indirect Dot-ELISA detection architecture of Fusarium oxysporum.Nitrocellulose filter is not only cheap than ELISA Plate, and can be cut into different sizes according to the test needs.When carrying out diagnosis of disease field and investigation, the cellulose nitrate membrane volume is little, is easy to carry, and can carry out a large amount of sample detection on very little volume, uses very convenient; Amount of samples is few, only needs 3～5 μ L to get final product.The substrate of reaction is deposited on the nitrocellulose filter with non-capacitive solid form, and the result can long preservation.Test shows, the Dot-ELISA detection method high specificity that this research institute sets up, and highly sensitive, this method has been simplified reaction time and the step of ELISA, will have broad prospects in the disease screening of cucumber fusarium axysporum.
3. conclusion
Process is identified the optimization and the specificity of the DOT-ELISA testing conditions of fusarium wilt (Fusarium solani), has obtained the diagnostic routine of field this disease of fast detecting, detects rate of accuracy reached to 95%.Can predict the fashion trend of disease at the initial stage of a disease,, reach the use amount that reduces agricultural chemicals, improve ecological benefits, reduce the purpose of peasant's loss to take corresponding precautionary measures.
Claims (10)
1. the method for quick of a cucumber fusarium axysporum, its feature mainly comprises: under the normal temperature condition, utilize indirect Dot-ELISA method, to the concrete trace routine of cucumber fusarium axysporum be:
(1) obtains antiserum with the mycelial soluble protein antigen immune of cucumber fusarium axysporum rabbit respectively;
(2) adopt saturated ammonium sulphate method and DEAE cellulose ion-exchange chromatography method antagonistic Serum to carry out purifying;
(3) antibody purification is carried out indirect Dot-ELISA method best operating condition screening.
(4) the on-the-spot detection: get healthy plant and plant tissue to be measured, tissue to be detected is cut into small pieces with eye scissors, put into extraction tube;
(5) in extraction tube, add reagent No. 1, be advisable with the submergence piece of tissue;
(6) with the glass grinding device piece of tissue to be detected is ground;
(7) get tissue fluid to be detected with getting the liquid pin, put on immobilon-p;
(8) treat that film is done after, carry out point sample 5-7 time with getting the liquid pin repeatedly, treat that last film is dried
(9) film is dipped in No. 2 reagent 1.5 hours;
(10) sway with No. 3 reagent and give a baby a bath on the third day after its birth time each 5min;
(11) film being dipped in antibody diluent dilution back concentration is that room temperature was placed 2 hours in the sickle-like bacteria immunity antiserum of 1.56 μ g/mL;
(12) sway with No. 3 reagent and give a baby a bath on the third day after its birth time each 5min;
(13) film is dipped in No. 5 reagent, placed 2 hours in room temperature;
(14) sway with No. 3 reagent and give a baby a bath on the third day after its birth time each 5min;
(15) film is dipped in No. 6 reagent, in room temperature lucifuge reaction 10min; Flowing water flushing cessation reaction, the visual method judged result: the test sample that shows of the black purple dot of all demonstrations is a droop, and the extremely shallow person of colourless or spot colors shows non-this kind disease of test sample.
2. according to claims 1, indirectly Dot-ELISA method best operating condition is that the mycelium exo-antigen is respectively 1.5h best off-period, the working concentration of mycelium exo-antigen antibody is respectively 3.125 μ g/mL and 6.25 μ g/mL, between the work of selection thalline exo-antigen antibody is the most in good time is 2.0h, the best effort concentration 600 of exo-antigen ELIAS secondary antibody *, the best incubation time of ELIAS secondary antibody is 2.0h; The sensitivity that detects the pure culture Fusarium oxysporum is 1500 times, i.e. 0.667 μ g/mL.
3. according to claims 1, No. 1 reagent is phosphate buffered solution (PBS): 0.01mol/L, and pH7.4 specifically prepares by NaCl 8.000g; KH
2PO
4, 0.200g; KCl, 0.200g; Na
2HPO
412H
2After O, 2.900g are dissolved in the 900mL deionized water, regulate pH to 7.4, constant volume is to 1000mL, behind 121 ℃ of following autoclaving 20min, and room temperature storage;
4. according to claims 1, immobilon-p is nitrocellulose filter (a NC film); Getting the liquid pin is the 1mL disposable syringe;
5. according to claims 1, No. 2 reagent are 5% skimmed milk power confining liquid, with skim milk, 0.5%BSA, the 1%BSA of PBS preparation 5% (m/V), 4 ℃ of storages; No. 3 reagent are lavation buffer solution: 0.01mol/L, and pH7.4 phosphate-tween damping fluid (PBST) promptly adds Tween-20 0.5mL, Sodium Mercurothiolate sodium 0.1g, room temperature storage among the 1000mL PBS; Antibody dilution buffer is PBST (containing 2%PVP);
6. according to claims 1, No. 5 reagent are the goat anti-rabbit igg solution of alkaline phosphatase (AP) mark, this solution is ELIAS secondary antibody solution, with AP mark goat anti-rabbit igg dilution, promptly 10Mm HEPES, 0.15MNaCl, pH7.5,0.1% Tween-20,0.1% the formulated solution dilution of crystallization bovine serum albumin(BSA) become 1000 times of liquid of concentration during use;
7. according to claims 1, No. 6 reagent are substrate solution (PNPP), and promptly the PNPP of 10mg is dissolved in the 10ml 0.05MpH9.8 carbonate buffer solution, and contains 0.5mM Mgcl
2
8. according to claims 1, the preparation of the used solution of antiserum purifying:
Saturated ammonium sulfate solution: remove ionized water 500.0mL, be heated to 80 ℃, take by weighing 400.0g (NH
4) SO
4, slowly add in the entry and constantly and stir, up to (NH
4) SO
4Behind dissolving and the solution becomes clear, put 4 ℃ and spend the night and separate out fully,, pH is transferred to 7.0 with ammoniacal liquor with before getting supernatant to crystallization.
The DE-52 cellulose: take by weighing 5g DE-52 and be dissolved in 25ml distilled water, boiling water is boiled three times repeatedly, removes the chromatographic column of packing into behind the impurity.
0.01mol/L phosphate buffered solution (PB): KH
2PO
4, 0.200g; Na
2HPO
412H
2O, 2.900g, be dissolved in the 900mL deionized water after, regulate pH to 7.4, constant volume is to 1000mL, room temperature storage.
20% sulfosalicylic acid solution: take by weighing the 2g sulfosalicylic acid and be dissolved in the 10mL distilled water room temperature preservation.
Barium chloride solution (2%): get 0.2g BaCl
2Be dissolved in the 10.0mL deionized water.
9. the method for quick of a cucumber fusarium axysporum, its principal character comprises that all testing processes are all carried out at normal temperatures, suitable field is on-the-spot to be detected.
10. the method for quick of a cucumber fusarium axysporum, its principal character comprises that the pathogen of cucumber fusarium axysporum is Fusariumoxysporium.
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| CN104160846A (en) * | 2014-07-15 | 2014-11-26 | 武汉市蔬菜科学研究所 | Seedling stage identification method for cowpea wilt resistance |
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| CN102520185A (en) * | 2011-11-18 | 2012-06-27 | 河南省农业科学院 | Serologic detecting kit for potyvirus on sweet potato and detecting method thereof |
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| CN103760346B (en) * | 2014-01-25 | 2015-06-24 | 云南省农业科学院生物技术与种质资源研究所 | Dot fluorescence immunoassay method for quantitatively detecting plant virus |
| CN104160846A (en) * | 2014-07-15 | 2014-11-26 | 武汉市蔬菜科学研究所 | Seedling stage identification method for cowpea wilt resistance |
| CN104160846B (en) * | 2014-07-15 | 2016-02-17 | 武汉市蔬菜科学研究所 | A kind of sprout period testifying method of cowpea anti-blight |
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