CN1904076A

CN1904076A - Detection method of soyabean phytophthora and special primer

Info

Publication number: CN1904076A
Application number: CNA2006100891054A
Authority: CN
Inventors: 文景芝; 刘春来; 杨明秀
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2006-08-03
Filing date: 2006-08-03
Publication date: 2007-01-31

Abstract

The present invention discloses a detection method of soybean phthoramycin and its special primer. Said special primer is a pair of primers formed from nucleotide sequence of sequence 1 and nucleotide sequence of sequence 2 in sequence table. Said soybean phthoramycin detection method includes the following steps: using genome DNA of material to be detected as template, using the above-mentioned a pair of primers to make PCR amplification and utilizing soybean phthoramycin as positive control, if in the amplification product the identical strip band is existed, said detected material contains soybean phthoramycin.

Description

The detection method of soyabean phytophthora and primer special

Technical field

The present invention relates to the detection method and the primer special of soyabean phytophthora.

Background technology

Soyabean phytophthora (Phytophthora sojae) can cause that soybean endangers CR Critical soybean phytophthora root rot in producing, and is the dangerous Quarantine Objects of I class that Chinese foreign is announced.Up to the present, existing many research methods are used for the detection of soyabean phytophthora to be identified, but there are many weak points in existing soyabean phytophthora detection technique, mainly is that the sensitivity and the accuracy of detection is low, detection time is long, and the incompatibility sanitary authority is the requirement of port rapid detection particularly.

Up to now, main morphological specificity according to its nourishing body and sporophore is identified in the classification of plant pathogenic fungi, and soyabean phytophthora is no exception.The detection authentication method of traditional soybean phytophthora is to wait by separation and Culture, microscopic examination form and simple physiological character mensuration to realize.The soyabean phytophthora poor growth is present in the soil with oospore, and condition is sprouted when suitable and produced zoospore and infect soybean root system and cause butt rot.Because disease sites often infects other pathogen of soybean root rot and saprophytic microorganism, therefore separation and Culture and purifying acquire a certain degree of difficulty, generally adopt in sick soil the plantation susceptible variety or with the germ zoospore in the leaf dish method trapping soil, separate also purifying pathogenic bacteria with the substratum that contains multiple antibiotic from the plant or the leaf dish of neopathy then.Though aforesaid method can effectively detect soyabean phytophthora, required time is long, program is loaded down with trivial details, sensitivity is low.

Along with serological technique and nucleic acid Study on Technology and application, making fungi detect authentication method has had great development aspect quick, sensitive.The relevant in recent years report that utilizes serological technique to detect phytophthora (comprising soyabean phytophthora) is more, but serological method then has many difficulties when practical application, mainly be that the polyclonal antibody antiserum titre is not high, specificity is not strong, be difficult to reach actual application level; Monoclonal antibody too single-minded again (may be to belong to the level level, also may be kind of level level even physiological strain level level) the omission phenomenon may occur when practical application, be difficult to use.

Summary of the invention

The purpose of this invention is to provide a kind of method and primer special that soyabean phytophthora detects that detect.

Provided by the present inventionly be used for the primer that soyabean phytophthora detects, a pair of primer of forming by the nucleotide sequence of sequence in the sequence table 1 and sequence 2.

Sequence 1 in the sequence table is made up of 18 deoxynucleotides, and sequence 2 is made up of 19 deoxynucleotides.

The method that detection soyabean phytophthora provided by the present invention detects, be that genomic dna with determinand is a template, use a pair of primer of forming by the nucleotide sequence of sequence in the sequence table 1 and sequence 2 to carry out pcr amplification, and with the positive contrast of soyabean phytophthora pure growth, as having equal band in the amplified production, then contain soyabean phytophthora in this determinand.

Preferred pcr amplification reaction system is: the genomic dna template 50ng of determinand, the upstream primer 1.0 μ l of 10pmol/ μ l, the downstream primer 1.0 μ l of 10pmol/ μ l, the dNTP 0.5 μ l of 10mM, 10 * PCR damping fluid, 5 μ l, the MgCl of 25mM ₂1.0 μ l, Taq archaeal dna polymerase 0.5 μ l, Tween-20 0.5 μ l, 0.1% BSA 5 μ l supply 50 μ L with distilled water.

Preferred PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 25sec subsequently, 56 ℃ of 25sec, 72 ℃ of 25sec, totally 30 circulations; 72 ℃ of 10min again.

Can be that 1.5% agarose gel electrophoresis detects in the amplified production whether the purpose band is arranged by concentration.

Use the inventive method, determinand can be soybean phytophthora thalline, soybean seeds, soybean plant strain or soil etc.

The present invention designs a pair of Auele Specific Primer according to by analyzing the sequence of soyabean phytophthora rDNA transcribed spacer (ITS), carries out pcr analysis with this primer, can the rapid detection soyabean phytophthora.The inventive method is a kind of quick, easy, accurate and highly sensitive Molecular Detection means, the detection that not only can be applied to bacterial strain is identified, the direct quarantine that also can be applicable to soybean seeds, soil and field disease plant detects, and can be widely used in departments such as customs, port, agricultural.

Description of drawings

Fig. 1 is the technical process of fungi total DNA extraction;

Fig. 2 is a bacteria total DNA extraction process flow process;

Fig. 3 is the technical process of diseased tissue total DNA extraction;

Fig. 4 is 20 total DNA electrophoretograms of bacterial strain;

Fig. 5 is the total DNA electrophoretogram of diseased tissues;

Fig. 6 is an oospore DNA electrophoretogram in the soil;

Fig. 7 is other fungi of phytophthora and common soil-borne disease fungal pathogens pcr amplification product electrophoretogram;

Fig. 8 is to separating the detection electrophoretogram of bacterium in soil;

Fig. 9 is to separating the detection electrophoretogram of fungi in soil;

Figure 10 detects electrophoretogram to the actinomycetes that separate in soil;

Figure 11 is the detection electrophoretogram to pure culture soyabean phytophthora zoospore;

Figure 12 A, 12B are the detection electrophoretogram of zoospore in the soil;

Figure 13 is to being inoculated in the detection electrophoretogram of the oospore in the soil;

Figure 14 is the catch an illness detection collection of illustrative plates of seed and stem tissue of soybean;

Figure 15 detects collection of illustrative plates for root tissue that soybean is caught an illness.

Embodiment

One, experiment material preparation process

1, experimental strain

1.1, the separation of soil bacterial strain

1.1.1 collecting location: soybean field, Institute of Plant Protection, academy of agricultural sciences, Heilongjiang Province experimental field, soybean field, farm, Northeast Agricultural University Xiangfang experimental field

1.1.2 acquisition method: regularly get 3 points in experimental field in same soybean field, dig out the soybean plant strain that has complete root system of some amount, be respectively charged in the sterilization bag, take back the laboratory, shake is removed loose attached to the soil on the root system gently, collect remaining soil with hairbrush and be rhizosphere soil,, and carry out immediately that fungi, bacterium and actinomycetes separate in the soil the pedotheque of 3 native mixings of collecting as this experimental plot.Simultaneously a part of rhizosphere soil is kept in-20 ℃ the refrigerator and is used for the soil Molecular Detection.

1.1.3 for the examination substratum

Fungi is used the Ma Dingshi substratum in the separation soil, and bacterium is used the beef extract-peptone nutrient agar, and actinomycetes are with improveing No. 1 substratum of Gao Shi.Common Radix Dauci Sativae agar (CA) substratum is all adopted in cultivation for each bacterial strain of examination.

Ma Dingshi substratum: KH ₂PO ₄1.0g glucose 10.0g

MgSO ₄0.5g agar 18g

Peptone 5.0g distilled water 1000mL

This substratum 1000mL adds 1% rose-bengal aqueous solution 3.3mL, the packing sterilization.Face and add 1% Streptomycin sulphate 0.3mL in every 1000mL substratum of time spent.

Beef extract-peptone nutrient agar: extractum carnis 3g peptone 5g

Agar 18g distilled water 1000mL

pH 7.0-7.2

Earlier that extractum carnis and peptone is soluble in water, acidity adjustment is to pH 7.0-7.2, and every 1000mL adds agar-agar 18g, the packing sterilization.

No. 1 substratum: KNO of improvement Gao Shi ₃1.0g KeSO ₄7H ₂O 0.01g

K ₂HPO ₄0.5g starch 20.0g

MgSO ₄7H ₂O 0.5g agar 18g

NaCl 0.5g distilled water 1000mL

After above-mentioned substance mixing and dissolving, the packing sterilization.Facing the time spent adds potassium bichromate solution in the substratum that has melted, to suppress the growth of bacterium and mould, add 3% potassium bichromate 1mL in every 300mL substratum.

Common Radix Dauci Sativae agar (CA): the 200g fresh carrot adds an amount of distilled water and smashs to pieces with tissue mashing machine, 8 layers of filtered through gauze are removed residue, add 18～20g agar in the filtrate and boil fusing, and adding distil water complements to 1000mL, packing, 121 ℃ of following autoclaving 30min.

The Radix Dauci Sativae liquid nutrient medium: the 200g fresh carrot adds an amount of distilled water to be smashed to pieces with tissue mashing machine, and 8 layers of filtered through gauze are removed residue, and filtrate complements to 1000mL with distilled water, packing, 121 ℃ of following high pressure steam sterilization 30min.

1.1.4 separation method

1.1.4.1 dilution-plate method separates soil microorganisms

Take by weighing the adding of 5g soil sample with 1/1000 balance and fill in the triangular flask of 45mL sterilized water, vibration 10min is evenly distributed in the solution soil sample, becomes soil suspension; Drawing then vibrates in 1mL soil suspension and the 9mL sterilized water makes its dilution evenly, is diluted to 10 according to 10 times of dilution methods ^-2~ 10 ^-7The soil suspension of concentration gradient.How much select proper concn to separate inoculation according to each quasi-microorganism quantity in soil.This test fungi adopts 10 ^-1, 10 ^-2Concentration gradient, actinomycetes adopt 10 ^-4, 10 ^-5Concentration gradient, bacterium adopts 10 ^-5, 10 ^-6Concentration gradient is respectively established 3 repetitions.Whole test is carried out under aseptic condition, to prevent sneaking into of inoculating microbe.

1.1.4.2 the purifying of soil microorganisms

The purifying of fungi: will tentatively distinguish the soil fungi that obtains according to the morphological specificity of bacterium colony, 25 ℃ of 1 weeks of constant temperature culture on the CA medium slant, with a small amount of mycelia piece of inoculating needle picking to the sterilization the culture dish that a certain amount of sterilized water is housed in, make spore suspension, and make suspension concentration be about 50/mL.With CA substratum fusing, to be cooled during to 40-50 ℃ every 100mL substratum add penicillin liquid (penicillin 1g+ sterile distilled water 4mL) 50 μ L with bacteria growing inhibiting.On the clean worktable of behaviour in sterilisable chamber, the aseptic Tissue Culture Plate in 96 holes is inverted, raise at the bottom of the plate, substratum is tiled in covers skim, after to be cooled the solidifying, drawing the 1mL spore suspension with micropipet evenly drips on above-mentioned substratum thin layer, cover at the bottom of the plate, put dark culturing 24h in 25 ℃ of incubators, microscopically is observed, and the spore of sprouting can form minute colony, with sterilization duckbilled tweezers single bacterium colony is scooped up and is placed on thermophilic cultivation (every ware can be put about 10) on the CA flat board, form macroscopic bacterium colony behind the 3d and be bacterial strain behind the purifying, it is chosen on the CA inclined-plane preserve.Asporogenic fungal bacterial strain is taked to carry out purifying with the method for inoculating needle picking mycelia tip.

The purifying of bacterium: will tentatively distinguish the soil bacteria that obtains according to the morphological specificity of bacterium colony, and press plate streaking partition method purifying.Dip in the transplanting ring of sterilization promptly that to get the inclined-plane bacterial colony streak culture on the CA agar plate, draw 3-5 bar line in proper order in a side of flat board earlier, again culture dish is changeed 60 °, will transplant the ring sterilization after, from second line end, mark 3-5 bar line in proper order.According to the speed of colony growth, after cultivation for some time, form little single bacterium colony on the flat board, it is chosen on the CA inclined-plane preserve.

Actinomycetic purifying: method is similar to bacterium.

From soil, isolate 38 fungal strains altogether, 18 strain bacteriums and 46 strain actinomycetes.

Strains tested totally 140 strains in the present invention's experiment: 21 strain soyabean phytophthoras (P.sojae), 2 strain phytophthora blight of pepper (P.capsici), 3 strain phytophthora infestans (P.infestans) and 2 strain phytophthoras (P.cinnamomi, P.citrophthora), reach the 10 strains common soil-borne disease fungal pathogens of bacterial strain in contrast, its host and source see Table 1, also comprise isolated 38 fungal strains from the soybean rhizosphere soil simultaneously, 18 strain bacteriums and 46 strain actinomycetes.

Table 1 strains tested and source thereof

Sequence number NO	Bacterial strain Isolates	Host Hosts	Source Locations	Sequence number NO	Bacterial strain Isolates	Host Hosts	Source Locations
Sequence number NO	Bacterial strain Isolates	Host Hosts	Source Locations	Sequence number NO	Bacterial strain Isolates	Host Hosts	Source Locations	1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41	P.sojae 855 ₁ P.sojae JK1 P.sojae 597-3 P.sojae H ₂ P.sojae Za P.sojae W ₁ P.capsici P.infestans P.infestans Botrytis cinerea Gibberella zeae Bipolaris sorokiniana Cercospora sojina Fusarium oxysporum ZJ.1-38 FJ.1-46 P.sojae R ₁ P.sojae R ₇ P.sojae R ₁₀ P.sojae R ₂₄ P.sojae R ₄₄	Soybean soybean soybean soybean soybean soybean-pumpkin potato potato cucumber wheat wheat soybean soybean * *	Harbin, Harbin, Heilongjiang Academy of Agricultural Sciences Heilongjiang Academy of Agricultural Sciences Harbin, Beijing, Keshan Beijing, Harbin, Huanan, 597 farm, Zhalantun, farm, Red River, land-reclaimable general bureau 597 farm, 855 farms	2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40	P.sojae 855 ₁ P.sojae F ₈ P.sojae Li P.sojae Ja P.sojae Aa P.capsici P.infestans P.citrophthora P.cinnamomi Magnaporthe grisea Rhizoctonia cerealis Verticillium lecanii Sclerotinia sclerotiorum Rhizoctonia solani XJ.1-18 P.sojae R ₃ P.sojae R ₉ P.sojae R ₁₃ P.sojae R ₁₅ P.sojae R ₁₃	Soybean soybean soybean soybean soybean capsicum tomato rice and wheat soybean *	Harbin, Heilongjiang Academy of Agricultural Sciences Heilongjiang Academy of Agricultural Sciences Beijing, Beijing, Jia Nan Arong Banner northeast agricultural university of northeast agricultural university Nanjing agricultural university Nanjing agricultural university Beijing, 855 farm Mishans, 597 farm

*: representative separates from soil: ZJ: represent fungi: XJ: represent bacterium; FJ: represent actinomycetes

1.2, the activation culture of bacterial strain

With all transferring on solid medium (CA) inclined-plane for the examination fungi, put (24-26 ℃) cultivation 3-4d in the constant incubator, the bacterium after the activation is transferred to cultivated in the Radix Dauci Sativae nutrient solution about (24-26 ℃) 7d, take out hypha body, blot with filter paper ,-20 ℃ of preservations are standby as far as possible.

To transfer on the CA flat board for 18 strain bacteriums of examination, put (24-26 ℃) cultivation 3-4d in the constant incubator, and the bacterium after the activation be transferred to be cultured to the bacterium logarithmic phase in the Radix Dauci Sativae nutrient solution, use in order to extracting bacterial genomes DNA.

2, used plant and instrument of test and reagent

Plant and instrument: HPG-280B illumination box (Ha Donglian), OLYMPUS inverted microscope (JAPAN), PTC-100 type PCR instrument (U.S. MJ company), UV-1601PC type ultraviolet-visible pectrophotometer, PHS-3C digital ph (Shanghai), CS501A type water-bath, HERAEUS type high speed freezing centrifuge, DYY-6C type electrophoresis apparatus (Beijing 6 1), HWS24 electric-heated thermostatic water bath (Shanghai), the 1/1000g electronic balance, the full-automatic high-pressure steam sterilizer, the full temperature vibrator of HZQ-Q (Ha Donglian), KQ-600B type ultrasonic cleaner (Kunshan, Jiangsu), Bio Imaging System GeneGeniusLP-400 ultraviolet gel imaging system (U.S.), micro sample adding appliance (Eppendorf P1000, P200, P20, P10, P2) etc.

Reagent: sodium acetate, potassium acetate, chloroform, primary isoamyl alcohol, dehydrated alcohol, Proteinase K, CTAB, SDS, Virahol, bromjophenol blue, boric acid, disodium salt sodium (EDTA), ethidium bromide (EB), agarose, trihydroxy-aminomethane (Tris), TaqDNA polysaccharase, dNTP, λ DNA Marker, DL2000 marker, Tris balance phenol, skim-milk etc.

3, the extraction of the total DNA of bacterial strain

3.1, the extraction of the total DNA of fungi

Extract the total DNA of fungi according to flow process shown in Figure 1.

3.2, the extraction of bacteria total DNA

(concentration is 1.8 * 10 to logarithmic phase with the bacterium shaking culture ⁸Individual/as mL), to get the 1mL bacterial suspension and join in the 1.5mL centrifuge tube, the centrifugal 5min of 7500rpm abandons supernatant.Extract bacteria total DNA according to flow process shown in Figure 2.

3.3, the extraction of the total DNA of actinomycetes

Extract the total DNA of actinomycetes according to following program:

1) thalline is prepared:, add liquid nitrogen and fully grind, and put it in the ampoul tube in mortar with the less lawn of aseptic bamboo let picking from solid medium.

2) add washings (50mmol/L Tris, pH7.7,25mmol/L EDTA, 0.1% PVP) 1mL in the ampoul tube of thalline is housed, 5s vibrates on vortex mixer;

3) the centrifugal 1min of 5700rpm abandons supernatant;

4) add 35 μ L lysates (50mmol/LTris, pH8,25mmol/L EDTA, 3% SDS, 1.2% PVP), vibration suspends, and is to handle 60s in the 600W microwave oven in power;

5) extract (10mmol/L Tris, pH8,1mmol/L EDTA, 013mol/LNaAc, 1.2% PVP) of 65 ℃ of preheatings of adding 400uL, vibration 5s;

6) add the extracting of the saturated phenol-chloroform solution of isopyknic Tris, the centrifugal 5min of 10000rpm;

7) supernatant is with isopyknic isopropanol precipitating, and 70% washing with alcohol is dissolved in after the drying in the 20 μ L TE solution.

4, the soybean phytophthora diseased tissue detects

The acquisition of root tissue 4.1 soybean catches an illness

1) for examination soybean varieties: Williams82 (disease-resistant variety), Sloan (susceptible variety)

2) preparation of zoospore suspension: the several ware zoospore suspension that obtain by the described method of 2.1.6 merge, pours in the 500mL triangular flask, and mixing, and calculate the wherein concentration of zoospore suspension.

3) cultivation of soybean seedling: the respectively Williams82 of picking full seed and Sloan soybean seeds number, put in the ceramic whiteware dish that fills less water, upward apply several layers of gauze and preserve moisture 25 ℃ of incubator vernalization 2-3d.When treating the long 2cm of the bud left and right sides, its branch is installed in the ampoule, add the tap water of equivalent in the bottle, every bottle is amplified bean seedlings 2 strains.The rearmounted fixed temperature and humidity illumination box of bottling is interior to be cultivated.

4) inoculation: treat that soybean seedling length to 1 compound leaf during phase, draws a certain amount of zoospore suspension with the 1mL pipettor, be inoculated in the bottle 1000 zoospores of every bottle graft kind.After inoculation finishes, bottle is put back in the fixed temperature and humidity illumination box.

5) sampling: 2 strains (i.e. soybean seedling in 1 bottle) are all got in each sampling, respectively get two big bean seedlings of kind before the inoculation respectively in contrast; Behind the inoculation zoospore, every the 15min sampling once, for avoiding attached to the seedling root but the zoospore of not invading test is exerted an influence, the seedling of taking out is all vibrated in ultrasonic cleaner earlier behind the 5min, with the tap water flushing root several seconds, blot root with filter paper, cut the seedling root and be put in the sealed bag, in-20 ℃ of refrigerator-freezers, preserve standby.

The acquisition of stem's tissue 4.2 soybean catches an illness

2) inoculation method: adopt hypocotyl wound inoculation method.

3) plantation of soybean seedling: respectively with the planting seed of above 2 kinds in the polypots that sterilization soil is housed, when treating that plant grows to true leaf and launches, select and wherein count strains inoculation soyabean phytophthoras, be susceptible contrast with susceptible variety Sloan, and establish blank.Take a sample behind the 4d with the plastic lousing 60h that preserves moisture in inoculation back.

4) sampling: upwards cut macroscopic brown along inoculation position and extend morbidity stem tissue,, and, preserve standby in-20 ℃ of refrigerators with in its sealed bag of packing into as stem's diseased tissues material of this test.

The acquisition of seed 4.3 soybean catches an illness

1) for the examination soybean varieties: close rich 25 (susceptible variety)

2) inoculation method: adopt the inoculation of bacteria suspension injection, the soybean phytophthora bacteria suspension for preparing is injected beanpod, 0.2mL/ pod with clean syringe.

3) inoculation period: respectively in soybean beanpod drum grain back but beanpod when still be green and beanpod by green commentariess on classics yellow two periods, the selection more consistent beanpod of growing is inoculated.

4) results backs strip off beanpod, get seed pack into standby in the sealed bag, the period of record inoculation.

4.4 the extraction of the total DNA of diseased tissue

Extract the total DNA of diseased tissue according to flow process shown in Figure 3, earlier 2%CTAB is extracted damping fluid before extracting and be preheated to 65 ℃.

5, the preparation of soyabean phytophthora zoospore, oospore suspension

5.1, water culture induces sporocyst to produce

The soyabean phytophthora that the inclined-plane is preserved is being transferred on the CA flat board under the aseptic condition, puts and cultivates 3-5d (easier the bringing out of young tender mycelia produces sporocyst) in 25 ℃ of constant incubators.Cut colony edge with aseptic punch tool, several piece bacterium dish is put into aseptic empty culture dish, the bacterium dish faces up, and pours the sterilization tap water into and does not have bacterium dish surface until just, and continuous irradiation is cultivated 24-48h and can be produced a large amount of sporocysts under fluorescent lamp.Change the tap water of once sterilizing every 6-8h during this time and produce to promote sporocyst, microscopy is observed the sporocyst production.

5.2, stimulate sporocyst to discharge zoospore

Having a large amount of sporangial bacterium dish to choose above-mentioned generation fills in the culture dish of sterilization tap water about 10mL, put in the 5-10 ℃ of refrigerator behind the 10-15min, taking-up places 10-30min under the thermophilic (20-26 ℃), or above-mentioned culture dish put under the room temperature, change sterile purified water once about every 30min, after changing water 3-4 time, culture dish put in 25 ℃ of incubators cultivate 12h, can stimulate sporocyst to discharge zoospore in a large number.

5.3, the preparation of oospore suspension

The soyabean phytophthora that the inclined-plane is preserved is being transferred on the CA flat board under the aseptic condition, puts that (about 20 ℃) can produce a large amount of oospore after cultivating for 2 weeks under the room temperature.Cut the agar block that contains oospore from the plate that covers with soyabean phytophthora, add sterile purified water 100mL, with refiner (5000rpm) homogenate 1.5～2min, homogenate is through 200 orders (aperture 76 μ m), 300 orders (aperture 54 μ m), 600 orders (aperture 25 μ m) screen filtration, after washing 200 orders and 300 eye mesh screens with sterile purified water 1000mL, sweep away the oospore of collecting on 600 eye mesh screens with a small amount of sterile purified water, be prepared into oospore suspension.

5.4, the counting of zoospore and oospore

Draw zoospore (oospore) suspension 5 μ L on clean slide with micro sample adding appliance, when discharging suspension, the sample injector suction nozzle is slightly dragged towards the right side, suspension is dragged into a thin short band on slide glass, take a sample altogether 5 times, under 10 * 10 power microscope visuals field, calculate the sum of zoospore (oospore), obtain the mean value of zoospore (oospore) number in the 5 μ l suspension.Calculation formula is as follows:

6 pure culture soyabean phytophthora zoospores detect

6.1 the acquisition of zoospore is with 5

6.2 the broken and detection of zoospore

Adopt the quartz sand crush method.In the aseptic EP pipe of 1.5mL, add 0.05g quartz sand (analytical pure), add 45 μ L aseptic deionized waters again, add 10 μ L zoospore suspension (5.5/μ L) subsequently, vortex 5min, the centrifugal 2min of 5000rpm, directly getting 0.3～10 μ L supernatant liquor then is that template is carried out the PCR detection.

The soyabean phytophthora zoospore detects in 7 soil

7.1 the acquisition of zoospore is with 5

7.2 the broken and detection of zoospore in the soil

Taking by weighing 0.3g soil with 1/1000 electronic balance joins in the aseptic EP pipe of 1.5mL, add the aseptic tap water of 835 μ L, add 165 μ L zoospore suspension (about 1000 of microscope direct census) subsequently, mixing leaves standstill several minutes, gets supernatant liquor 100 μ L and puts into another aseptic EP pipe, add 0.05g quartz sand (analytical pure), vortex 5min, the centrifugal 2min of 5000rpm, directly getting 0.3～10 μ L supernatant liquor is that template is carried out the PCR detection.

Carry out following test simultaneously for reducing error: the electronic balance with 1/1000 takes by weighing 6 parts of 0.3g soil respectively and joins in 6 aseptic EP pipes of 1.5mL, add 987.5 μ L respectively, 975 μ L, 950 μ L, 925 μ L, 875 μ L, the aseptic tap water of 750 μ L, add 12.5 μ L subsequently, 25 μ L, 50 μ L, 75 μ L, 125 μ L, 250 μ L zoospore suspension (4/μ L), mixing, leave standstill several minutes, get supernatant liquor 100 μ L respectively and put into another aseptic EP pipe, add 0.05g quartz sand (analytical pure), vortex 5min, the centrifugal 2min of 5000rpm, directly getting 1 μ L supernatant liquor respectively is that template is carried out the PCR detection.

Oospore detects in 8 soil

8.1 the acquisition of oospore is with 5

8.2 the extraction of oospore DNA in the soil

The method of DNA extraction main reference Volossiouk T (1995), this method is a kind of method of directly extracting DNA from the soil that contains a small amount of oospore, and the DNA that extracts does not need purifying, as long as after the total DNA that is obtained suitably diluted, can carry out next step PCR and detect.Its step is as follows:

1) the soil sample 1g that will contain the some amount oospore puts in the mortar, fully grinds about 5min or becomes even fine powdered up to soil with liquid nitrogen.

2) powdery soil after will grinding is suspended in (mixture of 0.1g skim-milk and 25mL water) in the 2mL skim-milk solution, carries out vortex simultaneously.

3) the centrifugal 10min of 12000rpm (4 ℃) removes the soil residue behind the vortex.

4) get supernatant and mix, carry out vortex simultaneously with 8mLSDS extraction damping fluid (0.3%SDS is added among 0.14M NaCl, the 50mM NaAc [pH5.1]).

5) add isopyknic Tris balance phenol (water-saturated phenol), intermittently vortex 2min, the centrifugal 10min of 12000rpm then under room temperature.

6) get the dehydrated alcohol that the supernatant liquor that contains DNA adds 2.5 times of volumes, a few hours or spend the night.

7) 4 ℃ of down centrifugal acquisition DNA precipitations, precipitation is with 70% washing with alcohol twice, and is centrifugal, drying.

8) dried precipitation is dissolved in the deionized water of 100 μ L, packing-20 ℃ preservation is standby.

Two, pcr amplification

1, special primer design

ITS region sequence according to the soybean blight bacterium of delivering on the GenBank, sibling species and sibling species rDNA, utilize DNAMAN software that these sequences are carried out diversity ratio and reach homology analysis, and utilize the synthetic oligonucleotide special primer of the distinctive one section conserved sequence design of the selected soybean phytophthora of OLIGO software right:

Upstream primer (18bp): 5 '-CTG GAT CAT GAG CCC ACT-3 ';

Downstream primer (19bp): 5 '-TCT CCA TCC ACC GAC TAC A-3 '.

2 PCR response procedures and reaction systems

The consumption of each amplification factor in the 50 μ L reaction systems:

Template DNA (50ng) 1.0 μ l, upstream and downstream primer (10pmol/ μ l) 1.0 μ l, dNTP (10mM) 0.5 μ l, 10 * PCR buffer, 5 μ l, MgCl ₂(25mM) 1.0 μ l, Taq archaeal dna polymerase 0.5 μ l, Tween-20 0.5 μ l, BSA (0.1%) 5 μ l, an amount of distilled water is supplied 50 μ L.

The PCR response procedures:

1. 94 ℃ of pre-sex change 5min, 2. 94 ℃ of sex change 25sec, 3. 56 ℃ of annealing 25sec, 4. 72 ℃ are extended 25sec (circulate 30 times), 5. at last at 72 ℃ of extension 10min down.

3 PCR product agarose gel electrophoresis detect

The preparation of (1) 1.5% sepharose: take by weighing agarose 1.05g, put into the 250mL triangular flask, add tbe buffer liquid 70mL, heating 1min makes it dissolving in microwave oven.Be cooled to about 60 ℃, add 5 μ LEB (5mg/mL), mixings gently.The gel bed is put well, earlier sepharose solution is poured into, insert comb again, make comb tooth be higher than base plate 0.5-1.0mm.Produce if any bubble, with the fragmentation of rifle head, treat that gel hardens fully after.Carefully extract comb, whether the sample for reference hole is complete, and gel piece is taken out, and puts into the electrophoresis chamber that electrophoretic buffer is housed.

(2) point sample: draw 10 μ L PCR products, mix, sample is added in the sample well, compare with DL2000 DNA Marker with 10 μ L liquid-transfering guns with an amount of bromjophenol blue solution.

(3) electrophoresis:, stop electrophoresis when bromjophenol blue swimming during to 3/4 place of whole gel film.

(4) take a picture: gel is put the observation of full automatic gel imaging system and take pictures the record electrophoresis result.

Three, experimentation and result

1, total DNA abundance detects

Agarose gel electrophoresis detects: good DNA mother liquor 1 μ L detects its abundance at 0.8% agarose gel electrophoresis that contains EB to get dissolving, with λ DNA (50ng/ μ L) as standard (Marker), constant voltage electrophoresis 40-50min, observe and take pictures with the full automatic gel imaging system, by relatively determinand and the λ DNA brightness deduction DNA concentration that obtains.If several concentration gradients are got partial mother liquid and are diluted to and are placed in 4 ℃ of refrigerators stand-byly about 50ng/ μ L, all the other mother liquors are placed in-20 ℃ of refrigerators and preserve.

1) strains tested DNA extraction

The sepharose that preparation 0.8% contains EB carries out total DNA electrophoresis to 140 pure culture bacterial strains that extracted, and the electrophoresis result of 20 bacterial strains is wherein seen Fig. 4; Among the figure, swimming lane 1-2: soybean phytophthora bacterial strain P.sojae 597-3, R24; 3-6: other phytophthora strain P.cinnamomi, P.citrophthora, P.capsici, P.infestans; 7-11: common soil-borne fungus Botrytis cinerea, Fusarium oxysporum, Magnaporthe grisea, Rhizoctonia solani, Sclerotinia sclerotiorum; 12-20: isolating 3 kinds of fungies, 3 kinds of bacteriums and 3 kinds of actinomycetes from soil.

As seen from the figure, occur apparent bands of a spectrum behind the DNA electrophoresis of the bacterial strain that detects, none degraded has obtained more complete genomic dna, is applicable to that all next step pcr amplification detects.

2) the catch an illness extraction of the total DNA of soyabean tissue

Adopt the 2%CTAB method to extract total DNA of root, stem and the seed tissue sample of the soybean that catches an illness, and the total DNA that is extracted carried out electrophoresis, the result as shown in Figure 5, among the figure, swimming lane 1-2:Williams82 root (not inoculating and inoculate 2h); 3-4:Sloan root (not inoculating and inoculate 1h); 5:Sloan stem (inoculation); 6-7: the kind skin and the embryo (not inoculation) that close rich 25 seeds; 8-9: the drum grain phase is closed the kind skin and the embryo (inoculation) of rich 25 seeds; 10-11: the ripening stage is closed the kind skin and the embryo (inoculation) of rich 25 seeds

As seen from the figure, apparent bands of a spectrum all appear in each swimming lane behind the electrophoresis, illustrate that total DNA amount and the integrity extracted are all fine, suitable next step test.

3) extraction of soyabean phytophthora oospore DNA in the soil

The method of genomic dna is extracted in employing from micro-soil, extract DNA from the 1g soil that contains 10 oospore, electrophoresis result as shown in Figure 6, among the figure, swimming lane 1-2: control soil; 3-4: inoculation soybean phytophthora oospore soil.

As seen from the figure, obtained apparent bands of a spectrum, obtained more complete genomic dna, for further the detection of oospore in the soil being laid a good foundation.

2, primer specificity check

2.1 detected result to other fungi of phytophthora and common soil-borne disease fungal pathogens

For verifying the specificity of designed primer, be template with total DNA of 7 strain phytophthora fungies and the common soil-borne disease fungal pathogens of 10 strains, with the positive contrast of DNA of soybean phytophthora pure culture bacterial strain, carry out pcr amplification, as shown in Figure 7, M:DL2000 marker; 1,13: the soybean phytophthora bacterial strain; 2-8:P.cinnamomi, P.citrophthora, P.capsici (pumpkin), P.capsici (capsicum), P.infestans (potato, Harbin), P.infestans (potato, Keshan), P.infestans (tomato); The 9-12:19-22 bacterial strain; The 14-19:23-28 bacterial strain.

As can be seen, have only the soybean phytophthora bacterial strain to produce a length and be about specific amplification products fragment about 288bp among the figure, all other bacterial strains for examination all do not have any amplified band and occur, and illustrate that this primer has stronger specificity.

2.2 detected result to the soil microorganisms pure culture

Adopt the soybean phytophthora special primer of design, with total DNA of separating 18 strain bacteriums in soil, 38 fungal strains, 46 strain actinomycetes pure cultures is template, with the positive contrast of DNA of soybean phytophthora bacterial strain pure culture, carry out pcr amplification, the result is shown in Fig. 8,9,10; Among Fig. 8, M:DL2000 marker; 1,8,9,14: the soybean phytophthora bacterial strain; Other swimming lane is for separating the bacterium in soil; Among Fig. 9, M:DL2000 marker; 1,7,8,9,14,15,22,28,29,30,35: the soybean phytophthora bacterial strain; Other swimming lane is for separating the fungi in soil; Among Figure 10, M:DL2000 marker; 1,8,9,10,15,16,22,28,29,35,36: the soybean phytophthora bacterial strain; Other swimming lane is for separating the actinomycetes in soil.

As can be seen, all amplify a length for the soybean phytophthora bacterial strain of examination and be about specific amplification products about 288bp, and all do not have the generation of amplification bands of a spectrum for all bacteriums, fungi and the actinomycetes strain of examination, further specify this special primer and soyabean phytophthora is had the specificity of planting.

3 soyabean phytophthora pure culture pcr amplification product sequencing analysis

With soyabean phytophthora pure culture DNA is template, increase according to the PCR program, obtain the PCR product, its sequence is shown in sequence in the sequence table 3, length is 287bp, the sequence surveyed is carried out sequence relatively by Blast, and the number of landing is that the homology of soybean phytophthora bacterial strain sequences such as AY423301, AY590273, AY590274, AF266769 reaches 98% on this sequence and the Genbank, illustrates that utilizing the designed special primer fragment that obtains that increases is the product of soyabean phytophthora.

The detection of 4 detection methods is used

4.1 detection to pure culture soyabean phytophthora zoospore

Adopting the quartz sand crush method, is that template is carried out pcr amplification with the broken liquid of quartz sand zoospore directly, the result as shown in figure 11, among the figure, M:DL2000 marker; 1-10: be respectively broken liquid 0.3,0.5,1,2,3,4,5,6,8, the 10 μ L of zoospore; 11: negative control; 12: positive control (soybean phytophthora mycelium DNA).

As can be seen from Figure 11, different template amounts all can obtain the specific amplification products of 288bp, and along with the increase of template amount, amplified production also has the trend that increases, and the theoretical precision of detection can reach 0.3 zoospore.

4.2 detection to zoospore in the soil

Adopt the soyabean phytophthora zoospore that uses the same method to being inoculated in the soil and detect, the result shown in Figure 12 A, among the figure, M:DL2000 marker; 1-10: be respectively broken liquid 0.3,0.5,1,2,3,4,5,6,8, the 10 μ L of zoospore in the soil; 11: negative control; 12: positive control (soybean phytophthora mycelium DNA).The result shows that different template amounts all can obtain the specific amplification products of 288bp, and the theoretical precision of detection can reach 0.3 zoospore.But a little less than the luminance factor pure culture zoospore of amplified production bands of a spectrum, and along with the increase of template amount, amplified production does not show the trend of showed increased.When particularly the template amount was 6 μ L and 8 μ L, the brightness of band was very weak, and the amount that amplified production is described is not a lot of relatively, and tracing it to its cause may be after the template amount increases, to have the soil ulmin of negative impact also to increase relatively to PCR in the soil.

In order to eliminate the testing error that inhibitory substance such as soil ulmin in the soil are brought, take the method for template amount unanimity, promptly carry out PCR when detecting the template amount all get 1 μ L, but its contained real zoospore quantity is different.Shown in Figure 12 B, M:DL2000 marker among the figure; 2-7: template content is respectively 0.05,0.1,0.2,0.3,0.5,1 zoospore; 1: negative contrast; 8: positive contrast (soybean phytophthora mycelium DNA).The result shows that each swimming lane has all produced the specific fragment of a 288bp, amplifies band but not all template amount can both be stable, when having only the template amount to contain content more than or equal to 0.3 zoospore, could produce stable expanding effect.

4.3 to being inoculated in the detected result of the oospore in the soil

The method of genomic dna is extracted in utilization from micro-soil, extract DNA as template from the 1g soil that contains 10 oospore, gets the template amount of 1-7 μ L and carries out the PCR detection, the results are shown in Figure 13, among the figure, and M:DL2000 marker; Swimming lane 3-9 template amount is respectively 1,2,3,4,5,6,7 μ L; Swimming lane 2 is control soil (not inoculating oospore); Swimming lane 10 negative contrasts.The result shows that each template amount all amplifies the specific amplification fragment of 288bp, and along with the increase of template amount, the amount of amplified production also has the trend of increase simultaneously.The theoretical precision that detects is minimum to reach 0.06 oospore.

4.4 to infecting the diseased tissues detected result of soyabean phytophthora

4.4.1 detection to seed and the stem tissue of catching an illness

The results are shown in Figure 14, among the figure, M:DL2000 marker; 1: positive contrast (soybean phytophthora mycelium DNA); 2,3: the kind skin and the embryo of healthy seed; 4,5: the kind skin and the embryo of drum grain phase inoculation morbidity seed; 6,7: the kind skin and the embryo of ripening stage inoculation morbidity seed; 8: the morbidity stem; (1-8: the template amount is 0.2 μ L; 9-16: template is identical with 1-8, and just the template amount is 0.5 μ L).The result shows that healthy soybean stem, seed are organized does not all have the amplified band generation; Catch an illness stem and seed tissue (comprising kind of skin and embryo two portions) and pure culture soybean phytophthora mycelium all can amplify the specific fragment of 288bp, but the embryo of the seed of ripening stage inoculation morbidity does not have amplified production, shows that the PCR detection method based on this special primer can be directly used in the soyabean phytophthora that detects in the soybean diseased tissue.In addition, the PCR detected result shows that also at drum grain phase inoculation soyabean phytophthora, germ can be invaded in the kind skin and embryo of soybean kernel; And when inoculating in the ripening stage, soyabean phytophthora can only invade kind of a skin portion, fails to reach in the embryo.

4.4.2 detection to the root tissue of catching an illness

In order to study intrusion and the field planting time of soyabean phytophthora zoospore at soybean root, and check the sensitivity of this PCR detection method, quantitatively inoculate the seedling root of Williams82 (disease-resistant variety) and Sloan (susceptible variety) respectively with soyabean phytophthora zoospore (1000/bottle), every the 15min sampling once, and to the sample of being gathered carry out the PCR detection.The result as shown in figure 15, M:DL2000 marker; 1,6: positive control (soybean phytophthora mycelium DNA); The healthy root of 2:Sloan; The healthy root of 7:Williams82; 3-5:Sloan root template amount 0.2,0.5, the 1 μ L (sample time for inoculation back 1h) that catches an illness; 8-10:Williams82 root template amount 0.5,1, the 2 μ L that catch an illness; (being inoculation back 2h sample time).

Susceptible variety Sloan seedling root inoculation back 1h can detect exist (the swimming lane 3-5) of soyabean phytophthora; And for disease-resistant variety Williams82, positive test symbol is then postponed till postvaccinal 2h and (swimming lane 8-10) just occurred, occur on the required minimum template amount from the male detected result, the template amount that disease-resistant variety Williams82 needs is 0.5 μ L, Duo 1.5 times than the template amount 0.2 μ L that the susceptible variety positive detection needs, show that under identical condition susceptible variety will be early than disease-resistant variety on the time that soyabean phytophthora detects, and required template amount also is less than disease-resistant variety.Will be early than the enantiopathy kind thereby further specify the soyabean phytophthora zoospore to the intrusion time of susceptible variety, the amount of growing surely is also more than disease-resistant variety, illustrate also that simultaneously the PCR detection method of setting up with this special primer has very high detection sensitivity to the soyabean phytophthora in the root tissue of catching an illness, detect sense, disease-resistant variety infect and the time of growing is postvaccinal 1-2h surely.

Above experimental result shows:

1, primer of the present invention has very strong specificity to soyabean phytophthora, can detect the soyabean phytophthora of pure culture specifically.

2, use this primer system, set up the detection method of soyabean phytophthora, can detect the soyabean phytophthora zoospore in pure culture and the soil specifically, the theoretical precision of detection can reach 0.3 zoospore; Also can detect the soyabean phytophthora oospore in the soil specifically, the theoretical precision of detection is minimum to reach 0.06 oospore; Can also detect the phytophthora in the soybean kernel of inoculating soybean root, hypocotyl and the different development stage inoculation acquisition of falling ill specifically; Soyabean phytophthora in the root tissue of catching an illness also had very high detection sensitivity.

3, the present invention's detection method of establishing soyabean phytophthora is finished (DNA extraction 4-5h, pcr amplification 3h, electrophoresis 1h) in 1d, and detected result high specificity, highly sensitive, good stability.

Sequence table

<160>3

<210>1

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

ctggatcatg agcccact 18

<210>2

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

tctccatcca ccgactaca 19

<210>3

<211>287

<212>DNA

＜213〉soyabean phytophthora (Phytophthora sojae)

<400>3

ctggatcatg gccccctttt taaacccctt cttaaatact gaatatactg tggggacgaa 60

agtctctgct tttaactaga tagcaacttt cagcagtgga tgtctaggct cgcacatcga 120

tgaagaacgc tgcgaactgc gatacgtaat gcgaattgca ggattcagtg agtcatcgaa 180

attttgaacg catattgcac ttccgggtta gtcctgggag tatgcctgta tcagtgtccg 240

tacatcaaac ttggctctct tccttccgtg tagtcggggg atggaga 287

Claims

1, a kind ofly is used for the primer that soyabean phytophthora detects, a pair of primer of forming by the nucleotide sequence of sequence in the sequence table 1 and sequence 2.

2, detect the method that soyabean phytophthora detects, be that genomic dna with determinand is a template, use a pair of primer of forming by the nucleotide sequence of sequence in the sequence table 1 and sequence 2 to carry out pcr amplification, and with the positive contrast of soyabean phytophthora pure growth, as having equal band in the amplified production, then contain soyabean phytophthora in this determinand.

3, method according to claim 2, it is characterized in that: the pcr amplification reaction system is: the genomic dna template 50ng of determinand, the upstream primer 1.0 μ l of 10pmol/ μ l, the downstream primer 1.0 μ l of 10pmol/ μ l, the dNTP 0.5 μ l of 10mM, 10 * PCR damping fluid, 5 μ l, the MgCl of 25mM ₂1.0 μ l, Taq archaeal dna polymerase 0.5 μ l, Tween-20 0.5 μ l, 0.1% BSA 5 μ l supply 50 μ L with distilled water.

4, method according to claim 2 is characterized in that: described PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 25sec subsequently, 56 ℃ of 25sec, 72 ℃ of 25sec, totally 30 circulations; 72 ℃ of 10min again.

5, method according to claim 2 is characterized in that: detecting amplified production is to carry out 1.5% agarose gel electrophoresis.

6, according to the arbitrary described method of claim 2-5, it is characterized in that: described determinand is soyabean phytophthora thalline, soybean seeds, soybean plant strain or soil.