CN104846094A - Quick detection method for a variety of pathogen molecules of strawberry root rot diseases and application of quick detection method - Google Patents
Quick detection method for a variety of pathogen molecules of strawberry root rot diseases and application of quick detection method Download PDFInfo
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- CN104846094A CN104846094A CN201510250463.8A CN201510250463A CN104846094A CN 104846094 A CN104846094 A CN 104846094A CN 201510250463 A CN201510250463 A CN 201510250463A CN 104846094 A CN104846094 A CN 104846094A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a quick detection method for a variety of pathogen molecules of strawberry root rot diseases and application of the quick detection method. The method is characterized in that DNA (Deoxyribose Nucleic Acid) of pathogen is subjected to PCR (Polymerase Chain Reaction) reaction by using a general specific primer of pathogenic fungus; when a single strip of about 500bp is represented by a PCR product, the PCR product is subjected to sequencedsequencing; the sequencing result is compared in an NCBI (National Center For Biotechnology Information) database, so that different pathogens which are contained in a disease sample tissue and cause the strawberry root rot diseases can be defined. The method has the advantages of simplicity in operation, short time and large flux; the method can be used for qualitatively detecting root part disease samples of seedlings in a strawberry nursery, so that the spread and the incidence of the strawberry root rot diseases can be effectively avoided, and the construction of strawberry disease-free seedling production bases and safe production of the strawberry disease-free seedling in China are guaranteed.
Description
Technical field
The invention belongs to fruit diseases control and technical field of plant quarantine, relate to the Strawberry Root Rot multiple nosophyte numerator method for quick of evil and application.
Background technology
Strawberry Root Rot is the large class disease general name jointly caused by soil-borne disease fungal pathogens.The Pathogens of Root Rot thing that in world wide, strawberry main producing region has been reported reaches more than 20 and plants, and is a kind of soil-borne disease of more difficult control.Common pathogenic bacteria has: what cause strawberry black root rot mainly contains sickle-like bacteria (Fusarium spp.), Fusarium oxysporum (F.oxysporum), dry thread Pyrenomycetes (Rhizoctoniasolani), pythium spp (Pythium sp.), plan pestalotia bacteria (Pestalotiopsis sp.); The pathogenic bacteria of Strawberry red stele root rot is caused to be phytophthora (Phytophthorafragariae C.J.Hickman).Strawberry Root Rot classical symptom cuts old complaint or peel root exterior skin to see garnet diseased tissues, forms distinct contrast with healthy position; Rapidly, over-ground part is wilting suddenly in underground part morbidity, and complete stool green grass or young crops is withered to die.Root rot sickness rate is 20% ~ 30%, and indivedual area reaches 50 ~ 80%, disease plant mortality ratio 100%.Strawberry black root is rotten and the harm of red center pillar root-rot in Diseases of Strawberry is the heaviest, have the greatest impact in recent years, has a strong impact on output and the benefit of strawberry, becomes the bottleneck problem of strawberry cultivating development.
The pathogenic fungi of Strawberry Root Rot is caused to have the feature of obvious latent infection.On strawberry, the latent infection of pine root fungus is very general phenomenon, when envrionment conditions is applicable to morbidity, Strawberry Root Rot will occur.The asexual sapling multiplication of general use in strawberry production, between each department, the mobility of seedling is also very frequent.If the seedling of transplanting there is the latent infection of root-rot pathogenic bacteria, often causes the trans-regional long propagation of pathogenic bacteria, cause root rot regionality to break out, bring tremendous economic to lose to strawberry production.Therefore, the generation of Strawberry Root Rot can be avoided from root with the aseptic seedling of the nursery base breeding occurred without root rot.But along with cultivated area expands, add that disease spread increasing extent is extensive, this method difficulty is very large.Therefore it is very important for setting up effective seedling detection technique, effectively can avoid propagation and the generation of Strawberry Root Rot.But due to the aobvious disease of the seedling that carries disease germs, and some methods of inspection of development at present are because be loaded down with trivial detailsly consuming timely not easy promotion and implementation, or bring extreme difficulty to the inspection of current pine root fungus.The cause of disease of Strawberry Root Rot is complicated, and chemical prevention and control method reasonable employment prerequisite is exactly to make the pathogenic micro-organism kind of this area Strawberry Root Rot clear, suits the remedy to the case and just can play effect of getting twice the result with half the effort.Meanwhile, the residue problem brought of chemical prevention and the destruction of ecotope be can not be ignored.The method of traditional pathogenicbacteria separation culture identification depends on again the knowwhy of the pathogenic bacteria classification of professional's rich experience and system, will affect best control opportunity like this adversely.At present, prevent and treat Strawberry Root Rot and also there is no effective method.Mainly take based on cultural control, chemical prevention is auxiliary comprehensive control measures.Therefore, Strawberry Root Rot should, to put prevention first, be strengthened emphatically detecting the quarantine of this disease.For preventing Strawberry Root Rot from importing Pest-or disease-free area into from epidemic-stricken area, guaranteeing the safety in production of domestic strawberry, setting up a set of stable, easy, quick, sensitive molecular detecting method and strawberry seedling detection of quarantining is very important.PCR detection method has the advantages such as highly sensitive, precision is high, sampling amount is small, detection time is short.By this technology to qualitative detection such as strawberry seedlings, for guaranteeing construction, the safety in production of China's strawberry disease-free seedlings production base, intercept simultaneously external danger in spite of illness strawberry seedling to import China into significant.
Summary of the invention
The object of the invention is to the defect overcoming existence of the prior art, a kind of Strawberry Root Rot evil nosophyte numerator method for quick is provided.The method is easy to operation, directly applies to production practice, for the highly sensitive rapid detection of the plant tissue that carries disease germs, ensures production to provide healthy aseptic seedling, be of great significance nosophyte numerator early warning tool.
Another object of the present invention is to the application that described Strawberry Root Rot nosophyte numerator method for quick is provided.
Its concrete technical scheme is:
The multiple nosophyte numerator method for quick of Strawberry Root Rot evil, comprises the following steps:
The separation of step 1. pathogenic bacteria
The pathogenic bacteria of tissue isolation to strawberry root is adopted to be separated, cut the fritter of the diseased tissues 4-5mm of the strong intersection of disease, be placed in 75% ethanol and soak 5s, then diseased tissues is moved in 0.1% mercuric chloride solution and soak 3-5min, again diseased tissues to be transferred in sterilized water rinsing 3 times, finally moved on to by tissue on PDA substratum, placed by culture dish in 25 DEG C of incubators and cultivate 3-5d, namely colony diameter 1cm carries out DNA extraction experiment;
Step 2. pathogenic bacteria DNA extracts on a small quantity
1) with a small amount of pathogenic bacteria mycelia on sterilizing toothpick scraping PDA flat board, load in the centrifuge tube of 2.0ml;
2) 500 μ l Lysis buffer are added, 1 broken pearl, Multi-example tissue grinder grinding 60-90s;
3) room temperature leaves standstill 10min;
4) 13200rpm, the centrifugal 10min of room temperature, get supernatant liquor 400 μ l and proceed in new centrifuge tube;
5) add 800 μ l dehydrated alcohols, put upside down mixing;-20 DEG C of standing 10min precipitate DNA;
6) 13200rpm, 4 DEG C of centrifugal 5min;
7) precipitation uses 75% washing with alcohol, with 50 μ l ddH after air drying
2o dissolves, and supernatant is the DNA solution of extraction, for downstream PCR template;
Step 3. designs pathogenic fungi universal primer
Upstream primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 '; Downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ';
Step 4.PCR increases
PCR reaction system is 25 μ L, and reaction solution is: each 1 μ L of 10 × buffer2.5 μ L, dNTP1 μ L, primer I TS1 and ITS4, Taq enzyme 0.5 μ L, DNA profiling 3 μ L, ddH2O16 μ L, pcr amplification program: 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 53 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations; 72 DEG C of total elongation 10min; The agar gel of 1% detects PCR result, when PCR primer presents about 500bp band, namely contain Strawberry Root Rot pathogenic bacteria in sample, random choose sample CY2, JN3, JM2, LW2PCR product is sent to Sangon Biotech's order-checking;
Step 5. sequential analysis
Sequencing result and ncbi database
Http: ∥ blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn & PAGE_TYPE=BlastSearch & LINK_LOC=blasthome sequence alignment, obtains clear and definite Strawberry Root Rot Species of Pathogens.
Strawberry Root Rot evil nosophyte numerator method for quick of the present invention, the application in strawberry seedling quarantine, the early diagnosis of Field strawberries root rot and the monitoring of germ and qualification process.
The invention has the beneficial effects as follows:
The present invention, according to the genome sequence causing Strawberry Root Rot pathogenic bacteria, designs general Auele Specific Primer.By this technology to qualitative detection such as strawberry seedlings, thus realize construction, the safety in production of China's strawberry disease-free seedlings production base, intercept the object that external dangerous strawberry is in spite of illness imported into simultaneously.The method is simple to operate, and consuming time few, flux is large.By the method, qualitative detection is carried out to the sick sample of Strawberry Nursery seedling root etc., effectively can avoid propagation and the generation of Strawberry Root Rot, ensure the construction of China's strawberry disease-free seedlings production base, safety in production.
Accompanying drawing explanation
Fig. 1 is for organizing PCR electrophorogram with the sick sample of regional strawberry root, and wherein, swimming lane M is DNA Marker 2000; Swimming lane 1-9 is that in following table, corresponding different areas strawberrying root tissue DNA is template PCR primer; Swimming lane ck-is negative control water is template PCR primer.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in more detail.
1. the separation of pathogenic bacteria
The pathogenic bacteria of tissue isolation to strawberry root is adopted to be separated.Cut the fritter of the diseased tissues 4-5mm of the strong intersection of disease, be placed in 75% ethanol and soak 5s, then diseased tissues is moved in 0.1% mercuric chloride solution and soak 3-5min, again diseased tissues to be transferred in sterilized water rinsing 3 times, finally tissue is moved on on PDA substratum, placed by culture dish in 25 DEG C of incubators and cultivate 3-5d, colony diameter 1cm can carry out DNA extraction experiment.
2. pathogenic bacteria DNA extracts on a small quantity
1) with a small amount of pathogenic bacteria mycelia on sterilizing toothpick scraping PDA flat board, load in the centrifuge tube of 2.0ml;
2) 500 μ l Lysis buffer are added, 1 broken pearl, Multi-example tissue grinder grinding 60-90s;
3) room temperature leaves standstill 10min;
4) 13200rpm, the centrifugal 10min of room temperature, get supernatant liquor 400 μ l and proceed in new centrifuge tube;
5) add 800 μ l dehydrated alcohols, put upside down mixing;-20 DEG C of standing 10min precipitate DNA.
6) 13200rpm, 4 DEG C of centrifugal 5min;
7) precipitation uses 75% washing with alcohol, with 50 μ l ddH after air drying
2o dissolves, and supernatant is the DNA solution of extraction, can be used for downstream PCR template.
3. design pathogenic fungi universal primer
Upstream primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 '; Downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' (step 3 can follow step 1,2 synchronously to carry out).
4.PCR increases
PCR reaction system is 25 μ L, and reaction solution is: 10 × buffer2.5 μ L, dNTP1 μ L, each 1 μ L of primer (ITS1 and ITS4), Taq enzyme 0.5 μ L, DNA profiling 3 μ L, ddH
2o16 μ L.Pcr amplification program: 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 53 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations; 72 DEG C of total elongation 10min.The agar gel of 1% detects PCR result, when PCR primer presents about 500bp band, namely contains Strawberry Root Rot pathogenic bacteria in sample.Random choose sample CY2, JN3, JM2, LW2PCR product is sent to Sangon Biotech's order-checking.
5. sequential analysis
Sequencing result and ncbi database
(http://blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn & PAGE_TYPE=BlastSearch & LINK_LOC=blasthome) sequence alignment, obtain clear and definite Strawberry Root Rot Species of Pathogens.
Experimental result as shown in figure 1 and table 1.
Table 1: different areas strawberrying sample message table
Note :+represent positive findings, there is false positive results in No. 4 samples.
CY2: with plan pestalotia bacteria (Pestalotiopsis sp) (FJ175373.1) homology consistence 99%, as shown in SEQ ID NO:1.
JN3: with alternaric bacteria (Alternaria alternata) (JF796073.1) homology consistence 99%, as shown in SEQ ID NO:2.
JM2: with colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides) (KM357285.1) homology consistence 99%, as shown in SEQ ID NO:3.
LW2: with sickle-like bacteria (Fusarium solani) (JN786598.1) homology consistence 99%, as shown in SEQ ID NO:4.
The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (1)
1. the multiple nosophyte numerator method for quick of Strawberry Root Rot evil, comprises the following steps:
The separation of step 1. pathogenic bacteria
The pathogenic bacteria of tissue isolation to strawberry root is adopted to be separated, cut the fritter of the diseased tissues 4-5mm of the strong intersection of disease, be placed in 75% ethanol and soak 5s, then diseased tissues is moved in 0.1% mercuric chloride solution and soak 3-5min, again diseased tissues to be transferred in sterilized water rinsing 3 times, finally moved on to by tissue on PDA substratum, placed by culture dish in 25 DEG C of incubators and cultivate 3-5d, namely colony diameter 1cm carries out DNA extraction experiment;
Step 2. pathogenic bacteria DNA extracts on a small quantity
1) with a small amount of pathogenic bacteria mycelia on sterilizing toothpick scraping PDA flat board, load in the centrifuge tube of 2.0ml;
2) 500 μ l Lysis buffer are added, 1 broken pearl, Multi-example tissue grinder grinding 60-90s;
3) room temperature leaves standstill 10min;
4) 13200rpm, the centrifugal 10min of room temperature, get supernatant liquor 400 μ l and proceed in new centrifuge tube;
5) add 800 μ l dehydrated alcohols, put upside down mixing;-20 DEG C of standing 10min precipitate DNA;
6) 13200rpm, 4 DEG C of centrifugal 5min;
7) precipitation uses 75% washing with alcohol, with 50 μ l ddH after air drying
2o dissolves, and supernatant is the DNA solution of extraction, for downstream PCR template;
Step 3. designs pathogenic fungi universal primer
Upstream primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 '; Downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ';
Step 4.PCR increases
PCR reaction system is 25 μ L, and reaction solution is: each 1 μ L of 10 × buffer2.5 μ L, dNTP1 μ L, primer I TS1 and ITS4, Taq enzyme 0.5 μ L, DNA profiling 3 μ L, ddH2O 16 μ L, pcr amplification program: 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 53 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations; 72 DEG C of total elongation 10min; The agar gel of 1% detects PCR result, when PCR primer presents 500bp band, namely contains Strawberry Root Rot pathogenic bacteria in sample, random choose sample CY2, JN3, JM2, LW2PCR product is sent to the order-checking of raw work biotechnology Shanghai limited-liability company;
Step 5. sequential analysis
Sequencing result and ncbi database
Http:// blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn & PAGE_TYPE=BlastSearch & LINK_LOC=blasthome sequence alignment, obtains clear and definite Strawberry Root Rot Species of Pathogens.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282398A (en) * | 2016-11-09 | 2017-01-04 | 广东省农业科学院植物保护研究所 | Citrus anthracnose bacterium and Citrus foot rot pathogen double check test kit and application thereof |
| CN106520935A (en) * | 2016-10-18 | 2017-03-22 | 新疆生产建设兵团第十二师农业科学研究所 | PCR rapid detection method of strawberry root rot pathogen Ilyonectria novozelandica |
| CN106687604A (en) * | 2014-09-11 | 2017-05-17 | 阿格洛法士公司 | Methods for pathogen detection and disease management on meats, plants, or plant parts |
| CN109852712A (en) * | 2019-01-27 | 2019-06-07 | 上海海洋大学 | A kind of simple and effective bacterial fungus bacterium colony PCR general program |
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| EP0955381A2 (en) * | 1994-04-25 | 1999-11-10 | Novartis AG | Detection of fungal pathogens using the polymerase chain reaction |
Non-Patent Citations (4)
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| J.RACEDO.S ET AL: "A strawberry disease caused by Acremonium strictum", 《EUR J PATHOL》 * |
| 盛茹媛: "草莓根腐病病原鉴定及分子检测技术研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106687604A (en) * | 2014-09-11 | 2017-05-17 | 阿格洛法士公司 | Methods for pathogen detection and disease management on meats, plants, or plant parts |
| CN106520935A (en) * | 2016-10-18 | 2017-03-22 | 新疆生产建设兵团第十二师农业科学研究所 | PCR rapid detection method of strawberry root rot pathogen Ilyonectria novozelandica |
| CN106282398A (en) * | 2016-11-09 | 2017-01-04 | 广东省农业科学院植物保护研究所 | Citrus anthracnose bacterium and Citrus foot rot pathogen double check test kit and application thereof |
| CN106282398B (en) * | 2016-11-09 | 2019-05-21 | 广东省农业科学院植物保护研究所 | Citrus anthracnose bacterium and citrus foot rot pathogen double check kit and its application |
| CN109852712A (en) * | 2019-01-27 | 2019-06-07 | 上海海洋大学 | A kind of simple and effective bacterial fungus bacterium colony PCR general program |
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