CN103695533B - One grows cotton main pathogen fungi ITS-RFLP rapid identification method - Google Patents
One grows cotton main pathogen fungi ITS-RFLP rapid identification method Download PDFInfo
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Abstract
The invention discloses one to grow cotton main pathogen fungi ITS-RFLP rapid identification method, mainly comprise the following steps: 1) cotton main pathogen hypha,hyphae extracting genome DNA, comprises verticillium dahliae, black and white Verticillium, dry thread Pyrenomycetes, fusarium moniliforme, trichothecium roseum and Fusarium oxysporum; 2) ribosome internal transcribed spacer (ITS) pcr amplification, uses fungi ITS universal primer ITS4 and ITS5 to carry out rapid amplifying; 3) digestion with restriction enzyme reaction, and band spectrum is differentiated, any use recognition site is that restriction endonuclease HhaI, HaeIII, TaqI, Sau3A ITS product to above-mentioned cotton main pathogen fungi of four bases carries out single endonuclease digestion, comparison ITS-RFLP band spectrum contrast table and standard electronic collection of illustrative plates, thus Rapid identification goes out the classification of pathogenic bacteria.Morphology physiology qualification that what the present invention was more traditional waste time and energy, and the ITS cloning and sequencing pathogen identification method of high cost, have fast, accurately, economical and the advantage of multiple pathogenic fungi can be identified simultaneously.
Description
Technical field
The present invention relates to plant pathology field, particularly, the present invention relates to one and to grow cotton main pathogen fungi ITS-RFLP rapid identification method.
Background technology
Cotton is worldwide important cash crop and textile industry raw material, is also the valuable cargo involved the interests of the state and the people.For a long time, generally occurring of cotton diseases and insect pests is the bottleneck problem of restriction cotton yield all the time, wherein seriously surely belongs to verticillium and blight, secondly be bell disease and seedling diseases, mostly is fungal disease.In order to science formulates rational prevention and control measure more, to the prerequisite being confirmed to be necessity the most of disease species, therefore quick and precisely pathogen identification just seems particularly important.
The traditional qualification of cotton pathogenic fungi is characterized as basis with morphology, cytology, physiology and ecology etc., but because a lot of mycobiology proterties is extremely similar, therefore, require that researcher has abundant practical experience, meanwhile, traditional qualification is comparatively time-consuming, effort also.Along with molecular biological development, ribosome internal transcribed spacer (Internaltranscribedspacer, ITS) amplification, cloning and sequencing, substantially increase the accuracy of Identification of The Fungal Species Causing, but still it is longer to there is experimental period, the problem that cost is higher.Complete ITS clone, order-checking, a series of program of comparison need about one week, and the cost of expert testimony of a sample is approximately 40 yuan.
Although adopt ITS-RFLP method qualification pathogenic fungi to have document report, due to the similarity on cotton pathogenic fungi gene, there is serious mutual interference, therefore how for the feature of cotton pathogenic fungi, suitable primer and restriction endonuclease is selected to remain those skilled in the art's technical problem urgently to be resolved hurrily to the accuracy improving qualification.
Summary of the invention
Technical problem to be solved by this invention is to provide one and grows cotton main pathogen fungi ITS-RFLP rapid identification method, fast and identify the kind of cotton pathogenic fungi accurately, for effective prevention and control of disease provide foundation.
The present invention solves the problems of the technologies described above adopted technical scheme: one grows cotton main pathogen fungi ITS-RFLP rapid identification method, select the pathogenic fungi of Main Cotton Diseases, comprise cotton verticillium wilt, blight, damping-off, red rot and the pathogenic bacteria verticillium dahliae corresponding to rouge and powder disease, black and white Verticillium, Fusarium oxysporum, dry thread Pyrenomycetes, fusarium moniliforme and trichothecium roseum, and measure the ITS sequence of above pathogenic fungi; Webcutter on-line analysis is utilized to select enzyme to cut zonation types different restriction enzyme HhaI, HaeIII, TaqI, Sau3A, in conjunction with pDRAW32 software, digestion products distribution situation is analyzed, and draw out the sepharose electronics restriction enzyme mapping of 2-4%, as the standard diagram that comparison uses;
Extract the genomic dna of the pathogenic fungi of cotton samples to be measured subsequently, the DNA adopting following primer pair to extract carries out pcr amplification;
ITS4：5’-TCCTCCGCTTATTGATATGC-3’：
ITS5：5’-GGAAGTAAAAGTCGTAACAAGG-3’：
Amplified production use restriction enzyme HhaI, HaeIII, TaqI, Sau3A respectively enzyme cut, and employing detects with the agarose gel electrophoresis of aforementioned sepharose same concentrations, and enzyme are cut result and the comparison of electronic standard collection of illustrative plates, qualification fungal pathogen.
It is characterized in that comprising the steps:
Table 1 Main Cotton Diseases and pathogenic fungi list
In a preferred embodiment of the present invention, described authentication step comprises the steps:
1) extraction of DNA: the cotton pathogenic fungi of separation and purification is seeded to PDA substratum, 28 DEG C of constant temperature quiescent culture, collect mycelia, collect the mycelia of cotton pathogenic fungi, adopt the extracting of CTAB-NaCl method, use ddH
2o dissolves genomic dna
2) pcr amplification: with the genomic dna of above-mentioned pathogenic fungi for template, adopts primer pair ITS45 '-TCCTCCGCTTATTGATATGC-3 ' and ITS55 '-GGAAGTAAAAGTCGTAACAAGG-3 ' to carry out pcr amplification.The PCR reaction system of 20 μ l: comprise 10 × PCRbuffer (Mg
2+) 2 μ l, concentration is the dNTP solution 1.6 μ l of 10mM, and concentration is each 1 μ l of 10uM primer I TS4/ITS5, genomic dna 1 μ l, and concentration is the Taq enzyme 0.2 μ l of 5U/ μ l, and the ddH of 13.2 μ l
2o.Amplified reaction program is: 94 DEG C of denaturation 2min, and afterwards, 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 40s, totally 25 circulations, and last 72 DEG C extend 10min, and obtain the PCR primer of ITS, product magnitude range is 500-750bp.
3) endonuclease reaction: use arbitrarily the PCR primer enzyme of restriction enzyme HhaI, HaeIII, TaqI, Sau3A to ITS to cut.The enzyme of 10 μ l cuts system: ITS template 6 μ l, 10 × restriction endonuclease buffer1 μ l, restriction endonuclease 0.5 μ l, ddH
2o2.5 μ l.Reaction conditions is: 37 DEG C of insulation 1h in PCR instrument, add 6 × loadingbuffer termination reaction of 2 μ l, 3% agarose gel electrophoresis constant voltage 75V, 2 hours, observe and take pictures under ultraviolet lamp, obtain ITS-RFLP band spectrum;
4) band spectrum is differentiated: by the ITS-RFLP band spectrum of above-mentioned acquisition and the comparison of standard electronic restriction enzyme mapping, first the size that comparison HhaI and Sau3A is corresponding and electronics collection of illustrative plates (as table 2 and Fig. 3), for trichothecium roseum (Tr), after cutting with HhaI enzyme, if there is four swimming bands, and be 295bp to the maximum, minimum is 23bp, then illustrate it is Tr, enzyme meanwhile in conjunction with Sau3A cuts result, having demonstrated two swimming bands, is 396bp and 190bp respectively, just confirms that bacterial strain to be identified is trichothecium roseum further.The invention provides 6 to grow cotton the band spectrum contrast table of 4 kinds of conventional restriction endonucleases of pathogenic fungi and electronics collection of illustrative plates, to realize the object of precise Identification.
Table 2 cotton pathogenic bacteria ITS-RFLP band spectrum contrast table (bp)
Beneficial effect of the present invention is mainly reflected in following three aspects:
1, operating process is simple, and workable, do not require that assessor has rich experience, qualification cycle is short, can Rapid identification cotton pathogenic fungi.
1) extracting genome DNA speed is fast.That the present invention adopts is CTAB-NaCl method extracting DNA, because the highly sensitive of ITS-PCR reaction is tested in downstream, therefore low to template DNA purity requirement, only need to use organic solvent extracting once to the albumen in lysate and polysaccharide impurity, then precipitate with ice-cold dehydrated alcohol, without the need to washing of precipitate, after drying, directly add ddH
2o, can complete DNA extracting in 1 and a half hours.
2) ITS Primer selection science.ITS4 selected by the present invention comes from the ribosomal 28S of fungi and guards section, ITS5 is then positioned at 18S, amplified production comprises two complete internal transcribed spacer (ITS), on the variable section that kind is different, the recognition site of different restriction enzyme is all not identical, makes the result of ITS-RFLP more credible.
3) pcr amplification ITS efficiency is high.In the present invention, the response procedures of ITS amplification is simple, because ITS sequence is shorter, all at below 720bp, as step 3) as shown in extend time 40s, amount to 25 circulations, can increase at the ITS completing pathogenic fungi within an hour.
4) ITS-RFLP method is novel and quick.The ITS amplified production of cotton pathogenic fungi reclaims without the need to purifying, with the arbitrary combination direct enzyme cutting ITS in the restriction enzyme of four listed by the present invention, enzyme is cut band spectrum and standard band spectrum contrast table and electronics restriction enzyme mapping to compare, just can identify the kind of pathogenic fungi.
In sum, the qualification work of cotton fungal pathogen, can complete at 5-6 hour, to compare substantially reduce qualification cycle with traditional Morphological Identification with ITS cloning and sequencing, more efficient and convenient.
2, the qualification of cotton pathogenic fungi is accurate, and false positive rate is low.The present invention is from 9 kind of four base restriction restriction endonuclease, screening obtains 4 and to grow cotton different restriction endonuclease HhaI, HaelII, TaqI, the Sau3A of the main pathogenic fungi restriction enzyme mapping to 6, and having made band spectrum contrast table and the electronics restriction enzyme mapping of standard I TS-RFLP, size is accurate, and collection of illustrative plates is clear.Assessor can select arbitrarily above four kinds of restriction enzymes or its combination according to the practical situation of oneself, and comparison standard diagram, can the classification of precise Identification cotton pathogenic bacteria.
3, with low cost.The present invention does not use any commercial reagents box and entrusts order-checking service, and relating to is all conventional molecular agents, and the qualification of a sample spends about 5 yuan, and disbursement is far below the method for the cloning and sequencing pathogen identification of 40 yuan/sample.
Accompanying drawing explanation
Fig. 1 shows 6 and to grow cotton pathogenic fungi extracting genome DNA electrophorogram
Fig. 2 show 6 grow cotton pathogenic fungi ITS increase electrophorogram
Fig. 3 shows the standard electronic collection of illustrative plates of the ITS-RFLP of four kinds of restriction enzymes
Embodiment
The extraction of embodiment 1 cotton pathogenic fungi genomic dna
1) from the fresh mycelia of scraping 50-100mg PDA solid medium, be put in 1.5ml centrifuge tube, add the DNA extraction damping fluid [formula: 50mmol/LHris-HCl (pH=7.5) of 200 μ l preheatings, 50mmol/LEDTA (pH=8.0), 1.4mol/LNacl, 20g/LCTAB], grind with miniature grinding rod, and then add the Extraction buffer of 450 μ l, 65 DEG C of water-bath 30min, every 10min reversion is once;
2) the saturated phenol of isopyknic 1:1 and chloroform is added, fully mixing of turning upside down, the centrifugal 15min of 12000g under room temperature;
3) carefully pipette supernatant liquor in new 1.5ml centrifuge tube, add isopyknic ice-cold dehydrated alcohol precipitation, room temperature places the centrifugal 10min collecting precipitation of 10min, 12000g, abandons supernatant, 37 DEG C of oven for drying;
4) ddH of 30 μ l is added
2o dissolves genomic dna, is placed in-20 DEG C of refrigerators for subsequent use.
5) electrophoresis detection, on the sepharose of the genomic dna getting 2 μ l together with 6 × loadingbuffer point sample to 0.8% of 1 μ l, 200V constant voltage electrophoresis 15min, is shown in Fig. 1.
Embodiment 2 cotton pathogenic fungi ITS increases
1) amplification reaction system: with the genomic dna of above-mentioned pathogenic fungi for template, adopts primer pair ITS45 '-TCCTCCGCTTATTGATATGC-3 ' and ITS55 '-GGAAGTAAAAGTCGTAACAAGG-3 ' to carry out pcr amplification.The PCR reaction system of 20 μ l: comprise 10 × PCRbuffer (Mg
2+) 2 μ l, concentration is the dNTP solution 1.6 μ l of 10mM, and concentration is each 1 μ l of 10uM primer I TS4/ITS5, genomic dna 1 μ l, and concentration is the Taq enzyme 0.2 μ l of 5U/ μ l, and the ddH of 13.2 μ l
2o.
2) amplified reaction program is: 94 DEG C of denaturation 2min, and afterwards, 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 40s, totally 25 circulations, and last 72 DEG C extend 10min, obtain the PCR primer of internal transcribed spacer.
3) electrophoresis detection, gets the PCR primer of the ITS of 5 μ l, mixes on the sepharose of 6 × loadingbuffer point sample to 0.8% of 1 μ l, and 200V constant voltage electrophoresis 10min, magnitude range is 500-750bp, sees Fig. 2.
The selection of embodiment 3 restriction enzyme
Screen suitable four base restriction restriction endonucleases.Grow cotton according to 6 the ITS sequence measurement result of pathogenic fungi, analyze the recognition site of 9 kind of four base restriction restriction endonuclease (AccII, AfaI, AluI, HaeIII, HhaI, Sau3A, MspI, TaqI and XspI) on different I TS one by one, calculate product length, take into full account the feature such as polymorphism and fungal species specificity, have finally chosen 4 kinds of common restriction endonuclease HhaI, HaeIII, TaqI, Sau3A.
The making of embodiment 4 cotton pathogenic fungi ITS-RFLP band spectrum contrast table and electronics collection of illustrative plates
1) ITS-RFLP band spectrum contrast table makes.Grow cotton the ITS sequence of pathogenic fungi for Main Basis with 6, utilize Webcutter on-line analysis to select enzyme to cut zonation types different restriction enzyme HhaI, HaeIII, TaqI, Sau3A, make ITS-RFLP band spectrum contrast table (table 2) according to digestion products clip size.
2) making of ITS-RFLP electronics collection of illustrative plates.In conjunction with pDRAW32 software, four kinds of digestion with restriction enzyme products distribution situations are analyzed, and draw out the agarose electronics restriction enzyme mapping of 3%, as the standard diagram that comparison uses, see Fig. 3.
Method of the present invention is adopted to identify 58 samples, through the method validation result of ITS cloning and sequencing, result shows method accuracy of the present invention more than 94%, and the average detected time is only 5.5 hours, cost is 5 yuan/sample, it is high that visible method of the present invention has accuracy, the advantage that cost is low, is applicable to extensive use.
The above is the preferred embodiments of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. one to grow cotton main pathogen fungi ITS-RFLP rapid identification method, select the pathogenic fungi of Main Cotton Diseases, comprise cotton verticillium wilt, blight, damping-off, red rot and the pathogenic bacteria verticillium dahliae corresponding to rouge and powder disease, black and white Verticillium, Fusarium oxysporum, dry thread Pyrenomycetes, fusarium moniliforme and trichothecium roseum, and measure the ITS sequence of above pathogenic fungi; Webcutter on-line analysis is utilized to select enzyme to cut zonation types different restriction enzyme HhaI, HaeIII, TaqI, Sau3A, in conjunction with pDRAW32 software, digestion products distribution situation is analyzed, and draw out the sepharose electronics restriction enzyme mapping of 2-4%, as the standard diagram that comparison uses;
Extract the genomic dna of the pathogenic fungi of cotton samples to be measured subsequently, the DNA adopting following primer pair to extract carries out pcr amplification;
ITS4：5’-TCCTCCGCTTATTGATATGC-3’；
ITS5：5’-GGAAGTAAAAGTCGTAACAAGG-3’；
Amplified production use restriction enzyme HhaI, HaeIII, TaqI, Sau3A respectively enzyme cut, and employing detects with the agarose gel electrophoresis of aforementioned sepharose same concentrations, and enzyme are cut result and the comparison of electronic standard collection of illustrative plates, qualification fungal pathogen.
2. rapid identification method according to claim 1, is characterized in that described authentication method comprises the steps:
1) extraction of DNA: the cotton pathogenic fungi of separation and purification is seeded to PDA substratum, 28 DEG C of constant temperature quiescent culture, collect mycelia, collect the mycelia of cotton pathogenic fungi, adopt the extracting of CTAB-NaCl method, use ddH
2o dissolves genomic dna;
2) pcr amplification: with the genomic dna of above-mentioned pathogenic fungi for template, adopts primer pair ITS45 '-TCCTCCGCTTATTGATATGC-3 ' and ITS55 '-GGAAGTAAAAGTCGTAACAAGG-3 ' to carry out pcr amplification; The PCR reaction system of 20 μ l: comprise containing Mg
2+10 × PCR damping fluid 2 μ l, concentration is the dNTP solution 1.6 μ l of 10mM, and concentration is each 1 μ l of 10uM primer I TS4/ITS5, genomic dna 1 μ l, and concentration is the Taq enzyme 0.2 μ l of 5U/ μ l, and the ddH of 13.2 μ l
2o; Amplified reaction program is: 94 DEG C of denaturation 2min, afterwards, 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 40s, totally 25 circulations, and last 72 DEG C extend 10min, and obtain the PCR primer of ITS, product size is 500-750bp;
3) endonuclease reaction: use arbitrarily the PCR primer enzyme of restriction enzyme HhaI, HaeIII, TaqI, Sau3A to ITS to cut; The enzyme of 10 μ l cuts system: ITS template 6 μ l, 10 × restriction endonuclease damping fluid 1 μ l, restriction endonuclease 0.5 μ l, ddH
2o2.5 μ l; Reaction conditions is: 37 DEG C of insulation 1h in PCR instrument, add 6 × sample-loading buffer termination reaction of 2 μ l, 3% agarose gel electrophoresis constant voltage 75V, 2 hours, observe and take pictures under ultraviolet lamp, obtain ITS-RFLP band spectrum;
4) band spectrum is differentiated: by the ITS-RFLP band spectrum of above-mentioned acquisition and the comparison of standard electronic restriction enzyme mapping, the size that first comparison HhaI and Sau3A is corresponding and electronics collection of illustrative plates.
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| CN105567849A (en) * | 2016-02-23 | 2016-05-11 | 蒋丹 | Fusarium oxysporum nucleotide sequence qualitative standard sample and preparing method thereof |
| CN106011269B (en) * | 2016-07-06 | 2019-12-10 | 中国农业科学院棉花研究所 | Method for rapidly identifying cotton verticillium wilt resistance by using real-time quantitative PCR |
| CN108277296A (en) * | 2018-04-17 | 2018-07-13 | 中国农业科学院植物保护研究所 | A kind of identification of the Fusarium strain for causing root rot and differentiating method |
| CN108841987A (en) * | 2018-07-05 | 2018-11-20 | 南京农业大学 | A kind of rapid detection method of Fusarium graminearum 2-cyano-3-amino-3-phenylancryic acetate resistant strain |
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| Genetic similarity among Cercospora apii-group species and their detection in host plant tissue by PCR/RFLP analyses of rDNA internal transcribed spacer(ITS);Siboe GM et al.;《J Gen App Microbiol》;20000430;第46卷(第2期);摘要，材料和方法，结果，讨论，表1，图1-6 * |
| rDNA-ITS在植物病原真菌分子检测中的应用;刘春来等;《东北农业大学学报》;20070225;第38卷(第01期);101-105 * |
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