CN106868138A - Primer and kit for identifying the pathogen of tomato neckrot root rot and droop - Google Patents
Primer and kit for identifying the pathogen of tomato neckrot root rot and droop Download PDFInfo
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- CN106868138A CN106868138A CN201710118646.3A CN201710118646A CN106868138A CN 106868138 A CN106868138 A CN 106868138A CN 201710118646 A CN201710118646 A CN 201710118646A CN 106868138 A CN106868138 A CN 106868138A
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Abstract
The invention discloses a kind of primer and kit for identifying the pathogen of tomato neckrot root rot and droop, for identifying that the primer sequence of pathogen of tomato neckrot root rot is SEQ ID NO：1 and SEQ ID NO：Shown in 2, for identifying that the primer sequence of pathogen of tomato wilt is SEQ ID NO：3 and SEQ ID NO：Shown in 4.Primer specificity of the present invention is strong, detection accuracy is high, can be used for Fusarium oxysporum tomato neckrot root rot specialized form, No. 1 detection of biological strain of Fusarium oxysporum tomato specialized form, the preventing and treating to tomato neckrot root rot and droop disease early stage quick detection, soil germ population distribution and disease is significant.
Description
Technical field
It is more particularly to a kind of for identifying tomato neckrot root rot and withering the present invention relates to technical field of molecular biology
The primer and kit of the pathogen of disease.
Background technology
Tomato (Solanum esculentum L.) is one of topmost vegetables of China, estimates national tomato planting face
Product is annual more than 10,000,000 mu, and the output value is more than 40,000,000,000 yuan.The cultivated area of tomato constantly expands, can be with anniversary kind
Plant, and yield more and more higher, variety design are more and more, widely used, deep to be liked by grower and consumer.Restriction at present
In addition to the more difficult prediction of market conditions, topmost be exactly heavier pest and disease damage to the factor of tomato production, and the yield and quality of tomato is difficult
There is bigger raising.Wherein, tomato neckrot root rot (Fusarium crown and root rot, FCRR) and droop
(Tomato Fusarium wilt) is 2 kinds of important soil-borne diseases in tomato production, is distributed widely in global tropical, sub- heat
Band and Temperate Region in China, are global important diseases, with host range it is wide, preventing and treating is difficult the features such as.Tomato neckrot root rot
It is by a kind of necrotrophic disease fungus Fusarium oxysporum tomato neckrot root rot specialized form (Fusarium oxysporum
F.sp.radicis-lycopersici, FORL) cause, a kind of tomato disease for newly rising in recent years, the incidence of disease increases year by year
Not in time, up to more than 80%, fatal rate causes Severe Reduction to the tomato incidence of disease up to more than 30% for aggravation, mistaken diagnosis or preventing and treating,
Serious economic loss (Cheng Lin etc., 2016) is caused to Planting household.Tomato wilt is by Fusarium oxysporum tomato specialized form
The fungoid vascular bundle diseases that (Fusarium oxysporum f.sp.lycopersici, FOL) causes.In recent years, with setting
The expansion and the increase in plantation age of tomato planting area are applied, by tomato neckrot pine root fungus, the microbial soil-borne disease of droop
Evil continuous cropping obstacle is on the rise, and the yield and quality to tomato causes serious threat, it has also become greenhouse tomato security industry metaplasia
The bottleneck of product.
So far, based on sickle-like bacteria morphology in terms of taxonomic identification method be difficult to be applied to the sickle easily undergone mutation
Knife bacterium, complex operation and identifies that the degree of accuracy is low, it is easy to cause mistaken diagnosis, affects preventing and treating opportunity, influence prevention effect adversely.With point
Sub- biology techniques are continued to develop, and the appraisal of Fusarium have also been introduced molecular systematics method, are usually used in identification
Main DNA sequence dna includes：Ribosomes the Internal Transcribed Spacer (ITS), intergenic spacer region (IGS), in polygalacturonase
Enzyme cutting (pg1, pg2, pg5) etc..Therefore, finding can distinguish Fusarium oxysporum tomato neckrot root rot specialized form (FORL) and point
The gene order of fusarium oxysporum tomato specialized form (FOL) pathogen, sets up a set of new molecular biology identification system, can be fast
Speed, exactly identification Fusarium oxysporum tomato neckrot root rot specialized form or/and Fusarium oxysporum tomato specialized form, the system
Set up to the anti-tool of tomato neckrot root rot and droop disease early stage quick detection, soil germ population distribution and disease
It is significant.Variety classes sickle-like bacteria biological characteristics is different, specifies species, the sociales of the pathogenic sickle-like bacteria in different regions
Group and pathogenicity, set up efficiently, fast and accurately Pathogen detection technology, can be that the formulation of its prophylactico-therapeutic measures lay the foundation.
The content of the invention
It is an object of the invention to provide a kind of primer for identifying the pathogen of tomato neckrot root rot and droop,
High specificity, detection accuracy is high, to tomato neckrot root rot and droop disease early stage quick detection, soil germ colony point
The preventing and treating of cloth and disease is significant.
Present invention also offers the kit comprising above-mentioned primer.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of primer for identifying the pathogen of tomato neckrot root rot, the pathogen is Fusarium oxysporum tomato neck
Rotten root rot specialized form, the primer sequence is as follows：
ForL- sense primers：5’-CCTTACTCGACATTTTTCAGA-3’；
ForL- anti-sense primers：5’-CATCAAGGCATAGTAAGAT-3’.
A kind of primer for identifying the pathogen of tomato wilt, the pathogen is Fusarium oxysporum tomato specialized form
No. 1 biological strain, the primer sequence is as follows：
For- sense primers：5’-GTATGACTCCTCTTATCATA-3’；
For- anti-sense primers：5’-GTGGAGTGAGCTTACCGTTA-3’.
A kind of kit for identifying the pathogen of tomato neckrot root rot, the pathogen is Fusarium oxysporum tomato
Neckrot root rot specialized form, including primer described in claim 1.
A kind of kit for identifying the pathogen of tomato wilt, the pathogen specially changes for Fusarium oxysporum tomato
No. 1 biological strain of type, including primer described in claim 2.
A kind of kit for identifying the pathogen of tomato neckrot root rot and droop simultaneously, tomato neckrot root rot
Pathogen be Fusarium oxysporum tomato neckrot root rot specialized form, the pathogen of tomato wilt is special for Fusarium oxysporum tomato
No. 1 biological strain of change type, including box body and 6 PCR pipes, be respectively provided with 2 PCR pipes 2 × Taq PCR MasterMix and
ddH2O；Draw the ForL- upstreams that detection Fusarium oxysporum tomato neckrot root rot specialized form is respectively provided with other 4 PCR pipes
Under thing, ForL- anti-sense primers, and For- sense primers, the For- of detection Fusarium oxysporum tomato No. 1 biological strain of specialized form
Trip primer.
Preferably,
ForL- upstream primer sequences are：5’-CCTTACTCGACATTTTTCAGA-3’；
ForL- downstream primer sequences are：5’-CATCAAGGCATAGTAAGAT-3’；
For- upstream primer sequences are：5’-GTATGACTCCTCTTATCATA-3’；
For- downstream primer sequences are：5’-GTGGAGTGAGCTTACCGTTA-3’.
The beneficial effects of the invention are as follows：Primer specificity is strong, and detection accuracy is high, can be used for Fusarium oxysporum tomato neckrot
Root rot specialized form, No. 1 detection of biological strain of Fusarium oxysporum tomato specialized form, to tomato neckrot root rot and droop disease
The preventing and treating of evil early stage quick detection, soil germ population distribution and disease is significant.
Brief description of the drawings
Fig. 1 is the gel electrophoresis figure after primer specificity detection sample DNA amplification of the present invention, in figure：1：Fusarium oxysporum
Tomato neckrot root rot specialized form；2：No. 1 biological strain of Fusarium oxysporum tomato specialized form.
Fig. 2 is the gel electrophoresis figure after isolated strains DNA cloning in Field diseased plant of the present invention, in figure：a：Fusarium oxysporum
Tomato neckrot root rot specialized form is detected；b：No. 1 biological strain detection of Fusarium oxysporum tomato specialized form；c：Fusarium oxysporum kind
No. 1 biological strain of eggplant neckrot root rot specialized form and Fusarium oxysporum tomato specialized form detects simultaneously.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, raw material and equipment for being used etc. is commercially available or commonly used in the art.
Method in following embodiments, unless otherwise instructed, is the conventional method of this area.
The primer synthesizes by Dalian treasured biotech company in the present invention, and the work of all sequences measure is given birth to by Shanghai
Thing Technology Co., Ltd. completes.
Embodiment：
First, design of primers
According to pgx4 in GenBank conserved sequence (GenBank numberings AB256820.1, AB256830.1,
AB256818.1、AB256798.1、AB256796.1、AB256797.1、AB256795.1、GU169176.1、
AB256825.1), design degenerate primer FL-F (5 '-GATGGTGGAACGGTATGACYC-3 ', SEQ ID NO：Shown in 5) with
FL-R (5 '-GATATCCTTRACACCATCACA-3 ', SEQ ID NO：Shown in 6), amplification length is 960bp, and its fragment is entered
Row clone, sequencing and analysis, and in 960bp interior sequences design Fusarium oxysporum tomato neckrot root rot specialized form and sharp spore sickle
Knife bacterium tomato No. 1 biological strain specific primer of specialized form.
Fusarium oxysporum tomato neckrot root rot specialized form specific primer sequence is as follows：
ForL- sense primers：5’-CCTTACTCGACATTTTTCAGA-3’(SEQ ID NO：Shown in 1)；
ForL- anti-sense primers：5’-CATCAAGGCATAGTAAGAT-3’(SEQ ID NO：Shown in 2)；
SEQ ID NO：1 by 21 base compositions, SEQ ID NO：2 are by 19 base compositions, expanding fragment length
210bp.Above-mentioned primer is detected suitable for Fusarium oxysporum tomato neckrot root rot specialized form PCR.Test sample is treated with above-mentioned primer pair
Product enter performing PCR amplification, and amplified production has the band of 210bp sizes, then contain Fusarium oxysporum tomato neckrot root rot in sample
Specialized pathogen bacterium.
Fusarium oxysporum knife tomato No. 1 biological strain specific primer sequence of specialized form is as follows：
For- sense primers：5 '-GTATGACTCCTCTTATCATA-3 ' (SEQ ID NO in sequence table：3)；
For- anti-sense primers：5 '-GTGGAGTGAGCTTACCGTTA-3 ' (SEQ ID NO in sequence table：4).
SEQ ID NO：3 by 20 base compositions, SEQ ID NO：4 are by 20 base compositions, expanding fragment length
455bp.Above-mentioned primer is applied to No. 1 biological strain PCR detection of Fusarium oxysporum tomato specialized form.Test sample is treated with above-mentioned primer pair
Product enter performing PCR amplification, and amplified production has the band of 455bp sizes, then contain No. 1 life of Fusarium oxysporum tomato specialized form in sample
Reason microspecies pathogen.
2nd, PCR detections
This step bacterial strain uses therefor plants micro- offer by academy of agricultural sciences of Zhejiang Province：Fusarium oxysporum tomato neckrot root rot specialized form and
No. 1 biological strain of Fusarium oxysporum tomato specialized form.
Detection method is carried out according to the following steps：
1st, the extraction of mycelia DNA：About 0.1 gram of mycelia is scraped from flat board with sterilizing toothpick, is placed in 2.0mL centrifuge tubes,
After routinely CTAB methods extract DNA, preserve respectively, it is standby.
2nd, PCR amplifications：After the μ L of each sample DNA 1 of step 1 extraction are separately added into each PCR pipe, 2 are sequentially added ×
Taq PCR MasterMix (commercially available, Tiangeng is biochemical) 5 μ L, each 0.25 μ L of upstream and downstream primer (0.25 μM of final concentration), plus ddH2O
To 10 μ L, by PCR response procedures：94 DEG C of predegeneration 3min, 94 DEG C of 50seconds, 56 DEG C of annealing 50seconds, 72 DEG C
60seconds, 35 circulations are expanded, 72 DEG C of extension 10min, 4 DEG C of preservations of product.
The upstream primer sequence for detecting Fusarium oxysporum tomato neckrot root rot specialized form is SEQ ID NO：1, downstream is drawn
Thing sequence is SEQ ID NO：2；No. 1 upstream primer sequence of biological strain of detection Fusarium oxysporum tomato specialized form is SEQ ID
NO：3, downstream primer sequence is SEQ ID NO：4.
3rd, the gel electrophoresis analysis of pcr amplification product：Each sample takes the μ L of amplified production 10 and by 0.25% bromjophenol blue respectively
Plus 40% μ L of sample-loading buffer 2 for being formulated of sucrose, mix, mixture is carried out electrophoresis point on 1.5% Ago-Gel
From, to bromjophenol blue reach gel 2/3 position at stop electrophoresis, gel through EB dye after observed with gel imaging system,
Photograph.
4th, No. 1 inspection of biological strain of Fusarium oxysporum tomato neckrot root rot specialized form and Fusarium oxysporum tomato specialized form
Survey：According to Gel electrophoresis results, analysis judges to be detected sample (see Fig. 1)：Fusarium oxysporum tomato neckrot root rot specialized form
210bp bands are amplified, No. 1 biological strain of Fusarium oxysporum tomato specialized form amplifies 455bp bands, with expected results phase
Symbol, shows that primer of the invention can be used for Fusarium oxysporum tomato neckrot root rot specialized form and Fusarium oxysporum tomato specialized form
No. 1 Standard PCR detection of biological strain.
3rd, actual sample detection
This step bacterial strain uses therefor is obtained by being separated on Zhejiang tomato Zhu Zai areas tomato neckrot root rot diseased plant and droop diseased plant
Bacterial strain.
Detection method is carried out according to the following steps：
1st, the extraction of mycelia DNA：About 0.1 gram of mycelia is scraped from flat board with sterilizing toothpick, is placed in 2.0mL centrifuge tubes,
After routinely CTAB methods extract DNA, preserve respectively, it is standby；
2nd, PCR amplifications：After the μ L of each sample DNA 1 of step 1 extraction are separately added into each PCR pipe, 2 are sequentially added ×
Taq PCR MasterMix (commercially available, Tiangeng is biochemical) 5 μ L, each 0.25 μ L of upstream and downstream primer (0.25 μM of final concentration), plus ddH2O
To 10 μ L, by PCR response procedures：94 DEG C of predegeneration 3min, 94 DEG C of 50seconds, 56 DEG C of annealing 50seconds, 72 DEG C
60seconds, 35 circulations are expanded, 72 DEG C of extension 10min, 4 DEG C of preservations of product.
The upstream primer sequence for detecting Fusarium oxysporum tomato neckrot root rot specialized form is SEQ ID NO：1, downstream is drawn
Thing sequence is SEQ ID NO：2；No. 1 upstream primer sequence of biological strain of detection Fusarium oxysporum tomato specialized form is SEQ ID
NO：3, downstream primer sequence is SEQ ID NO：4.
3rd, the gel electrophoresis analysis of pcr amplification product：Each sample takes the μ L of amplified production 10 and by 0.25% bromjophenol blue respectively
Plus 40% μ L of sample-loading buffer 2 for being formulated of sucrose, mix, mixture is carried out electrophoresis point on 1.5% Ago-Gel
From, to bromjophenol blue reach gel 2/3 position at stop electrophoresis, gel through EB dye after observed with gel imaging system,
Photograph.
4th, No. 1 inspection of biological strain of Fusarium oxysporum tomato neckrot root rot specialized form and Fusarium oxysporum tomato specialized form
Survey：According to Gel electrophoresis results, analysis judges to be detected sample (see Fig. 2)：Fusarium oxysporum tomato neckrot root rot specialized form
210bp bands are amplified, No. 1 biological strain of Fusarium oxysporum tomato specialized form amplifies 455bp bands, with expected results phase
Symbol, shows that primer of the invention can be used for Fusarium oxysporum tomato neckrot root rot specialized form and Fusarium oxysporum tomato specialized form
No. 1 Standard PCR detection of biological strain.
Also multiplex PCR detection can be carried out, while detecting Fusarium oxysporum tomato neckrot root rot specialized form and sharp spore reaping hook
No. 1 biological strain of bacterium tomato specialized form.
A kind of kit for identifying the pathogen of tomato neckrot root rot and droop simultaneously, tomato neckrot root rot
Pathogen be Fusarium oxysporum tomato neckrot root rot specialized form, the pathogen of tomato wilt is special for Fusarium oxysporum tomato
No. 1 biological strain of change type, including box body and 6 PCR pipes, be respectively provided with 2 PCR pipes 2 × Taq PCR MasterMix and
ddH2O；Draw the ForL- upstreams that detection Fusarium oxysporum tomato neckrot root rot specialized form is respectively provided with other 4 PCR pipes
Thing (SEQ ID NO：1), ForL- anti-sense primers (SEQ ID NO：2), and detection No. 1 life of specialized form of Fusarium oxysporum tomato
Manage For- sense primers (the SEQ ID NO of microspecies：3), For- anti-sense primers (SEQ ID NO：4).
Embodiment described above is a kind of preferably scheme of the invention, not makees any formal to the present invention
Limitation, also has other variants and remodeling on the premise of without departing from the technical scheme described in claim.
SEQUENCE LISTING
<110>Zhejiang Academy of Agricultural Science
<120>Primer and kit for identifying the pathogen of tomato neckrot root rot and droop
<130> 2017.02
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
ccttactcga catttttcag a 21
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
catcaaggca tagtaagat 19
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
gtatgactcc tcttatcata 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
gtggagtgag cttaccgtta 20
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<400> 5
gatggtggaa cggtatgacy c 21
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
gatatccttr acaccatcac a 21
Claims (6)
1. a kind of primer for identifying the pathogen of tomato neckrot root rot, the pathogen is Fusarium oxysporum tomato neckrot
Root rot specialized form, it is characterised in that the primer sequence is as follows：
ForL- sense primers：5’-CCTTACTCGACATTTTTCAGA-3’；
ForL- anti-sense primers：5’-CATCAAGGCATAGTAAGAT-3’.
2. a kind of primer for identifying the pathogen of tomato wilt, the pathogen is Fusarium oxysporum tomato specialized form 1
Number biological strain, it is characterised in that the primer sequence is as follows：
For- sense primers：5’-GTATGACTCCTCTTATCATA-3’；
For- anti-sense primers：5’-GTGGAGTGAGCTTACCGTTA-3’.
3. a kind of kit for identifying the pathogen of tomato neckrot root rot, the pathogen is Fusarium oxysporum tomato neck
Rotten root rot specialized form, it is characterised in that including primer described in claim 1.
4. a kind of kit for identifying the pathogen of tomato wilt, the pathogen is Fusarium oxysporum tomato specialized form
No. 1 biological strain, it is characterised in that including primer described in claim 2.
5. a kind of kit for identifying the pathogen of tomato neckrot root rot and droop simultaneously, tomato neckrot root rot
Pathogen is Fusarium oxysporum tomato neckrot root rot specialized form, and the pathogen of tomato wilt specially changes for Fusarium oxysporum tomato
No. 1 biological strain of type, it is characterised in that including box body and 6 PCR pipes, 2 × Taq PCR are respectively provided with 2 PCR pipes
MasterMix and ddH2O；Detection Fusarium oxysporum tomato neckrot root rot specialized form is respectively provided with other 4 PCR pipes
ForL- sense primers, ForL- anti-sense primers, and detect the For- upstreams of Fusarium oxysporum tomato No. 1 biological strain of specialized form
Primer, For- anti-sense primers.
6. kit according to claim 5, it is characterised in that
ForL- upstream primer sequences are：5’-CCTTACTCGACATTTTTCAGA-3’；
ForL- downstream primer sequences are：5’-CATCAAGGCATAGTAAGAT-3’；
For- upstream primer sequences are：5’-GTATGACTCCTCTTATCATA-3’；
For- downstream primer sequences are：5’-GTGGAGTGAGCTTACCGTTA-3’.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710118646.3A CN106868138A (en) | 2017-03-01 | 2017-03-01 | Primer and kit for identifying the pathogen of tomato neckrot root rot and droop |
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|---|---|---|---|
| CN201710118646.3A CN106868138A (en) | 2017-03-01 | 2017-03-01 | Primer and kit for identifying the pathogen of tomato neckrot root rot and droop |
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| CN106868138A true CN106868138A (en) | 2017-06-20 |
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ID=59169335
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710118646.3A Pending CN106868138A (en) | 2017-03-01 | 2017-03-01 | Primer and kit for identifying the pathogen of tomato neckrot root rot and droop |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110499386A (en) * | 2019-09-18 | 2019-11-26 | 扬州大学 | A kind of method of quick detection tomato wilt |
| CN111286555A (en) * | 2020-04-02 | 2020-06-16 | 浙江省农业科学院 | SNP molecular marker based on pgx4 gene, application thereof in fusarium oxysporum detection, detection method and kit |
| CN112176090A (en) * | 2020-10-23 | 2021-01-05 | 青岛农业大学 | Molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof |
| CN113943826A (en) * | 2020-07-16 | 2022-01-18 | 中国科学院遗传与发育生物学研究所 | Molecular marker closely linked with tomato neck rot and root rot resistance gene Frl and application thereof |
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| CN102559512A (en) * | 2011-12-16 | 2012-07-11 | 北京市农林科学院 | Fusarium oxysporum tomato neck rot root rot specialization type and application thereof in cultivation of tomato neck rot rood rot disease-resistant varieties |
| TWI391490B (en) * | 2009-04-01 | 2013-04-01 | Nat Univ Chung Hsing | Primer, method and kit for detecting fusarium wilt pathogen (fusarium oxysporum f. sp. niveum) of watermelon |
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| TWI391490B (en) * | 2009-04-01 | 2013-04-01 | Nat Univ Chung Hsing | Primer, method and kit for detecting fusarium wilt pathogen (fusarium oxysporum f. sp. niveum) of watermelon |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110499386A (en) * | 2019-09-18 | 2019-11-26 | 扬州大学 | A kind of method of quick detection tomato wilt |
| CN111286555A (en) * | 2020-04-02 | 2020-06-16 | 浙江省农业科学院 | SNP molecular marker based on pgx4 gene, application thereof in fusarium oxysporum detection, detection method and kit |
| CN113943826A (en) * | 2020-07-16 | 2022-01-18 | 中国科学院遗传与发育生物学研究所 | Molecular marker closely linked with tomato neck rot and root rot resistance gene Frl and application thereof |
| CN113943826B (en) * | 2020-07-16 | 2023-05-05 | 中国科学院遗传与发育生物学研究所 | Molecular marker closely linked with tomato neck rot resistance gene Frl and application thereof |
| CN112176090A (en) * | 2020-10-23 | 2021-01-05 | 青岛农业大学 | Molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof |
| CN112176090B (en) * | 2020-10-23 | 2022-06-10 | 青岛农业大学 | Molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof |
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Application publication date: 20170620 |