CN112176090B - Molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof - Google Patents
Molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof. The nucleotide sequence of the molecular marker FrlDel30-1 is shown in SEQ ID NO.1, and the molecular marker is a molecular marker for identifying the neck rot and root rot specialization type of fusarium oxysporum tomatoes; the nucleotide sequence of the molecular marker FrlDel30-2 is shown in SEQ ID NO.2, the molecular marker is a molecular marker for identifying the physiological race3 of fusarium oxysporum tomato fusarium wilt pathogenic bacteria, and a primer pair for detecting the molecular marker comprises FrlDel 30F: 5'-GAGCGGGAGTTGAATTCTTG-3', respectively; FrlDel 30R: 5'-AAGAGCCTGCTCCA GTTGAA-3' are provided. The molecular marker can simply, quickly and accurately distinguish fusarium oxysporum tomato neck rot root rot specialized strains and fusarium oxysporum tomato fusarium wilt pathogenic bacteria physiological race3 pathogenic bacteria, and has important effects on identification of types of fusarium oxysporum in susceptible tomatoes and detection of diseased parts or tissues of the susceptible tomatoes.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to a molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof.
Background
The Fusarium oxysporum-induced tomato neck Rot and Root Rot (FCRR), commonly known as "death disease", is another very destructive tomato main disease after outbreak of destructive diseases such as Root knot nematode disease and yellow leaf curl virus disease. The tomato neck rot and root rot especially pose serious threats to the safe production of facility tomatoes in China, wherein the incidence rate of the tomatoes in the sunlight greenhouse can reach 80% of the total content of the active ingredients is more than 30%. At present, there are at least 120 fusarium oxysporum specialization types and fusarium oxysporum physiological races, and the pathogenic bacteria causing the tomato FCRR are mainly fusarium oxysporum tomato neck rot root rot specialization types (f)Fusarium oxysporum f. sp. radicis-lycopersiciForl). The research on tomato FCRR is relatively few in China, and particularly, no very clear method exists for identifying pathogenic bacteria of the disease, so that the screening and the utilization of tomato disease-resistant materials are limited. The fusarium oxysporum tomato neck rot root rot specialization type is very close to the fusarium wilt physiological race3 in phenotype and is difficult to distinguish, the differentiation and identification are mainly carried out depending on the pathogenicity to a special host or a narrower host range, and no well-known Forl differential host material exists at present, so that the neck rot root rot transformation type of the fusarium oxysporum tomato cannot be determined by the traditional inoculation identification method.
The molecular marker technology has been widely used for the related research of plant pathogenic fungi, and provides a good foundation for identifying and detecting pathogenic bacteria. In recent years, many molecular markers have been successfully applied to the identification and diagnosis of various plant pathogenic fungi, such as banana wilt physiological races, fusarium oxysporum spinach specialized type, cucumber specialized type, towel gourd specialized type, bitter gourd specialized type, and the like. However, according to the existing identification method or identification specific primer, the identification result is mostly negative or the technology has defects, so that the development of more efficient and stable molecular markers capable of accurately identifying and detecting the pathogenic bacteria of the tomato neck rot and root rot disease is more needed.
Disclosure of Invention
The invention provides a molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof. The molecular marker can accurately identify the tomato neck rot root rot pathogen, and is applied to identifying the type of the fusarium oxysporum pathogen in susceptible tomatoes and detecting the diseased parts of the susceptible tomatoes.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a molecular marker FrlDel30-1 for identifying fusarium oxysporum tomato neck rot root rot, wherein the nucleotide sequence of the molecular marker FrlDel30-1 is shown in SEQ ID NO. 1.
Furthermore, the molecular marker FrlDel30-1 is a molecular marker for identifying the specialization type of neck rot and root rot of fusarium oxysporum tomatoes.
Further, the molecular marker FrlDel30-1 is 204bp in length.
The invention also provides a molecular marker FrlDel30-2 for identifying the neck rot and root rot of fusarium oxysporum tomatoes, and the nucleotide sequence of the molecular marker FrlDel30-2 is shown as SEQ ID NO. 2.
Further, the molecular marker FrlDel30-2 is a molecular marker for identifying the type 3 physiological race of fusarium oxysporum tomato fusarium wilt pathogenic bacteria.
Further, the molecular marker FrlDel30-2 has a length of 234 bp.
Further, the molecular marker FrlDel30-1 or the molecular marker FrlDel30-2 is of an InDel type.
The invention also provides a primer pair for detecting the molecular marker FrlDel30-1 or the molecular marker FrlDel30-2, wherein the primer pair comprises:
FrlDel 30F: 5'-GAGCGGGAGTTGAATTCTTG-3', respectively; and
FrlDel30R：5’-AAGAGCCTGCTCCAGTTGAA-3’。
the invention also provides application of the molecular marker FrlDel30-1 or the molecular marker FrlDel30-2 in identifying types of fusarium oxysporum pathogenic bacteria in susceptible tomatoes.
Further, the identification method comprises the following steps: extracting pathogenic bacteria genome DNA of the susceptible tomato plant, and performing PCR amplification and electrophoresis on the pathogenic bacteria genome DNA by using the primer pair FrlDel30F and FrlDel 30R; if a specific strip of 204bp appears in the sample, the strip is the molecular marker FrlDel30-1, and the infected tomato is fusarium oxysporum tomato neck rot root rot obligate pathogen; if a 234bp specific band appears in the sample, the band is the molecular marker FrlDel30-2, and the infection of the susceptible tomato is fusarium oxysporum tomato wilt pathogenic bacteria physiological race 3.
Furthermore, the reaction system of the PCR reaction is a PCR reaction systemThe series (20. mu.L) was: including a concentration of 10. mu. mol.L -11. mu.L of each of the forward and reverse primers of (1); 1.2mM dNTPs; 2.5mM MgCl2；2μL 2 × MasterMix（1.2mmol dNTPs，2.5mmol MgCl20.5units Taq); 22 μ L of DNA template (30-50 ng. μ L)-1），ddH2And O is supplemented to 20 mu L.
Further, the procedure of PCR amplification is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extension at 72 ℃ for 10 min.
The invention also provides application of the molecular marker FrlDel30-1 or the molecular marker FrlDel30-2 in detection of diseased tissues or diseased parts of tomato.
Further, the disease part comprises a root part, a stem base to a cotyledon part and a cotyledon to a true leaf part.
Compared with the prior art, the invention has the following advantages:
on the basis of carrying out identification of the tomato neck rot root rot and analysis and research of a transcriptome of the tomato neck rot root rot, the invention successfully obtains an InDel molecular marker which has the specificity, consistency and stability of fusarium oxysporum tomato neck rot root rot specialized strain and can identify fusarium oxysporum tomato neck rot root rot specialized pathogenic bacteria and fusarium oxysporum tomato fusarium wilt pathogenic bacteria physiological race3 pathogenic bacteria, and designs a primer pair. The molecular marker can easily identify fusarium oxysporum tomato neck rot root rot specialized pathogenic bacteria and fusarium oxysporum tomato wilt pathogenic bacteria physiological races 3, so the molecular marker has an important role in identifying fusarium oxysporum tomato neck rot root rot specialized strains, lays a foundation for establishing a specific, rapid, high-sensitivity, stable and reliable molecular detection technical system of the fusarium oxysporum tomato neck rot root rot specialized pathogenic bacteria, can also detect pathogenic parts and pathogenic tissues of susceptible tomatoes, further deeply researches the pathogenesis of the tomatoes, and lays a foundation for overcoming fusarium oxysporum tomato neck rot in the future. In addition, the method can overcome the defects of large workload and long period of the conventional identification and differentiation of fusarium oxysporum tomato neck rot root rot specialized pathogenic bacteria, and realizes the rapid and accurate identification and detection of pathogenic substances.
Drawings
FIG. 1 is a comparison of sequence characteristics of fusarium oxysporum tomato neck rot root rot obligate genome (JH 651385.1) and tomato fusarium wilt pathogen race3 genome (CM 012187.1).
FIG. 2 is a PCR amplification map of the molecular marker FrlDel30 of the invention against different strains of Fusarium oxysporum solani; wherein, M: DNA ladder D2000; 1-2: are fusarium oxysporum tomato neck rot root rot specialized strain ForlWF001 and fusarium oxysporum tomato wilt pathogenic strain FolYT 002.
FIG. 3 is a comparison of the nucleotide sequences of PCR amplified fragments of the molecular marker FrlDel30 of the invention against different strains of Fusarium solani.
FIG. 4 shows the bacterial infection of different parts of tomato MoneyMaker seedlings inoculated with FCRR pathogenic bacteria; wherein, the upper left is root, the upper right is stem base to cotyledon part, and the middle lower is cotyledon to true leaf part; a: CK MoneyMaker; b: MoneyMaker.
FIG. 5 is a detection amplification map of the molecular marker FrlDel30 of the invention on total DNA of different part tissues of a tomato neck rot root rot seedling plant 15 days after artificial inoculation; wherein, M: DNA ladder D2000; 1-3: inoculating cotyledon to true leaf part, stem to cotyledon part and root neck part of the seedling; 4: FolYT 002; 5: blank control.
FIG. 6 is a detection amplification map of the molecular marker FrlDel30 of the invention on stem base isolation pathogenic bacteria genome DNA of tomato neck rot root rot plants collected from different regions; wherein, M: DNA ladder; 1-8: the source of the sample was Shandong province; 9-10: the source of the sample was Beijing; 11-12: the source of the sample was Henan province; 13-14: the source of the sample is Heilongjiang; 15-16: the source of the sample was Hebei province; 17: folrace 3 pathogen; CK: blank control.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples.
Example 1:
discovery and development of molecular markers
Fusarium oxysporum tomato neck rot root rot specialized transcriptome data are used: a physiological race3 genome (CM 012187.1) of pathogenic bacteria of the tomato fusarium wilt and a fusarium oxysporum tomato neck rot root specialized genome (JH 651385.1) are used as references, and fusarium oxysporum tomato neck rot root rot specialized transcriptome reads map is added into the two reference genomes. A plurality of InDel sites are found through comparison, wherein 1 InDel site with 30bp exists between the first chromosome of CM012187.1 and the first chromosome of JH651385.1, and a pair of PCR primers comprising an upstream primer FrlDel30F and a downstream primer FrlDel30R are designed aiming at the site (CM 012187.1: chromome 1, 1293308-1293541; JH 651385.1: chromome 1, 1040360-1040536), and the primer sequences are as follows:
FrlDel30F：5’-GAGCGGGAGTTGAATTCTTG-3’；
FrlDel30R：5’-AAGAGCCTGCTCCAGTTGAA-3’。
the pair of primers can amplify a 204bp fragment in fusarium oxysporum tomato neck rot root rot specialized genome DNA, and the sequence is shown as SEQ ID NO. 1; and a 234bp fragment can be amplified in the genome DNA of the fusarium solani tomato wilt pathogenic bacterium physiological race3, and the sequence is shown as SEQ ID NO. 2. The obtained two sequences are used as InDel markers to distinguish fusarium oxysporum tomato neck rot root rot specialization strains from fusarium oxysporum tomato blight pathogenic bacteria physiological race3 strains, and the markers are named FrlDel30-1 and FrlDel30-2 respectively.
Second, verification of molecular marker in fusarium oxysporum
The specificity verification of the InDel marker is carried out by respectively taking fusarium oxysporum tomato neck rot root rot specialized strain ForlWF001 (Forl, sourced from Shandong Shouguang) and fusarium oxysporum tomato blight pathogenic strain FolYT002 (Fol race3, sourced from Shandong Haiyang) as materials. The primer FrlDel30F/R is used for carrying out PCR amplification and electrophoresis on the extracted DNA, the electrophoresis result is shown in figure 2, then TA clone sequencing is carried out on the PCR product, the sequencing result is shown in figure 3, and the molecular markers FrlDel30-1 and FrlDel30-2 can be used for distinguishing fusarium oxysporum tomato neck rot root rot specialized strains and fusarium oxysporum tomato blight pathogen physiological race3 strains.
Wherein, the PCR reaction system (20 μ L) is: including a concentration of 10. mu. mol.L -11. mu.L of each of the forward and reverse primers of (1); 1.2mM dNTPs; 2.5mM MgCl2；2μL 2 × MasterMix（1.2mmol dNTPs，2.5mmol MgCl20.5units Taq); 22 μ L of DNA template (30-50 ng. μ L)-1），ddH2And O is supplemented to 20 mu L.
The procedures for PCR amplification were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 45s, and 35 cycles; extending for 10min at 72 ℃; the storage temperature was 4 ℃.
Example 2
Separation and culture of pathogenic bacteria after inoculation of fusarium oxysporum on tomatoes to specialize in neck rot and root rot
1. Selecting a disease-susceptible tomato variety 'MoneyMaker' to soak seeds for germination, sowing the seeds into a 50-hole plug tray filled with a commercial seedling substrate, placing the plug tray in an artificial climate chamber for culture in the daytime (the illumination is 600 mu mol.m)-2•S-1) The temperature is 25 ℃, the temperature is 18 ℃ at night, the temperature is 12 hours, and the humidity is 55 percent. Filtering the strain ForlWF001 cultured by PDB shake bacteria with six layers of gauze, centrifuging at 1000 rpm for 15 min, discarding supernatant, retaining precipitate, and adjusting spore suspension to 10% concentration by using sterilized water instead of supernatant7cfu∙ml-1. When the tomato grows to be 'two leaves and one heart', the tomato seedlings are pulled out from the matrix slightly, the roots are washed clean by sterilized water, the tomato seedlings are soaked in the spore suspension for 30 min and then planted back into the matrix, and the disease occurrence condition is observed after 15 days.
2. To investigate whether the increasingly emerging disease symptoms of plants are associated with the extensive colonization of the fungus within tomato, plant fungal tissue reisolation tests were also performed: selecting the parts of the tomato 'MoneyMaker' seedlings inoculated with the ForlWF001 strain and cultured for 15 days at different heights, performing tissue re-separation on the parts from root parts, stem parts to cotyledon parts and cotyledon parts to true leaves, culturing on a PDA culture medium for 5 days, and simultaneously calculating the area and the fungal reproduction rate by using a method of measuring the hypha diameter in a cross mode by taking the MoneyMaker seedlings not inoculated with FCRR pathogenic bacteria as a reference.
As a result, as shown in FIG. 4, no fungal growth was observed in the control group; after the inoculation treatment, the roots, stems to cotyledons, cotyledons to true leaves of the plants are colonized by pathogenic bacteria, and the propagation rates of the fungi are 75.95%, 21.8% and 2.47% respectively.
Detection of molecular marker FrlDel30 in tomato inoculated plants
Respectively extracting DNA of pathogenic bacteria cultured from the root, stem base to cotyledon and cotyledon to true leaf of a tomato neck rot root rot disease plant inoculated with the fusarium oxysporum tomato neck rot root rot specialized strain, and amplifying by using a primer pair marked by InDel FrlDel 30. The result is shown in figure 5, after 15 days of inoculating the strain, a specific PCR product of 204bp is amplified on most plant parts in samples from the root, stem base to cotyledon and cotyledon to true leaf parts of a tomato neck rot plant, which indicates that a susceptible plant is a fusarium oxysporum tomato neck rot specialized plant, a fragment of 234bp is also amplified, indicates that the susceptible plant is a tomato blight pathogen physiological race3 plant, and a specific PCR product is not amplified in a clear water contrast way, thereby proving that the molecular marker FrlDel30 can be used for detecting the artificially inoculated susceptible tomato neck rot plant.
Example 3: detection of molecular markers in field diseased plants
The primer pair of the InDel marked FrlDel30 is used for carrying out PCR amplification on pathogenic bacteria genome DNA separated from stem bases of tomato neck rot root rot disease-causing plants collected from Shandong province, Beijing City, Henan province, Hebei province and Heilongjiang province, genome DNA of tomato blight physiological race3 and clear water contrast, the result is shown in figure 6, wherein 16 samples amplify 204bp specific strips, 17 samples amplify 234bp specific strips, no amplification is carried out in the clear water contrast, and the 16 samples of pathogenic bacteria are fusarium oxysporum tomato neck rot specialization types.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao university of agriculture
<120> molecular marker for identifying fusarium oxysporum tomato neck rot root rot and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 204
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gagtgggagt tgaattcttg gactcagatt cctcaggtgc gtcatctgcc tcagcattct 60
cctcctcaac gtccatgctg tcctcctggt cggtaagatc attcacttct tcagctgctt 120
ctgggactgc ttcagcctca gtcgcaacat cttcctcaac ctcggcgaca gtctcgatgg 180
cagcttcaac tggagcaggc tctt 204
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<211> 234
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<213> Artificial Sequence (Artificial Sequence)
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gagcgggagt tgaattcttg gactcagatt cctcaggtgc gtcatctgcc tcagcattct 60
cctcctcaac gtccatgctg tcctcctggt cggtaagatc attcacttct tcagctgctt 120
ctgggactgc ttcagcctca gtcgcaacat cttcttcaac ctcggcgaca gtctcgactt 180
ccacctgagg ctcttcaaga gtctcgatgg cagcttcaac tggagcaggc tctt 234
Claims (2)
1. The application of the molecular marker FrlDel30-1 and the molecular marker FrlDel30-2 in identifying the types of fusarium oxysporum pathogens in susceptible tomatoes is characterized in that the types of the fusarium oxysporum pathogens are fusarium oxysporum tomato neck rot specialization type pathogens or fusarium oxysporum tomato fusarium wilt pathogen physiological races 3, the nucleotide sequence of the molecular marker FrlDel30-1 is shown as SEQ ID No.1, and the nucleotide sequence of the molecular marker FrlDel30-2 is shown as SEQ ID No. 2;
the identification method comprises the following steps: extracting the genome DNA of pathogenic bacteria of a disease-sensitive tomato plant, and performing PCR amplification and electrophoresis on the genome DNA by using a primer pair FrlDel30F and FrlDel 30R;
FrlDel30F：5’-GAGCGGGAGTTGAATTCTTG-3’；
FrlDel30R：5’-AAGAGCCTGCTCCAGTTGAA-3’；
if a specific strip of 204bp appears in the sample, the infected tomato is infected by fusarium oxysporum tomato neck rot root rot specialized pathogen;
if a 234bp specific strip appears in the sample, the infected tomato is infected by fusarium oxysporum tomato wilt pathogenic bacteria physiological race 3.
2. The application of the molecular marker FrlDel30-1 and the molecular marker FrlDel30-2 in detecting diseased tissues or diseased parts of susceptible tomatoes is characterized in that the types of fusarium oxysporum pathogenic bacteria in the diseased tissues or diseased parts are fusarium oxysporum tomato neck rot specialization type pathogenic bacteria or fusarium oxysporum tomato wilt pathogenic bacteria physiological races 3, the nucleotide sequence of the molecular marker FrlDel30-1 is shown as SEQ ID No.1, and the nucleotide sequence of the molecular marker FrlDel30-2 is shown as SEQ ID No. 2; the disease parts comprise roots, stems from bases to cotyledons and cotyledons to true leaves.
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| ZA2021/03925A ZA202103925B (en) | 2020-10-23 | 2021-06-08 | Molecular marker for identifying fusarium crown and root rot of tomato and use thereof |
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| CN105755133A (en) * | 2016-04-08 | 2016-07-13 | 山东寿光蔬菜种业集团有限公司 | Tomato neck/root rot molecular marker as well as marking method and application thereof |
| CN106868138A (en) * | 2017-03-01 | 2017-06-20 | 浙江省农业科学院 | Primer and kit for identifying the pathogen of tomato neckrot root rot and droop |
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| CN105755133A (en) * | 2016-04-08 | 2016-07-13 | 山东寿光蔬菜种业集团有限公司 | Tomato neck/root rot molecular marker as well as marking method and application thereof |
| CN106868138A (en) * | 2017-03-01 | 2017-06-20 | 浙江省农业科学院 | Primer and kit for identifying the pathogen of tomato neckrot root rot and droop |
| 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 |
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| Title |
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| Fusarium oxysporum Fo47 chromosome I，ACCESSION：CP052038;Wang,B.等;《genbank》;20200715;第1-2页 * |
| 山东省番茄尖孢镰刀菌的分离及鉴定;张秀等;《山东农业科学》;20191230(第12期);全文 * |
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