CN112442548A - LAMP primer group containing kit for detecting tobacco target spot disease, application and detection method thereof - Google Patents
LAMP primer group containing kit for detecting tobacco target spot disease, application and detection method thereof Download PDFInfo
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Abstract
The invention provides a kit for detecting tobacco target spot disease, which comprises an LAMP primer group, and an application and a detection method thereof, and relates to the technical field of molecular biology. The LAMP primer group comprises an upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3 and a downstream outer primer B3, wherein the nucleotide sequences of the upstream inner primer FIP, the downstream inner primer BIP, the upstream outer primer F3 and the downstream outer primer B3 are shown in SEQ ID Nos. 1-4. The 4 primers were designed using the ITS sequence of the AG3 fusion population of Targeted tobacco pathogen as a target molecule. Therefore, the 4 primers have high specificity and sensitivity to the tobacco target spot disease, and the kit for detecting the tobacco target spot disease and the detection method thereof comprising the LAMP primer group can realize the rapid detection of the tobacco target spot disease and have the advantages of high sensitivity and good specificity.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a kit containing an LAMP primer group for detecting tobacco target spot disease, and an application and a detection method thereof.
Background
The tobacco target spot is an important tobacco leaf disease caused by the basidiospore of the pangamia citrulli, and the asymptomatic condition of the tobacco target spot is rhizoctonia solani. The early disease is easy to be confused with other leaf diseases such as alternaria alternata and the like, is not easy to be found, can be rapidly developed once being epidemic, is difficult to prevent and causes great loss. Therefore, establishing a method for quickly detecting the target leaf spot of the tobacco is very important for early prevention and control of the disease.
The current detection method for the disease mainly comprises pathogen separation and identification, and common PCR and fluorescent quantitative PCR identification. The methods have high requirements on specialization, complicated process and long time consumption, or have high requirements on instruments and equipment, and can not meet the requirements on efficient and economical detection.
Therefore, it is necessary and urgent to develop a method for detecting tobacco target spot disease with strong sensitivity, high specificity, accurate detection, simple and rapid operation and low requirement for detection equipment to avoid the problem of confusion with other leaf diseases such as tobacco brown spot disease in the early stage of the disease.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide an LAMP primer group, a kit for detecting tobacco target spot disease and a detection method, wherein the kit comprises the LAMP primer group, and the kit for detecting the tobacco target spot disease and the detection method comprise the LAMP primer group.
In order to achieve the purpose, the invention particularly provides the following technical scheme:
the LAMP primer group provided by the invention comprises an upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3 and a downstream outer primer B3, wherein:
the nucleotide sequence of the upstream inner primer FIP is shown as SEQ ID No. 1;
the nucleotide sequence of the downstream inner primer BIP is shown as SEQ ID No. 2;
the nucleotide sequence of the upstream outer primer F3 is shown as SEQ ID No. 3;
the nucleotide sequence of the downstream outer primer B3 is shown as SEQ ID No. 4.
The LAMP primer group provided by the invention is applied to detection of the tobacco target spot.
The kit for detecting the tobacco target spot comprises a primer solution containing the LAMP primer group.
Further, the molar ratio of the upstream inner primer FIP, the downstream inner primer BIP, the upstream outer primer F3 and the downstream outer primer B3 in the primer solution is 1-2 μ M: 1-2 μ M: 0.1-0.4 μ M: 0.1-0.4 μ M: 0.2-0.5 μ M: 0.2 to 0.5 μ M.
Further, the kit also comprises LAMP reaction buffer solution, dNTPs and Mg2+A strand displacing DNA polymerase, and a fluorescent dye having a binding ability to dsDNA.
Further, the reaction system of the kit comprises:
primer solution, LAMP reaction buffer solution, dNTPs and MgSO4Strand displacement DNA polymerase, a fluorescent dye having binding ability to dsDNA, and ultrapure water;
further, the fluorescent dye having binding ability to dsDNA is SYBR Green I.
The invention provides a detection method of tobacco target spot, which comprises the following steps:
providing a DNA extract of a sample to be detected, carrying out LAMP amplification reaction on the DNA extract of the sample to be detected by using the kit for detecting the tobacco target spot to obtain an amplification product, and then carrying out electrophoresis or fluorescence detection on the amplification product to judge whether the sample to be detected is infected with the tobacco target spot.
Further, the LAMP amplification reaction comprises the following steps:
firstly, amplifying for 35-45 min at 60-66 ℃, and then inactivating the strand displacement DNA polymerase for 2-3 min at 94-96 ℃ to obtain an amplification product.
Compared with the prior art, the invention has the beneficial effects that:
the LAMP primer group provided by the invention comprises an upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3 and a downstream outer primer B3, wherein the nucleotide sequences of the upstream inner primer FIP, the downstream inner primer BIP, the upstream outer primer F3 and the downstream outer primer B3 are shown in SEQ ID No. 1-4. The 4 primers are designed by taking the ITS sequence of the fusion group of the tobacco Tartary blight bacterium AG3 as a target molecule, so the 4 primers have high specificity and sensitivity to the tobacco Tartary blight. In addition, the invention also provides a kit for detecting the tobacco target spot disease and a detection method thereof, wherein the kit comprises the LAMP primer group, can realize the rapid detection of the tobacco target spot disease, and has the advantages of high sensitivity and good specificity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the results of detecting various common tobacco pathogens according to example 4 of the present invention;
FIG. 2 is a diagram showing the results of detecting genomic DNA of Tartary sclerotinia rot of tobacco at different concentrations, which is provided in example 5 of the present invention;
FIG. 3 is a comparison chart of fluorescence detection of tobacco leaves infected with tobacco target spot, healthy tobacco leaves and other diseases, such as alternaria alternate, according to example 6 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the invention, a LAMP primer set comprises an upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3 and a downstream outer primer B3, wherein:
the nucleotide sequence of the upstream inner primer FIP is shown as SEQ ID No. 1;
the nucleotide sequence of the downstream inner primer BIP is shown as SEQ ID No. 2;
the nucleotide sequence of the upstream outer primer F3 is shown as SEQ ID No. 3;
the nucleotide sequence of the downstream outer primer B3 is shown as SEQ ID No. 4.
Loop-mediated isothermal amplification (LAMP) is a novel circulating isothermal amplification technology developed in 2000, and has the characteristics of strong specificity, high sensitivity, high amplification speed, low requirement on experimental equipment, easiness in observing detection results and the like, and is completed under an isothermal condition. The LAMP technology designs 4 specific primers aiming at 6 regions of a target gene, and under the action of strand displacement DNA polymerase (Bst DNA polymerase), the amplification is carried out at the constant temperature of 60-65 ℃, and the nucleic acid amplification of 109-1010 times can be realized within about 15-60 minutes. After the reaction is finished, whether magnesium pyrophosphate precipitate is generated or not is observed by naked eyes, and whether the target sequence exists or not can be judged. Fluorescent indicators can also be added into the reaction liquid, so that the reaction result can be observed more conveniently, visually and reliably by naked eyes. Compared with common PCR and fluorescent quantitative PCR detection, the technology has stronger applicability and is easy to popularize. Although LAMP has been applied to detection of other phytopathogens, no related report for detecting tobacco target spot disease exists at present.
The LAMP primer group provided by the invention comprises an upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3 and a downstream outer primer B3, wherein the nucleotide sequences of the upstream inner primer FIP, the downstream inner primer BIP, the upstream outer primer F3 and the downstream outer primer B3 are shown in SEQ ID No. 1-4. The 4 primers are designed by taking the ITS sequence of the tobacco leaf spot pathogen and different fusion group sequences of other rhizoctonia as target molecules. Therefore, the 4 primers have higher specificity and sensitivity to the tobacco target spot.
According to one aspect of the invention, the LAMP primer group is applied to detection of tobacco target spot. Specifically, the application comprises the step of carrying out PCR reaction or LAMP reaction on the LAMP primer group so as to amplify or detect the tobacco target leaf spot pathogen.
According to one aspect of the present invention, a kit for detection of tobacco target spot disease comprises a primer solution containing the above LAMP primer set.
In a preferred embodiment of the present invention, the molar ratio of the upstream inner primer FIP, the downstream inner primer BIP, the upstream outer primer F3 and the downstream outer primer B3 in the primer solution is 1-2 μ M: 1-2 μ M: 0.1-0.4 μ M: 0.1-0.4 mu M;
in a preferred embodiment, the molar ratio of the primers in the primer solution can increase the rate and stability of the LAMP amplification reaction of the target gene.
In a preferred embodiment of the invention, the kit further comprises LAMP reaction buffer, dNTPs and Mg2 +A strand displacing DNA polymerase, and a fluorescent dye having a binding ability to dsDNA.
In a preferred embodiment of the present invention, the reaction system of the kit comprises:
primer solution, LAMP reaction buffer solution, dNTPs and MgSO4Strand displacement DNA polymerase, a fluorescent dye having binding ability to dsDNA, and ultrapure water;
in a preferred embodiment of the invention, the fluorescent dye having binding ability to dsDNA is SYBR Green I.
In a preferred embodiment, after the fluorescent dye is added, the color of the LAMP reaction product of the test sample is observed under natural light, and if the color is green fluorescence, the result is positive, that is, the sample is infected with the tobacco target spot disease, and if the color is orange fluorescence, the result indicates that the test sample is not infected with the tobacco target spot disease.
Preferably, the volume of the reaction system is 25 μ L;
wherein 10uM FIP and BIP primers 4 uL, 10uM F3 and B3 primers 0.5 uL, 10mM dNTPs 2.5 uL, 8U/uL Bst DNA polymerase 1 uL, 50mM MgSO4mu.L, 10 × Isothermal amplification buffer 2.5. mu.L, template DNA 1. mu.L, and made up to 25. mu.L with sterilized ultrapure water.
According to one aspect of the present invention, a method for detecting tobacco target spot disease, the method comprising the steps of:
providing a DNA extract of a sample to be detected, carrying out LAMP amplification reaction on the DNA extract of the sample to be detected by using the kit for detecting the tobacco target spot to obtain an amplification product, and then carrying out electrophoresis or fluorescence detection on the amplification product to judge whether the sample to be detected is infected with the tobacco target spot.
The detection method of the tobacco target spot disease provided by the invention is characterized by providing a DNA extract of a sample to be detected, carrying out LAMP amplification reaction on the DNA extract of the sample to be detected by using the kit for detecting the tobacco target spot disease to obtain an amplification product, and then carrying out electrophoresis or fluorescence detection on the amplification product to judge whether the sample to be detected is infected with the tobacco target spot disease. The method has the advantages of high sensitivity and good specificity, can be used for quickly diagnosing and identifying the target spot disease of the tobacco in the field at the early stage of the disease, and effectively solves the problem that the disease is easy to be confused with other leaf diseases such as tobacco brown spot and the like and difficult to identify at the early stage of the disease. The detection method can realize the rapid detection of the tobacco target leaf spot germs, has simple operation, high sensitivity, strong specificity and high detection speed, and has important significance for the early diagnosis and identification of the tobacco target leaf spot and the effective prevention and control of the disease.
Meanwhile, the detection method has the characteristics of strong detection specificity, rapidness, sensitivity, stability, high efficiency, simple instrument requirement, simplicity and convenience in operation, low cost and easiness in popularization and use in a basic layer, and compared with the conventional detection method, the detection primer combination can be used for specifically detecting the tobacco target spot and can well distinguish other rice and corn sheath blight diseases and tobacco brown spot diseases in a tobacco field.
In the above preferred embodiment, the LAMP amplification reaction comprises the steps of:
firstly, amplifying for 35-45 min at 60-66 ℃, and then inactivating the strand displacement DNA polymerase for 2-3 min at 94-96 ℃ to obtain an amplification product.
The technical solution of the present invention will be further described with reference to the following examples.
Example 1 LAMP detection specific primer combination design of tobacco target spot pathogen:
the ITS sequence of tobacco leaf spot pathogen and other different fusion group sequences of rhizoctonia are subjected to sequence comparison by using DNAMAN software, the fragments with low homology with other pathogen sequences in the leaf spot pathogen are screened, and then the partial sequences are designed by using online software Primer Exploore V5 (LAMP Primer of the leaf spot pathogen, including outer Primer F3/B3, front inner Primer FIP (composed of F1 reverse complementary sequence F1c and F2), and rear inner Primer BIP (composed of B1 reverse complementary sequence B1c and B2).
Wherein:
the nucleotide sequence of the upstream inner primer FIP is shown as SEQ ID No. 1;
the nucleotide sequence of the downstream inner primer BIP is shown as SEQ ID No. 2;
the nucleotide sequence of the upstream outer primer F3 is shown as SEQ ID No. 3;
the nucleotide sequence of the downstream outer primer B3 is shown as SEQ ID No. 4.
The synthesized primers are respectively diluted to 10 mu mol/L by using sterilized double distilled water, and the primers are placed in a refrigerator at the temperature of minus 20 ℃ and are kept away from light for later use.
Example 2
A method of detecting tobacco target spot disease, the method comprising the steps of:
1. extraction of genomic DNA of test strains
Taking a proper amount of a sample to be detected, putting the sample to be detected into a centrifuge tube containing 400 mu L of DNA lysate (0.15M Tris-HCl, 0.04M EDTA-Na2, 0.2M NaCl, 3mM SDS, pH 8.0), adding a steel ball, shaking for 1min at 65Hz, centrifuging at 12000rpm for 2min, taking 200 mu L of supernatant, adding 400 mu L of absolute ethyl alcohol, standing at-20 ℃ for 30min, centrifuging at 12000rpm for 2min, discarding the supernatant, drying in the air, adding 30 mu L of sterile double distilled water to dissolve DNA, and obtaining a genome DNA extract.
Of course, in other embodiments, the extraction of the template DNA is an optional step, and the detection can be performed directly using the prepared DNA template.
2. Establishment of LAMP reaction detection system
The main reagents are as follows: BstDNA polymerase large fragment was purchased from NEB, UK; DNA marker was purchased from Bao bioengineering Dalian GmbH; dNTPs were purchased from Thermo Scientific. The primers were synthesized by Biotechnology (Shanghai) Ltd.
The established LAMP detection method reaction system comprises:
1. mu.L of the above DNA template,
10×isothermal amplification buffer 2.5μL
50mM MgSO4 4μL、
10mM dNTP Mix 2.5μL、
0.5. mu.L of Bst DNA polymerase 8U/. mu.L,
10 mu mol/L inner primer FIP/BIP 4 mu L,
10 μmol/L of the outer primer F3/B30.5 μ L,
Add ddH2O to make up to 25. mu.L, centrifuge to mix well the reagents.
The reaction conditions are as follows: the temperature is 65 ℃, the reaction time is 60min, and the Bst DNA polymerase is inactivated at 80 ℃ for 10min after the reaction is finished.
3. And (3) observing a reaction result: after the reaction, 0.5. mu.L of SYBR Green I dye was added, and the color change was directly observed with naked eyes after mixing. The reaction solution of the tobacco target spot pathogen DNA is bright green, and other pathogenic bacteria and the negative control reaction solution are transparent light brown yellow.
Example 3
A method for detecting tobacco target spot disease, said method comprising, in addition to step 3:
and detecting by adopting an electrophoresis detection result. The method comprises the following specific steps: carrying out agarose gel electrophoresis on the reaction product, and if a trapezoidal band appears in the electrophoresis result, judging that the detection result is positive; otherwise, the test result is negative, and the same procedure as in example 2 is repeated.
Example 4
The detection method provided in example 2 is adopted to identify different common tobacco germs, water is used as negative control, the information of the different common tobacco germs is shown in Table 1, and the detection results are shown in FIG. 1 (in the figure, 1: tobacco leaf blight, 2: tobacco leaf blight, 3: tobacco leaf blight, 4: rice sheath blight, 5: rice sheath blight, 6: corn sheath blight, 7: corn sheath blight, 8: tobacco brown spot, 9: tobacco black shank, 10: tobacco anthracnose, 11: tobacco fusarium root rot, and 12: negative control water).
As is clear from the results shown in FIG. 1, green fluorescence was observed only in the reaction tube using the DNA of tobacco Taraxacum, and the agarose gel electrophoresis detected that a ladder-shaped band characteristic to LAMP appeared, while the color development of the reaction tube using the DNA of other fungi such as Rhizoctonia solani, Rhizoctonia zeae, and Alternaria alternata as templates was orange and no amplified band appeared in the agarose gel electrophoresis. The result shows that the primer has strong specificity and can specifically detect the tobacco target leaf spot bacteria.
Example 5
By adopting the detection method provided by the embodiment 2, the genome DNA of the tobacco leaf blight bacteria of the sample to be detected is diluted with sterilized double distilled water at different concentrations to obtain the samples to be detected with DNA concentrations of 1000 ng/muL, 100 ng/muL, 10 ng/muL, 1 ng/muL, 100 pg/muL, 10 pg/muL, 1 pg/muL, 100 fg/muL, 10 fg/muL and 1 fg/muL respectively as templates to carry out LAMP reaction, and meanwhile, sterile double distilled water is used as a negative control template to carry out synchronous experiments.
The detection results are shown in FIG. 2 (in the figure: 1: 1000 ng/. mu.L, 2: 100 ng/. mu.L, 3: 10 ng/. mu.L, 4: 1 ng/. mu.L, 5: 100 pg/. mu.L, 6: 10 pg/. mu.L, 7: 1 pg/. mu.L, 8: 100 fg/. mu.L, 9: 10 fg/. mu.L, 10: 1 fg/. mu.L);
a is LAMP sensitivity detected based on SYBR Green I staining color change; among them, 1 to 5 tubes were yellow-green and showed positive.
The concentrations of the template DNAs were 1000 ng/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, and 100 pg/. mu.L, respectively. Tubes 6-10 were light orange, indicating negative results.
The template DNA concentrations were 10 pg/. mu.L, 1 pg/. mu.L, 100 fg/. mu.L, 10 fg/. mu.L and 1 fg/. mu.L, respectively.
B is agarose gel electrophoresis picture of LAMP detection sensitivity.
M is Marker (2kbp), and the template concentrations in lanes 1-5 are 1000 ng/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, and 100 pg/. mu.L, respectively. Lanes 6-10 template DNA concentrations were 10pg/μ L, 1pg/μ L, 100fg/μ L, 10fg/μ L and 1fg/μ L, respectively.
As can be seen from FIG. 2, the samples were positive from the template concentration of 1000 ng/. mu.L to 100 pg/. mu.L, the samples were negative from the template concentration of 10 pg/. mu.L to 1 fg/. mu.L and the negative control. This indicates that the detection method of the present application can detect DNA concentrations as low as 100 pg/. mu.L with extremely high sensitivity.
Example 6
And detecting the disease spots on the tobacco leaves infected with the tobacco target spot. Taking healthy tobacco leaves and tobacco leaves infected with other diseases of tobacco brown spot as a control. DNA was extracted from the lesion and healthy tissue as templates, and LAMP reaction was carried out by establishing an LAMP reaction detection system in the method of example 2.
The detection results are shown in fig. 3 (in the figure, 1: tobacco target spot disease-affected lamina, 2: tobacco target spot disease-affected lamina, 3: tobacco target spot disease-affected lamina, 4: healthy tobacco lamina, 5: healthy tobacco lamina, 6: tobacco alternaria alternata disease-affected lamina, 7: tobacco alternaria alternata disease-affected lamina), and it can be seen from fig. 3 that the detection displays of 3 samples to be detected are all positive results, the reaction product in the reaction tube is bright green, and the electrophoresis result is a trapezoidal strip. The results of 2 healthy leaves and 1 tobacco brown spot plant sample are negative. The experiment further shows that the method has high detection specificity on the tobacco leaf blight bacteria, has good application prospect, and can be widely used for detecting the tobacco leaf blight bacteria.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Chenzhou city company, tobacco company, Hunan province
Kit containing LAMP primer group and used for detecting tobacco target spot disease, application and detection method thereof
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence
<400> 1
taggtgaacc tgcggaag 18
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence
<400> 2
gtttatatga gttgagtaga caga 24
<210> 3
<211> 44
<212> DNA
<213> Artificial sequence
<400> 3
agagaaaggt gtgcacatgc tttaatgaag agtttggttg tagc 44
<210> 4
<211> 43
<212> DNA
<213> Artificial sequence
<400> 4
cacctgtgaa cttgtgagac aagggttcaa tgacttatca tct 43
Claims (9)
1. An LAMP primer group, which comprises an upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3 and a downstream outer primer B3, wherein:
the nucleotide sequence of the upstream inner primer FIP is shown as SEQ ID No. 1;
the nucleotide sequence of the downstream inner primer BIP is shown as SEQ ID No. 2;
the nucleotide sequence of the upstream outer primer F3 is shown as SEQ ID No. 3;
the nucleotide sequence of the downstream outer primer B3 is shown as SEQ ID No. 4.
2. The application of the LAMP primer group of claim 1 in the detection of tobacco target spot.
3. A kit for detecting tobacco target spot disease, characterized by comprising a primer solution containing the LAMP primer set according to claim 1.
4. The kit for detecting the tobacco target spot disease according to claim 3, wherein the molar ratio of the upstream inner primer FIP, the downstream inner primer BIP, the upstream outer primer F3 and the downstream outer primer B3 in the primer solution is 1-2 μ M: 1-2 μ M: 0.1-0.4 μ M: 0.1 to 0.4 μ M.
5. The kit for detecting tobacco target spot disease according to claim 3, wherein the kit further comprises LAMP reaction buffer, dNTPs, Mg2+A strand displacing DNA polymerase, and a fluorescent dye having a binding ability to dsDNA.
6. The kit for detecting the tobacco target spot according to claim 3, wherein the reaction system of the kit comprises:
primer solution, LAMP reaction buffer solution, dNTPs and MgSO4Strand displacement DNA polymerase, fluorescent dye having binding ability to dsDNA, and ultrapure water.
7. The kit for detecting tobacco target spot according to claim 5 or 6, wherein the fluorescent dye having binding ability to dsDNA is SYBR Green I.
8. A method for detecting tobacco target spot disease, which is characterized by comprising the following steps:
providing a DNA extract of a sample to be detected, carrying out LAMP amplification reaction on the DNA extract of the sample to be detected by using the kit for detecting the tobacco target spot disease as claimed in any one of claims 3-7 to obtain an amplification product, and then carrying out electrophoresis or fluorescence detection on the amplification product to judge whether the sample to be detected is infected with the tobacco target spot disease.
9. The method for detecting tobacco target spot according to claim 8, wherein the LAMP amplification reaction comprises the following steps:
firstly, amplifying for 35-45 min at 60-66 ℃, and then inactivating the strand displacement DNA polymerase for 2-3 min at 94-96 ℃ to obtain an amplification product.
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| CN113652377A (en) * | 2021-09-17 | 2021-11-16 | 湖南农业大学 | Bacterial strain for antagonizing pathogenic bacteria of tobacco target spot and application thereof |
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