A leaf spot pathogen LAMP detection primer, kit and LAMP rapid detection method

Abstract

The invention provides a LAMP detection primer for a leaf spot pathogen, and relates to the technical field of plant pathogen detection. The primer comprises a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP and a reverse inner primer BIP. The primer series are as follows: F3: 5'-ACCAGTGCGGTGGTATCG-3'; B3: 5'-CAAGAGCGCCTGTCACTC-3'; FIP: 5'-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3'; BIP: 5'-CATACGACGACTCGACAAGCGCCC ACCACCCAAAAAAACGAC-3'.

Description

Technical Field

[0001] The invention relates to the technical field of plant pathogen detection, and in particular to a LAMP detection primer, a kit and a LAMP rapid detection method for leaf spot pathogens. Background Art

[0002] Daylily (Hemerocallis citrina Baroni), also known as daylily, analgesic, forget-me-not, is a perennial herbaceous plant of the Liliaceae family. Its edible part is the buds that have not yet bloomed. It is rich in nutrients, containing a variety of sugars, proteins, vitamins, inorganic salts and a variety of essential amino acids for the human body. It is one of my country's traditional specialty vegetables. This plant is widely planted in China, North Korea, South Korea and Japan, and has ornamental and vegetable value. In addition, the bud metabolites of daylily contain rich rutin, hesperidin, colchicine, etc., which are effective in treating symptoms such as anxiety, edema, and depression. It has the effects of antibacterial and anti-inflammatory, clearing heat and dampness, improving eyesight and calming the mind, and beautifying and nourishing the skin. It can be used as a Chinese herbal medicine and a modern medicinal plant. With the rise of daylily cultivation and the development of the specialty vegetable industry, the planting area has gradually expanded, and the occurrence of its diseases has become increasingly serious, causing huge economic losses to the industry.

[0003] Many leaf and stalk diseases often occur on daylilies, including leaf blight, leaf spot, brown spot, stem blight, yellow leaf disease, anthracnose, and red rot, most of which are caused by anthracnose ( Colletotrichum sp . )、Fusarium( Fusarium sp . )、wheel spot bacteria( Pestallozzia sp . ), Phytophthora macrophylla ( Macrophoma sp . ), Phytophthora solani ( Phoma sp . ) and Discosporium ( Gloeosporium sp . )wait.

[0004] Currently, PCR technology is commonly used to detect pathogens, but this technology has low detection efficiency and is not conducive to plant protection. Summary of the invention

[0005] The purpose of the present invention is to provide a LAMP detection primer, a kit and a LAMP rapid detection method for leaf spot pathogens, so as to realize rapid detection of leaf spot pathogens and improve detection efficiency.

[0006] The present invention is achieved by the following technical solution: the primers include a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP and a reverse inner primer BIP, and the primer series is as follows:

[0007] F3: 5'-ACCAGTGCGGTGGTATCG-3';

[0008] B3: 5'-CAAGAGCGCCTGTCACTC-3';

[0009] FIP: 5'-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3';

[0010] BIP: 5'-CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC-3'.

[0011] Provided is a LAMP detection kit for leaf spot pathogens, comprising the above primers.

[0012] In order to better realize the present invention, further, the kit also includes dNTPs, 8 U / μL Bst DNA polymerase, 10×isothermal amplification buffer and MgSO4.

[0013] Provided is a LAMP detection reaction system for leaf spot pathogens, the reaction system comprising: 4 μL of 10 uM FIP and BIP primers, 0.5 μL of 10 uM F3 and B3 primers, 2.5 μL of 10 mM dNTPs, 1 μL of 8 U / μL Bst DNA polymerase, 4 μL of 50 mM MgSO4, 2.5 μL of 10×isothermal amplification buffer, and the mixture is filled up to 25 μL with sterile ultrapure water.

[0014] Provided is a LAMP detection method for leaf spot pathogens, comprising the following steps:

[0015] S1: Obtain the conserved region of the EF-1α gene of the leaf spot pathogen and design primers online using Primer Explore V5;

[0016] S2: prepare 25 μL of detection reaction system;

[0017] S3: performing LAMP amplification on the total DNA of the sample to be tested in the detection reaction system described in S2, and taking the reaction product after the reaction is completed;

[0018] S4: using agarose gel electrophoresis to detect the amplification results of the reaction products, or / and using SYBR Green I dye to observe the changes in the LAMP reaction solution;

[0019] S5: If the LAMP reaction product appears as continuous diffuse bands in agarose gel electrophoresis, it is positive and the leaf spot pathogen is present; and / or the reaction solution appears emerald green, which is positive and the leaf spot pathogen is present; light orange is negative and the leaf spot pathogen is not present.

[0020] In order to better implement the present invention, further, the reaction conditions in the detection reaction system are: temperature is 60° C., and reaction time is 60 min.

[0021] In order to better implement the present invention, further, the operation steps of obtaining the leaf spot pathogen in S1 are as follows:

[0022] Step 1: Cut the tissue at the junction of the diseased and healthy leaves and disinfect it, then rinse it with sterile water, dry it with sterile filter paper, place it on a PDA plate containing streptomycin, and culture it in the dark at 26℃;

[0023] Step 2: The grown colonies were transferred to PDA medium, purified by single spore separation, and then stored on PDA slant at 4°C for future use to obtain isolated and purified strains.

[0024] In order to better realize the present invention, further, the identification of the leaf spot pathogen includes the following steps:

[0025] Sa: The isolated and purified strain was inoculated into PDA medium, cultured in the dark at 26°C for 7 days, and then the mycelium was scraped and fungal DNA was extracted using the CTAB method;

[0026] Sb: The ribosomal intergenic region and partial sequence of the translation elongation factor gene were amplified using primers ITS4 / ITS5 and EF1-728F / EF1-986R;

[0027] Sc: construct a PCR reaction system, which includes 25 μL of 2×Es Taq Master Mix, 2 μL of total DNA template, 2 μL of 10 μM upstream and downstream primers, and 19 μL of ddH2O, and perform an extension reaction to obtain the reaction product;

[0028] Sd: The reaction products were detected by agarose gel electrophoresis and then sequenced. The obtained sequences were compared with the NCBI database by BLASTN. The phylogenetic tree was constructed by the neighbor-joining method in MEGA6 software, and the bootstrap method was used for 1000 repeated tests.

[0029] In order to better implement the present invention, further, the PCR reaction conditions are 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min, a total of 35 cycles; finally 72°C extension for 10 min, and storage at 4°C.

[0030] The invention discloses an application of a kit in identifying pathogenic bacteria of plant leaf spot disease, wherein the plant is day lily.

[0031] The beneficial effects of the present invention are:

[0032] The present invention designs a set of LAMP detection primers that can specifically detect leaf spot pathogens based on the EF-1α sequence of the leaf spot pathogen. F. armeniacum The detection primers can be used to quickly detect pathogens and improve the efficiency of detection.

[0033] The present invention sets LAMP detection primers and quickly detects leaf spot pathogens by combining LAMP detection primers and LAMP detection technology. The LAMP detection technology has strong detection specificity, short detection time, high sensitivity, low requirements for instruments and equipment, and the results are visualized and easy to judge.

[0034] The LAMP detection provided by the present invention can meet the requirements of detecting pathogens of infected plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solution of the present invention, the accompanying drawings required for use in the present invention will be briefly introduced below. It should be understood that the following accompanying drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these accompanying drawings without paying creative work.

[0036] Figure 1 This is an experimental diagram of the LAMP reaction specificity detection provided by the present invention;

[0037] Figure 2 The actual sample leaf spot pathogen detection diagram provided by the present invention;

[0038] Figure 3 Leaf spot pathogen test provided by the present invention Figure 1 ;

[0039] Figure 4 Leaf spot pathogen test provided by the present invention Figure 2 ;

[0040] Figure 5 A morphological diagram of the leaf spot pathogen provided by the present invention;

[0041] Figure 6 The phylogenetic tree provided by the present invention. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be described below in conjunction with the accompanying drawings of the present invention.

[0043] Example:

[0044] The present invention provides a LAMP detection primer for leaf spot pathogens:

[0045] Primers were designed online using Primer Explore V5 based on the conservative region of the EF-1α sequence gene of the leaf spot pathogen. The primers included two outer primers F3 and B3 and two inner primers FIP and BIP. The primers used were completed by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are as follows:

[0046] F3: 5'-ACCAGTGCGGTGGTATCG-3';

[0047] B3: 5'-CAAGAGCGCCTGTCACTC-3';

[0048] FIP: 5'-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3';

[0049] BIP: 5'-CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC-3'.

[0050] The LAMP detection reaction system was constructed, which included 4 μL of 10 uM FIP and BIP primers, 0.5 μL of 10 uM F3 and B3 primers, 2.5 μL of 10 mM dNTPs, 1 μL of 8 U / μL Bst DNA polymerase, 4 μL of 50 mM MgSO4, 2.5 μL of 10×isothermal amplification buffer, 1 μL of template DNA, and was filled to 25 μL with sterile ultrapure water. During the LAMP detection reaction, the reaction conditions were: temperature 60°C, reaction time 60 min.

[0051] After the reaction was completed, 3 μL of the reaction product was taken and the amplification results were detected by 1% agarose gel electrophoresis. In addition, 1 μL of SYBR Green I dye was added to observe whether the LAMP reaction solution changed in color.

[0052] Reaction results:

[0053] In electrophoresis observation, the LAMP reaction product appeared as continuous diffuse bands in 1% agarose gel electrophoresis, which was positive and the leaf spot pathogen was present. The reaction solution was observed to be emerald green, which was positive and the leaf spot pathogen was present. Light orange was negative.

[0054] Experimental example - LAMP detection primer specificity detection;

[0055] LAMP detection primer specificity detection: Daylily leaf spot pathogens, Zizania latifolia sheath rot pathogens ( Fusarium andiyazi ), Colletotrichum citri var. Colletotrichum gloeosporioides ) Rhizoctonia solani ), Echinococcus sorghumus ( Epicoccum nigrum ) The detection was performed according to the LAMP detection method of the embodiment, and the specificity of the LAMP primers was determined by adding SYBRGreen I dye for staining and observing the results by 1% agarose gel electrophoresis. The detection results are as follows: Figure 1 As shown in the figure, a is SYBR Green I detection of LAMP products, b is gel electrophoresis detection of LAMP products, M is DL2000 marker; the numbers 1 to 3 are day lily leaf spot pathogens; 4 is Zizania latifolia sheath rot pathogen; 5 is Colletotrichum gloeosporioides; 6 is Rhizoctonia solani; 7 is Echinococcus kaoliang; 8 is sterile water negative control.

[0056] The test results are as follows: the electrophoresis bands of the LAMP product using the DNA of the pathogen of leaf spot disease of day lily as a template are diffuse and the color turns green, indicating a positive reaction and the presence of leaf spot pathogens, while the electrophoresis bands of the LAMP detection product using the DNA of other pathogenic fungi as templates are not visible and the color remains orange, indicating a negative reaction, thus demonstrating that the LAMP detection system can specifically detect the pathogen of leaf spot disease of day lily.

[0057] Experimental example - Sensitivity test of LAMP and conventional PCR test

[0058] The standard DNA of the pathogen of daylily leaf spot disease at 1000 ng / μL was diluted to 1fg / μL according to a 10-fold concentration gradient and used as reaction templates. The conventional PCR detection system and LAMP system were used for detection. According to the color change of the amplified product and the agarose gel electrophoresis observation results, the minimum detection limit of the LAMP reaction system was 100 pg / μL, while the minimum detection limit of the ordinary PCR reaction was 1ng / μL, indicating that the sensitivity of the LAMP detection method is 10 times that of the conventional PCR detection method.

[0059] Experimental Example - Detection of Pathogens in Actual Samples

[0060] Here’s how to do it:

[0061] Total DNA was extracted from the leaves of daylily artificially inoculated with the pathogen of daylily leaf spot disease 4 days after inoculation and used as a template for LAMP amplification. The results showed that the daylily leaf spot disease pathogen was not detected in the leaves of daylily not inoculated with the pathogen, while the pathogen was detected in the inoculated plants. Figure 2As described in a, a in Figure 2 is the LAMP detection of artificially inoculated daylily leaves; M is DL 2000 marker; in addition, 1 is a positive daylily leaf spot disease strain; 2 and 3 are leaves inoculated with daylily leaf spot disease pathogens; 4 and 5 are healthy plants that have not been inoculated with pathogens; and 6 is a sterile water negative control.

[0062] In addition, tissue separation was performed on leaf samples of two plants that showed symptoms of daylily leaf spot disease and were collected in Jinyun County, Lishui City, Zhejiang Province. Through identification of strain morphology and molecular biology, it was determined that the daylily leaf spot pathogen was isolated from the two samples, while no pathogen was isolated from the leaves of two other healthy plants. At the same time, LAMP detection was performed on the same samples, and the results showed that both diseased samples produced positive reactions, which was consistent with the results of pathogen isolation, while no positive reaction occurred in the healthy tissues of daylily. This shows that the LAMP technology can be used for rapid diagnosis of field samples of daylily leaf spot disease, such as Figure 2 As shown in b, b is the LAMP test of leaves of diseased plants sampled in the field; 1~3 are leaves of diseased plants in the field; 4 and 5 are leaves of healthy plants in the field; 6 is the sterile water negative control.

[0063] The present invention also provides an operation for obtaining the leaf spot disease pathogen and an identification of the leaf spot disease pathogen, which are morphological identification and molecular biological identification. The specific operations are as follows:

[0064] Leaves with obvious leaf spot symptoms were collected, leaves with typical symptoms were picked for observation, and the symptoms were recorded. The pathogen was isolated by tissue separation method. The tissue of 5×5 mm at the junction of the diseased and healthy leaves was cut and placed in 75% alcohol for 30 seconds, 2% sodium hypochlorite for 1 minute, and then rinsed with sterile water for 3 times. After drying with sterile filter paper, it was placed on a PDA plate containing streptomycin (50 μg / ml) and cultured in the dark at 26°C. The grown colonies were transferred to new PDA medium, purified by single spore separation, and stored on PDA slants at 4°C for later use.

[0065] Culture medium: Potato dextrose agar (PDA) culture medium: 20 g glucose, 200 g potato, 20 g agar, 1000 mL distilled water; no agar is potato dextrose (PD) liquid culture medium; carnation leaf-piece agar (CLA) culture medium: several carnation leaves, 20 g agar powder, 1 L distilled water.

[0066] Pathogenicity determination of the pathogen of daylily leaf spot disease:

[0067] Koch's postulates were verified by live inoculation of spore liquid. Representative test strains were selected and cultured in PD medium at 26°C and 180 r / min for 5 days. The spore liquid was collected after filtering with sterile gauze. The concentration of 1×10 was prepared with sterile water using a hemocytometer. 6 / mL of conidia suspension, selected healthy 40cm high potted daylily plants for pathogenicity determination, wiped the surface of the plant leaves with 70% ethanol for disinfection, and used a pipette to absorb the spore droplets on the leaves, 3~4 drops per leaf, 10 μL per point. Inoculated with sterile water as a control. All inocula were cultured at a constant temperature of 25℃ and a relative humidity of 80%~90%, and the disease was observed day by day. After the leaves became diseased, the pathogens were isolated and identified again on the diseased leaves. The inoculation experiment was repeated twice, and 3 potted plants were repeated for each treatment.

[0068] Pathogenicity test results:

[0069] Disease was observed on leaves 4 days after inoculation. Daylily leaf spot mainly affects leaves and appears on the leaf surface or leaf margins. Initially, it appears as water-soaked light yellow spots, which later expand into spindle-shaped spots with brown edges, gray centers, and yellow halos. Figure 3 As shown. In the later stage of the disease, the lesions expand, causing perforation and leaf rupture. The lesions can also extend to the stems, and brown, spindle-shaped lesions also appear on the stems, which then expand around the stems and cause the stems to wilt. Usually, under high humidity conditions in the field, white or red mold layers can be seen on the infected leaves and stems. Tissue separation was performed on the collected diseased leaves and stems, and 20 strains were isolated and purified from 20 disease samples.

[0070] like Figure 4 As shown, Figure 4 Figure a shows the symptoms of the disease 6 days after potted live inoculation; b shows the typical lesions on the inoculated leaves; CK is the control, and the specific manifestations in a are water-soaked to yellow-brown necrotic spindle spots with obvious yellow halos. Later, the lesions expand and cause perforation and leaf wilting. The inoculation symptoms are similar to the symptoms of natural disease in the field, while the control inoculated leaves do not show symptoms. Conidia of the pathogen can be microscopically detected from the inoculated lesions, and the pathogen can be isolated from the lesions again. The pathogen used for inoculation was identified by morphology and molecular identification as F. armeniacum , which shows that F. armeniacum It is the pathogen of daylily leaf spot disease.

[0071] In addition, the pathogens were identified by morphology:

[0072] The isolated and purified strains were inoculated on PDA plates, and the strains were inoculated on CLA plates and cultured in the dark at 26°C. After spores were produced, the morphological characteristics of large and small conidia and thick-walled spores were observed under an optical microscope. They were kidney-shaped or oval, with thick walls, 0 to 1 septa, and 7.7 to 14.1 × 2.7 to 5.0 μm in size. After 7 days of culture on CLA medium or at the lesions of diseased plants, large conidia were easily produced. They were sickle-shaped, thin-walled, with a long and pointed apical cell and a notched basal cell, 3 to 5 septa, and 20.5 to 38.5 × 2.8 to 5.1 μm in size. Thick-walled spores were nearly round, with smooth walls, and often strung together like Figure 5 -c~f, based on the morphological characteristics of the pathogen, the pathogen can be preliminarily identified as Fusarium armeniacum .like Figure 5 As shown, a is the front of the colony on the PDA plate; b is the back of the colony on the PDA plate; c is a large conidiophore; d is a large conidia; e is a small conidia; and f is a thick-walled spore.

[0073] In addition, there is also molecular biological identification of pathogens. The specific operations are as follows:

[0074] The isolated and purified strain was inoculated into PDA medium and cultured in the dark at 26℃ for 7 days. Mycelium was scraped and fungal DNA was extracted using the CTAB method (Lee and Taylor, 1990). Universal primers ITS4 / ITS5 (White et al., 1990) and EF1-728F / EF1-986R (Carbone et al., 1999) were used to amplify the intergenic region (ITS) of the ribosomal transcription factor (EF-1α) gene.

[0075] The PCR reaction system was 50 μL: 25 μL 2×Es Taq Master Mix, 2 μL total DNA template, 2 μL each of 10 μM upstream and downstream primers, and 19 μL ddH2O.

[0076] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min, for a total of 35 cycles; and finally extension at 72°C for 10 min and storage at 4°C.

[0077] The amplified products were detected by 1% agarose gel electrophoresis and then sequenced by Shanghai Bioengineering Co., Ltd. The obtained sequences were compared with the NCBI database by BLASTN. The phylogenetic tree was constructed by the neighbor-joining (NJ) method in MEGA 6 software, and the bootstrap method was used for 1000 replicate tests (Tamura et al., 2013).

[0078] Specifically, the genomic DNA of the representative strain HHCJ-4 was used as a template to amplify ITS and EF-1α The fragments of about 550 bp and 300 bp were obtained. The PCR products were purified and sequenced, and the sequences were submitted to GenBank (accession numbers: MN647064 and MN650204, respectively); BLASTN homology comparison showed that the sequences were consistent with the known F. armeniacum The strain sequences have 99.8% to 100% nucleotide similarity. F. armeniacum voucherRIFA 180 (accession number KF624790) and F. armeniacum MRC 2190 (accession number MH582288), etc.

[0079] In addition, the ITS and EF-1α The sequence is also consistent with the Fusarium-ID database F. armeniacum The strains share 98.9% to 99.8% nucleotide similarity (Geiser et al., 2004).

[0080] Using HHCJ-4 and different strains of Fusarium EF-1α The sequences were aligned and the phylogenetic tree was constructed. The results of evolutionary analysis showed that HHCJ-4 and F. armeniacum The strains clustered in one branch, e.g. Figure 6 Therefore, based on the morphological characteristics of the pathogen, EF-1α sequence homology comparison and phylogenetic analysis, the pathogen of daylily leaf spot was identified as F. armeniacum .

[0081] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims. Sequence Listing <110> Hunan Agricultural University <120> A leaf spot pathogen LAMP detection primer, kit and LAMP rapid detection method <141> 2022-03-18 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 accagtgcgg tggtatcg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 caagagcgcc tgtcactc 18 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <400> 3 ggatcgatgg gaagaagggc gcaagcgaac catcgagaag t 41 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <400> 4 catacgacga ctcgacaagc gcccaccacc caaaaaaacg ac 42

Claims

1. A LAMP detection method for the pathogen of daylily leaf spot disease, characterized in that: The LAMP detection method is based on a LAMP detection reaction system for leaf spot pathogens containing specific primers; the LAMP detection reaction system includes: 4 μL of 10 μM FIP and BIP primers, 0.5 μL of 10 μM F3 and B3 primers, 2.5 μL of 10 mM dNTPs, 1 μL of 8 U / μL Bst DNA polymerase, 4 μL of 50 mM MgSO4, 2.5 μL of 10×isothermal amplification buffer, and the mixture is filled to 25 μL with sterile ultrapure water; the specific primers include a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP and a reverse inner primer BIP, and the primer series is as follows: F3: 5'-ACCAGTGCGGTGGTATCG-3'; B3: 5'-CAAGAGCGCCTGTCACTC-3'; FIP-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3'; BIP-CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC-3'; The LAMP detection method comprises the following steps: S1: Obtain the conserved region of the EF-1α gene of the leaf spot pathogen and design primers online using Primer Explore V5; S2: Prepare 25 μL detection reaction system; S3: performing LAMP amplification on the total DNA of the sample to be tested in the detection reaction system described in S2, and taking the reaction product after the reaction is completed; S4: Detect the reaction product by agarose gel electrophoresis; or / and observe the changes of LAMP reaction solution by SYBR GreenI dye; S5: If the LAMP reaction product appears as continuous diffuse bands in agarose gel electrophoresis, it is positive, indicating the presence of leaf spot pathogens; and / or the reaction solution appears emerald green, which is positive, indicating the presence of leaf spot pathogens, and light orange, which is negative, indicating the absence of leaf spot pathogens; The reaction conditions in the reaction system are: temperature of 60°C and reaction time of 60 min; The operation steps of obtaining leaf spot pathogens in S1 are as follows: Step 1: Cut the tissue at the junction of the diseased and healthy leaves of the daylily for disinfection, rinse with sterile water, dry it with sterile filter paper, place it on a PDA plate containing streptomycin, and culture it in the dark at 26°C; Step 2: The grown colonies were transferred to PDA medium, purified by single spore separation, and then stored on PDA slant at 4°C for future use to obtain isolated and purified strains.

2. A LAMP detection kit for the pathogen of daylily leaf spot disease, characterized in that: The specific primers include a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP and a reverse inner primer BIP. The primer series is as follows: F3: 5'-ACCAGTGCGGTGGTATCG-3'; B3: 5'-CAAGAGCGCCTGTCACTC-3'; FIP-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3'; BIP-CATACGACGACTCGACAAGCGCCCACCACCCAAAAAAACGAC-3'; the LAMP detection kit also includes dNTPs, Bst DNA polymerase, 10×isothermal amplification buffer and MgSO4.

3. Use of the LAMP detection kit according to claim 2 in the identification of plant leaf spot pathogens, characterized in that: The plant is daylily.

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