Primers for lamp detection of rhizoctonia solani, detection kit and application thereof

Abstract

The application belongs to the technical field of plant pathogenic microorganism detection, and discloses a LAMP (Loop-Mediated Isothermal Amplification) detection primer of Rhizoctonia solani, a detection kit and application thereof, wherein the Rhizoctonia solani is used as a target gene, and four specific primers F3-RS, B3-RS, FIP-RS and BIP-RS for a loop-mediated isothermal amplification detection method are designed. Tubulin The application also prepares a kit containing the primers, and provides application of the kit in detection of tomato damping-off, and the detection result can be directly observed by naked eyes to determine whether the Rhizoctonia solani exists in the sample to be detected. The primers have a sensitivity of 100 ag / μL and high specificity, and the Rhizoctonia solani can be sensitively detected in the soil of a tomato planting area and the tomato leaves with no obvious disease phenotype in the early stage of infection. The application is suitable for early, rapid, sensitive and visual detection of tomato damping-off.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogenic microorganism detection technology, specifically to primers, detection kits and their applications for detecting Rhizoctonia solani LAMP. Background Technology

[0002] Rhizoctonia solani is a globally distributed soil-borne plant pathogen that can infect various crops, including tomatoes, potatoes, cotton, and rice. In greenhouse tomato cultivation, damping-off caused by this fungus is characterized by rapid onset, rapid spread, and severe damage. The pathogen can survive for extended periods in soil and diseased plant debris, exhibiting strong saprophytic competitiveness. Combined with the high temperature and humidity and frequent continuous cropping conditions in greenhouses, once an outbreak occurs, it is often difficult to control effectively, seriously threatening tomato yield and quality. Therefore, rapid and accurate detection of Rhizoctonia solani in the early stages of disease invasion, before symptoms appear, is a crucial prerequisite for implementing precise early warning and green control measures.

[0003] Current detection technologies for Rhizoctonia solani still have significant shortcomings. Traditional morphological identification relies on isolation, culture, and microscopic observation, which is time-consuming and requires a high level of experience, making it unsuitable for rapid field diagnosis. While conventional PCR methods have improved specificity, they require precise temperature control equipment, specialized operation, and a relatively long amplification time, making them difficult to promote in grassroots or field settings.

[0004] Loop-mediated isothermal amplification (LAMP) technology, due to its advantages such as isothermal amplification, rapid reaction, and visualized results, is theoretically suitable for early field detection. However, the reported LAMP detection methods for Rhizoctonia solani still have shortcomings: Firstly, they lack sensitivity in the early stages of infection. Most existing methods require the pathogen to accumulate to a certain biomass before stable detection, and in the very early stages when no visible lesions appear on tomato leaves or rhizomes, the presence of trace amounts of Rhizoctonia solani is often not effectively identified. This leads to missed opportunities for optimal chemical intervention or agricultural regulation. Secondly, the specificity of existing detection methods is insufficient to meet the detection needs of complex field samples. In greenhouse tomato cultivation systems, Rhizoctonia solani often coexists with various other pathogens, such as Fusarium oxysporum, Corynebacterium multiflorum, Pseudomonas syringae, and Pectinobacter carotenoides. These pathogens have a high degree of overlap with the ecological niche of Rhizoctonia solani in soil, roots, or leaf tissues. However, existing LAMP primers are prone to non-specific amplification or false positive signals when detecting co-infected samples, which seriously affects the reliability of the results.

[0005] In summary, to overcome the shortcomings of existing detection technologies in terms of early sensitivity, specificity, and system stability, there is an urgent need to develop a novel LAMP detection system that targets Rhizoctonia solani, can detect trace amounts of the target, and can effectively distinguish between the aforementioned coexisting pathogens. This system should also be able to be visualized and interpreted in the early stages of tomato infection and in asymptomatic tissues, thus providing reliable technical support for early warning and green control of tomato damping-off. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide LAMP detection primers, detection kits, and their applications for Rhizoctonia solani, in order to achieve specific and rapid detection of Rhizoctonia solani and solve the problem of insufficient sensitivity in the early detection of this pathogen in tomatoes.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A primer for detecting Rhizoctonia solani using LAMP consists of an outer primer and an inner primer. The outer primer is a forward outer primer F3-RS and a reverse outer primer B3-RS. The inner primer is a forward inner primer FIP-RS and a reverse inner primer BIP-RS.

[0009] The nucleotide sequences of each primer are as follows:

[0010] F3-RS: 5'-CGAGTTCTTGTTCTGGACGT-3';

[0011] B3-RS: 5'-CTTTGCCCCTTTGACCGC-3';

[0012] FIP-RS: 5'-CCTCACCGTAGCTGCCTTCTTCGCATCTGCTCCTCGACTT-3';

[0013] BIP-RS: 5'-AGCAGCCATCATGTTCTTGGCATGGCTCTGTTCAATACCGC-3'.

[0014] During the research, three sets of LAMP primers were designed using Rhizoctonia solani ITS and Tubulin genes as target genes. Unexpectedly, it was discovered that the LAMP-specific primers of this invention have outstanding advantages of high specificity and high sensitivity for early rapid detection of Rhizoctonia solani. In particular, the presence of the pathogen can be detected in tomatoes at the early stage of Rhizoctonia solani infection, when the bacterial load is extremely low and no disease phenotype is present.

[0015] The present invention also provides a tomato damping-off LAMP detection kit prepared using the above primers, the components of which include: the primers, LAMP reaction buffer, thermophilic DNA polymerase for LAMP detection, and nucleic acid fluorescent dye;

[0016] The primers contain equal amounts of the two outer primers and equal amounts of the two inner primers.

[0017] The molar ratio of inner primer to outer primer is 3~1:1~3, preferably 2:1;

[0018] As a limitation of this invention, the LAMP reaction buffer contains 8 mM Mg 2+ 10×Bst II reaction buffer;

[0019] The thermophilic DNA polymerase used for LAMP detection is Bst II DNA polymerase.

[0020] As a further limitation of the present invention, the nucleic acid fluorescent dye is SYBR Green I.

[0021] Among them, SYBR Green I is used to stain the LAMP reaction product with the added nucleic acid fluorescent dye from orange (orange-yellow) to green (yellow-green).

[0022] Preferably, the nucleic acid fluorescent dye can also be replaced with hydroxynaphthol blue (HNB) or calcein / manganese ion.

[0023] This invention also provides a method for detecting tomato damping-off using the above-mentioned tomato damping-off LAMP detection kit. Specifically, the method involves: extracting DNA from the sample to be tested as a template, mixing the LAMP reaction system, performing the LAMP reaction, and adding a nucleic acid fluorescent dye to the reaction product. If a positive color change occurs, the sample to be tested is a positive sample for tomato damping-off.

[0024] As a limitation of the present invention, the LAMP reaction system has the following composition: 1×Bst II Buffer, 8 mg / mL MgSO₄. 2+ 0.2~1.0U / μL Bst II DNA polymerase, 0.5~1.5 mM dNTPs, 0.5 μM FIP-RS, 0.5 μM BIP-RS, 0.25 μM F3-RS, 0.25 μM B3-RS, 1 μL template, and sterile ultrapure water to bring the volume to 20 μL.

[0025] Preferably, it contains 0.4 U / μL Bst II DNA polymerase and 1 mM dNTPs.

[0026] As a further limitation of the present invention, the LAMP reaction conditions are 52~66℃ for 20~70 min, followed by inactivation at 85℃ for 5 min.

[0027] As a further limitation of the present invention, the LAMP reaction conditions are: reaction at 56°C for 30 min, followed by inactivation at 85°C for 5 min.

[0028] This invention also provides an application of the above-mentioned Rhizoctonia solani LAMP detection primers in the early identification of tomato damping-off and the detection of Rhizoctonia solani.

[0029] This invention also provides the application of the above-mentioned tomato damping-off LAMP detection kit in the early identification of tomato damping-off and the detection of Rhizoctonia solani in soil and plant leaves.

[0030] By adopting the above-described technical solution, the beneficial effects achieved by this invention compared to the prior art are as follows:

[0031] (1) The LAMP detection primers for Rhizoctonia solani of the present invention can achieve very early and ultra-high sensitivity detection of Rhizoctonia solani, and are especially suitable for the detection of Rhizoctonia solani in DNA extracted by simple methods, providing a key window period for disease early warning.

[0032] (2) The detection kit prepared by the LAMP detection primers of Rhizoctonia solani of the present invention not only retains the advantages of visual visualization and convenience of the LAMP method, but also can complete the detection of Rhizoctonia solani within 30 min with a sensitivity of up to 100 ag / μL, which is significantly better than the LAMP method and conventional PCR method whose detection limit is still at the pg or fg level, thus achieving rapid and efficient detection.

[0033] (3) The LAMP detection primers for Rhizoctonia solani of the present invention exhibit high specificity. In the detection of Rhizoctonia solani, Corynebacterium multiflorum, Pseudomonas syringae, Staphylococcus aureus, Pectinobacter carotenoides, Fusarium graminearum, Fusarium graminearum, and Fusarium oxysporum, Rhizoctonia solani is specifically detected only for Rhizoctonia solani, and the results are accurate and reliable.

[0034] This invention is applicable to the early identification of damping-off disease in tomatoes and the detection of Rhizoctonia solani, and can be used for early monitoring and prevention of vegetable diseases. Attached Figure Description

[0035] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0036] Figure 1 This is a schematic diagram of the target sequence in Embodiment 1 of the present invention;

[0037] Figure 2The graph shows the detection results of LAMP reaction systems with different enzyme amounts in this invention.

[0038] Figure 3 The graph shows the detection results of LAMP reaction systems with different dNTP contents in this invention.

[0039] Figure 4 The graph shows the detection results of LAMP reaction systems with different inner and outer primer ratios in this invention.

[0040] Figure 5 The graph shows the detection results at different LAMP reaction temperatures in this invention.

[0041] Figure 6 The graph shows the detection results for different LAMP reaction times in this invention.

[0042] Figure 7 This is a graph showing the detection results of LAMP reactions with different template concentrations in this invention;

[0043] Figure 8 This is a graph showing the PCR reaction detection results of different template concentrations in Comparative Example 1 of the present invention;

[0044] Figure 9 This is a diagram showing the results of targeted monitoring of damping-off pathogen in tomatoes in a greenhouse cultivation area using the kit from Example 3 of the present invention.

[0045] Figure 10 This is a graph showing the specific detection results in Comparative Example 2 of the present invention;

[0046] Figure 11 This is a graph showing the specificity detection results of different primers in Comparative Example 3 of this invention;

[0047] Figure 12 The image shows the LAMP detection results of samples obtained by different extraction methods in Comparative Example 4 of this invention. Detailed Implementation

[0048] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustrative and understanding purposes only and are not intended to limit the scope of the invention.

[0049] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0050] In this example, 1.5% agarose gel electrophoresis and SYBR Green I fluorescence staining were used to determine the LAMP reaction results.

[0051] The strains involved in the examples—Corynespora cassiicola, Botryosphaeria dothidea, Pseudomonas syringae DC3000, Pectobacterium carotovorum, and Gibberella zeae—were all derived from the Institute of Biology, Hebei Academy of Sciences; Fusarium pseudograminearum was derived from Hebei University of Science and Technology; and Fusarium oxysporum and Rhizoctonia solani were derived from Hebei Agricultural University. All fungal strains were cultured on PDA solid medium, and bacteria on LB medium at 28°C.

[0052] BstDNA polymerase, Bst Buffer, and dNTP Mixture were purchased from Bomeide Biotechnology; the genomic DNA extraction kit was purchased from Beijing Polymer Biotechnology Co., Ltd.; SYBR Green I was purchased from Shanghai Ruichu Biotechnology Co., Ltd. The clean bench was purchased from Suzhou Antai Air Technology Co., Ltd., the benchtop high-speed refrigerated centrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd., the constant temperature metal bath was purchased from CoYoTebio, and the biochemical incubator was purchased from Shanghai Yiheng Scientific Instruments Co., Ltd.

[0053] Example 1

[0054] This embodiment describes a LAMP detection primer for Rhizoctonia solani, consisting of an outer primer and an inner primer. The outer primers are a forward outer primer F3-RS and a reverse outer primer B3-RS; the inner primers are a forward inner primer FIP-RS and a reverse inner primer BIP-RS.

[0055] The nucleotide sequences of each primer are as follows:

[0056] F3-RS: 5'-CGAGTTCTTGTTCTGGACGT-3', denoted as SEQ ID NO.1;

[0057] B3-RS: 5'-CTTTGCCCCTTTGACCGC-3', denoted as SEQ ID NO.2;

[0058] FIP-RS: 5'-CCTCACCGTAGCTGCCTTCTTCGCATCTGCTCCTCGACTT-3', recorded as SEQ IDNO.3;

[0059] BIP-RS: 5'-AGCAGCCATCATGTTCTTGGCATGGCTCTGTTCAATACCGC-3', denoted as SEQ ID NO.4.

[0060] The design and synthesis process of the above primers is as follows:

[0061] Using the Tubulin gene sequence of *Rhizoctonia solani* publicly available on the NCBI website as the target gene sequence, sequence alignment was used to identify gene fragments with low homology to other pathogenic fungi as target sequences. Figure 1 Access the PrimerExplorerV5 website (http: / / primerexplorer.jp) and design a set of LAMP primers based on the B1c, B2, B3, F1c, F2, and F3 regions of the target sequence: forward outer primer F3-RS and reverse outer primer B3-RS; the inner primers are forward inner primer FIP-RS and reverse inner primer BIP-RS. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0062] Example 2

[0063] This embodiment describes a tomato damping-off LAMP detection kit prepared using the primers from Example 1. The kit includes: forward outer primer F3-RS, reverse outer primer B3-RS; inner primers are forward inner primer FIP-RS and reverse inner primer BIP-RS, containing 8 mM Mg... 2+ The reaction mixture consisted of 10×Bst II reaction buffer, Bst II DNA polymerase, the nucleic acid fluorescent dye SYBR Green I, a Tubulin-synthesized DNA fragment (target sequence) as a positive control, and nuclease-free water as a negative control.

[0064] The usage method of this embodiment is as follows:

[0065] S1. Template extraction: Genomic DNA of Rhizoctonia solani was extracted using a DNA extraction kit and diluted with sterile ultrapure water to 1 ng / μL as a template for later use.

[0066] S2. Mixing LAMP reaction system: Take the reagents from the tomato damping-off LAMP detection kit and mix them according to the following original concentrations and volumes: 10×Bst II Buffer 2 μL (final concentration containing 8 mM Mg) 2+), 8 U / μL Bst II DNA polymerase 1 μL, 10 mM dNTPs 2 μL, 10 μM FIP-RS 1 μL, 10 μM BIP-RS 1 μL, 10 μM F3-RS 0.5 μL, 10 μM B3-RS 0.5 μL, template 1 μL, sterile ultrapure water 11 μL.

[0067] The final concentrations of each substance in the system are: 1×Bst II Buffer, 8 mM Mg 2+ 0.4 U / μL Bst II DNA polymerase, 1 mM dNTPs, 0.5 μM FIP-RS, 0.5 μM BIP-RS, 0.25 μM F3-RS, 0.25 μM B3-RS, 1 μL template, and sterile ultrapure water were added to bring the volume to 20 μL to obtain the LAMP reaction system.

[0068] S3. Perform the LAMP reaction: Incubate the LAMP reaction system at 56℃ for 30 min, and then inactivate it at 85℃ for 5 min.

[0069] S4. Reading the test results: After the LAMP reaction is complete, add 0.5 μL of SYBR Green I to the reaction product and visually observe the color development results. If the reaction product is observed to be green fluorescent, it is considered positive, indicating that Rhizoctonia solani is present in the sample. If orange is observed, it is considered negative, indicating that Rhizoctonia solani is not present.

[0070] The results showed that the reaction product was green fluorescent, indicating a positive result, which means that Rhizoctonia solani was present in the sample. This means that the primers and kit of the present invention can successfully detect Rhizoctonia solani.

[0071] In other embodiments, the amount of SYBR Green I used in the 20 μL LAMP reaction system can be any integer value from 0.5 to 1 μL.

[0072] This invention verified the optimal implementation method by adjusting the composition of the LAMP reaction system, the LAMP reaction conditions, and the template concentration in Example 2, as detailed below:

[0073] (I) Different LAMP reaction system compositions and their implementation effects

[0074] Based on Example 2, this invention adjusts the amounts of the main components Bst II DNA polymerase and dNTPs, as well as the ratio of inner and outer primers in the reaction system. The adjustments for each single factor are as follows: the final concentration of Bst II DNA polymerase is 0.2 U / μL, 0.4 U / μL, 0.6 U / μL, 0.8 U / μL, or 1.0 U / μL; the final concentration of dNTPs is 0.5 mmol / L, 0.75 mmol / L, 1 mmol / L, 1.25 mmol / L, or 1.5 mmol / L; and the ratio of inner and outer primers is 3:1, 2:1, 1:1, 1:2, or 1:3. With other component amounts and conditions remaining the same as in Example 2, the single-factor conditions are varied to conduct the LAMP reaction. Sterile ultrapure water is used as a negative control. After the reaction, the optimal LAMP reaction system for *Rhizoctonia solani* is established by gel electrophoresis and fluorescent dye detection.

[0075] The results are as follows Figures 2-4 As shown, Figure 2 The results showed that LAMP reactions using different concentrations of Bst II DNA polymerase yielded positive amplification results when the Bst II DNA polymerase concentration was between 0.2 and 1.0 U / μL, with specific ladder-like bands observed on electrophoresis. SYBR Green I staining indicated a green band, while the negative control (0 U / μL) showed no amplification band and stained orange. LAMP reactions using different concentrations of dNTPs also yielded the following results: Figure 3 As shown, when dNTPs are used at amounts of 0.5-1.5 mM, the amplification result is positive, and electrophoresis shows specific ladder-shaped bands. SYBR Green I staining is green. The negative control (0 mM) shows no amplification band and is stained orange. LAMP reactions were performed using different ratios of inner and outer primers, and the results are as follows. Figure 4 As shown, both the inner and outer primer ratios at the indicated ratios amplified positive results, with specific ladder-like bands observed on electrophoresis and stained green with SYBR Green I. The negative control (CK) showed no amplification bands and stained orange. Considering both amplification efficiency and cost, the optimal amounts of Bst II DNA polymerase, dNTPs, and the inner-outer primer ratio were determined to be 0.4 U / μL, 1 mM, and 2:1.

[0076] Therefore, the LAMP reaction system for Rhizoctonia solani was established as follows: final concentration of 1×Bst II Buffer, 8 mM Mg 2 +0.4 U / μL Bst II DNA polymerase, 1 mM dNTPs, 0.5 μM FIP-RS, 0.5 μM BIP-RS, 0.25 μM MF3-RS, 0.25 μM B3-RS, 1 μL DNA template, and sterile ultrapure water to bring the volume to 20 μL.

[0077] (II) Different LAMP reaction conditions and their implementation effects

[0078] Based on Example 2, this invention adjusts the reaction conditions as follows: reaction temperatures are 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, 66℃, or 68℃; reaction times are 10, 20, 30, 40 min, 50 min, 60 min, or 70 min. With other component amounts and conditions remaining the same as in Example 2, the single-factor conditions are varied to conduct the LAMP reaction. Sterile ultrapure water is used as a negative control. After the reaction, the optimal LAMP reaction conditions for *Rhizoctonia solani* are determined by gel electrophoresis and fluorescent dye detection.

[0079] The results are as follows Figure 5 As shown, amplification at 52-66℃ yielded a positive result, with electrophoresis showing specific ladder-shaped bands. SYBR Green I staining indicated a green band, while the negative control (CK) showed no amplification band and stained orange. Figure 6 Amplification results were positive between 20 and 70 minutes, showing specific ladder-like bands on electrophoresis. SYBR Green I staining was green. The negative control (CK) and the 10-minute amplification zone showed no amplification bands and stained orange. Amplification at 68℃ and for 10 minutes yielded poor results. Therefore, the optimal LAMP reaction conditions for Rhizoctonia solani are: 56℃ for 30 minutes followed by inactivation at 85℃ for 5 minutes.

[0080] (III) Different LAMP reaction template concentrations and their implementation effects

[0081] Genomic DNA of Rhizoctonia solani was diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, 100 ag / μL, 10 ag / μL, and 1 ag / μL, respectively. Using these different concentrations of DNA as templates, LAMP amplification was performed using the LAMP detection method established in Example 2. The results are as follows: Figure 7As shown, the lowest template concentration for a positive reaction is 100 ag / μL, indicating that the detection sensitivity of Rhizoctonia solani is 100 ag / μL. Compared with conventional PCR methods that amplify different template concentrations, the lowest template concentration for a positive reaction is 100 pg / μL, indicating that the LAMP detection using the primers of this invention has extremely high sensitivity.

[0082] Comparative Example 1

[0083] This comparative example used conventional PCR technology, with different concentrations of Rhizoctonia solani genomic DNA from (III) as templates. The outer primers F3-RS and B3-RS from the LAMP primer set were used as upstream and downstream primers for conventional PCR. The reaction system was adaptively adjusted based on the Rhizoctonia solani LAMP reaction system obtained in Example 2, maintaining the same volume. The results of conventional PCR amplification are shown below. Figure 8 The results showed that the minimum template concentration for a positive PCR reaction was 100 pg / μL, which was far less sensitive than the LAMP reaction of this invention.

[0084] In this comparative example, the reaction system for conventional PCR technology was as follows: 10 μL of Permix Taq (TaKaRa Taq Versin 2.0plus dye), 1 μL each of 10 μM F3-RS, 10 μM B3-RS and template, and 7 μL of sterile ultrapure water; the reaction program was as follows: 95℃ pre-denaturation for 5 min; 30 cycles of 95℃ denaturation for 30 s, 51℃ annealing for 30 s and 72℃ extension for 30 s; and a final extension at 72℃ for 10 min.

[0085] Example 3

[0086] In this embodiment, the kit from Example 2 is used for targeted monitoring of damping-off pathogen in tomato grown in greenhouse cultivation areas. The specific detection method is as follows:

[0087] Five pathogen monitoring points were set up in the greenhouse cultivation area. LAMP tests were conducted on air, tomato leaves, and soil samples from the tomato root colonization area during the tomato seedling stage, flowering and fruit setting stage, fruiting stage, and maturity stage.

[0088] The sampling method for air samples in the protected cultivation area is as follows: Reaction tubes containing template-free LAMP reaction solution were placed between the rows at the monitoring points, with the caps open for 30-60 minutes to allow air near the monitoring points to diffuse into the reaction tubes, serving as air templates for the LAMP reaction. Phenotypic healthy young leaves from the middle of tomato plants on both sides of the air sampling points were selected as leaf samples, and soil from the root colonization area of ​​the tomato plants was selected as soil samples. Genomic DNA was extracted from both leaves and soil samples as templates for the LAMP reaction. Two replicates were set up at each sampling point, resulting in a total of 10 samples, numbered 1-10. The 10 samples, along with positive and negative control samples, were subjected to the optimal LAMP reaction conditions and system described above for Rhizoctonia solani.

[0089] The results are as follows Figure 9 As shown in Table 1.

[0090] Table 1. Correspondence between Rhizoctonia solani pathogen detection and various factors during tomato growth period.

[0091]

[0092] The results showed that all tomato seedlings in the greenhouse exhibited a healthy phenotype, with no Rhizoctonia solani pathogen detected in the air or leaves. However, the pathogen was detected in the soil of the root colonization area, indicating that Rhizoctonia solani was present in the greenhouse soil during the seedling stage but had not spread into the air or infected the plant leaves. During the flowering and fruiting stage, Rhizoctonia solani was not detected in the air or leaves, but it was detected in the soil. At this time, a small number of plants in the greenhouse showed yellowing and wilting of the lower, older leaves, but this was not a characteristic phenotype of damping-off. During the fruiting stage, Rhizoctonia solani was detected in air samples from two areas, and it was also detected in a small number of leaves without a susceptible phenotype. Rhizoctonia solani was detected in all soil samples. A small number of fruits showed premature reddening and mold, indicating that with increased agricultural operations during this period, Rhizoctonia solani gradually spread from the soil into the air.

[0093] Damping-off, caused by Rhizoctonia solani, is a soil-borne disease that primarily affects the base of seedling stems, with less infection on leaves, consistent with leaf detection. High soil and air humidity easily induces the disease. At maturity, Rhizoctonia solani is widely detected in both air and soil, with a small amount still detected on leaves, indicating further spread of the bacterium into the air. This poses a significant potential risk of disease in the next season's tomato seedlings, necessitating thorough disinfection of the soil and air before transplanting.

[0094] The above results indicate that the kit in Example 2 can detect pathogens significantly earlier than the appearance of the diseased phenotype in tomatoes. It can be used for early detection of pathogens. Combined with plant disease phenotype, environmental factors, and agricultural operations, the seedling stage is the key period for monitoring Rhizoctonia solani pathogen. The frequency of LAMP detection can be increased to detect pathogens in a timely manner, assess the risk level of damping-off disease, and thus achieve early warning of damping-off disease.

[0095] Comparative Example 2

[0096] In this comparative example, the LAMP detection primers for Rhizoctonia solani from Example 1 were used to detect different strains or negative controls to verify the specificity of the primers of the present invention. The specific detection method was the same as in Example 2, wherein the detection objects were numbered sequentially as follows: 1: negative control; 2: Rhizoctonia solani; 3: Corynebacterium multiflorum; 4: Pseudomonas syringae; 5: Staphylococcus aureus; 6: Pectinobacter soft rot; 7: Fusarium graminearum; 8: Fusarium pseudograecum; 9: Fusarium oxysporum.

[0097] The results are as follows Figure 10 As shown, only *Rhizoctonia solani* strain 2 showed a positive amplification result, exhibiting a specific ladder-shaped band on electrophoresis and turning yellow with SYBR Green I staining. The negative control and other non-target pathogens showed no amplification bands and stained orange. These results indicate that the primers of this invention have high specificity, specifically detecting *Rhizoctonia solani* without detecting other non-target pathogens, and can be used for LAMP detection of *Rhizoctonia solani*.

[0098] The LAMP reaction results were further evaluated using 1.5% agarose gel electrophoresis, and the results were consistent with those of SYBR GreenI staining.

[0099] Comparative Example 3

[0100] In this comparative example, three sets of LAMP primers were designed targeting the ITS and Tubulin gene sequences of Rhizoctonia solani. The sequences of each primer are shown in Table 2. Each primer was used to perform LAMP amplification with Rhizoctonia solani genomic DNA as a template, following the method in Example 2. Positive results were obtained in all cases.

[0101] Further verification of specificity revealed that only the LAMP-RS primer combination of this invention has the effect of specifically detecting Rhizoctonia solani, as detailed below:

[0102] Following the method described in Example 2, LAMP amplification was performed using genomic DNA from *Rhizoctonia solani*, *Corynebacterium multiflorum*, *Botrytis cinerea*, and *Botrytis cinerea* as templates. The LAMP reaction results were evaluated using SYBR Green I fluorescence staining. The results are as follows: Figure 11 .

[0103] Table 2 Primer sequences and target genes

[0104]

[0105] Figure 11 A represents the detection results of primer LAMP-RS. The DNA template sources in each lane are as follows: CK is sterile ultrapure water, lane 1 is Botrytis cinerea, lane 2 is Corynebacterium multiflorum, lane 3 is Staphylococcus aureus, and lane 4 is Rhizoctonia solani.

[0106] Figure 11 B represents the detection results of primer LAMP-RSI1. The DNA template sources in each lane are as follows: CK is sterile ultrapure water, lane 1 is Rhizoctonia solani, lane 2 is Botrytis cinerea, lane 3 is Corynebacterium multiflorum, and lane 4 is Staphylococcus aureus.

[0107] Figure 11 C represents the detection result of primer LAMP-RSI2. The detection objects are numbered sequentially as follows: the DNA template sources in each lane are: CK is sterile ultrapure water, lane 1 is Rhizoctonia solani, lane 2 is Botrytis cinerea, lane 3 is Cladosporium multiflorum, and lane 4 is Staphylococcus aureus.

[0108] Depend on Figure 11 It is known that although all primers can amplify the genome of Rhizoctonia solani, compared with the primer LAMP-RS of this invention, the other primers have poor specificity and cannot distinguish Rhizoctonia solani from Corynebacterium multiflorum, Botrytis cinerea, and Botrytis cinerea, and cannot be used to accurately identify Rhizoctonia solani.

[0109] Comparative Example 4

[0110] To verify the applicability of the LAMP detection kit of Example 2 of this invention to DNA templates obtained under simple field extraction conditions, comparative analyses were performed on plant leaf samples and soil samples. All LAMP amplification results were interpreted using both electrophoresis bands and SYBR Green I fluorescence staining.

[0111] (1) For plant leaf samples, four DNA extraction methods were used to extract DNA templates: conventional kit method, isopropanol precipitation method, centrifugation method, and non-centrifugation method. The details are as follows:

[0112] The conventional kit method used the plant genomic DNA extraction kit from Beijing Polymer Biotechnology Co., Ltd., and DNA extraction was performed according to the kit instructions.

[0113] The isopropanol precipitation method is as follows: Take 1-2 leaves into a 1.5 ml tube, add 700 μl of Edwards Buffer (Tris 31.512 g / L, NaCl 14.625 g / L, EDTA 2.30625 g / L, SDS 5 g / L, pH adjusted to 7.5 with HCl), grind, centrifuge at 12000 rpm for 4 min at 4℃, take the supernatant, add an equal volume of isopropanol and mix well, precipitate at -20℃ for 30 min, centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant, wash the precipitate with 80% ethanol, centrifuge at 12000 rpm for 1 min, repeat once, let the precipitate air dry, dissolve in 30 μl of sterile ultrapure water, and store at -20℃ for later use.

[0114] The simplified centrifugation extraction method is as follows: Take one tender young leaf from a plant, immerse it in 75% alcohol for 45 seconds for surface disinfection, then wash it three times with sterile water, cut it into small pieces, transfer it to a 1.5 ml centrifuge tube, add 50 μl of 50 mM NaOH solution, boil in a water bath for 10 min, add 5 μl of Tris-HCl buffer (pH 7.5), centrifuge at 12000 rpm for 1 min, and take the supernatant as the extracted leaf DNA, and store it at -20℃ for later use;

[0115] The simplified extraction method without centrifugation is as follows: the simplified centrifugation method removes the step of centrifuging at 12000 rpm for 1 min, while the other steps remain unchanged.

[0116] LAMP amplification was performed using DNA extracted by the four methods described above as templates, and the results are as follows: Figure 12 As shown in Figure A, where CK represents the negative control; 1 represents the simplified extraction method; 2 represents the kit method; 3 represents the non-centrifugation simplified extraction method; and 4 represents the isopropanol precipitation method. Electrophoresis results showed that DNA extracted by all four methods could amplify ladder-like bands. The isopropanol precipitation method yielded the best amplification effect, while the centrifugation simplified extraction method and the kit method showed comparable amplification effects. The non-centrifugation simplified extraction method showed slightly weaker amplification effects. However, the color reactions corresponding to all four extraction methods showed positive results, indicating that even the DNA template obtained by the most simplified non-centrifugation simplified extraction method can be visually detected using the kit prepared with the primers of this invention.

[0117] (2) The LAMP amplification effects of soil DNA extracted by the simplified extraction method and the kit method were compared for soil samples. The simplified soil DNA extraction method involved placing 500 mg of soil in a 1.5 ml centrifuge tube, adding 50 μl of 50 mM NaOH solution, boiling in a water bath for 10 min, adding 5 μl of Tris-HCl buffer (Tris 121 g / L, pH adjusted to 7.5 with HCl), centrifuging at 12000 rpm for 1 min, and collecting the supernatant as the extracted soil DNA. This was stored at -20℃ for later use. The kit method used the MPBiomedicals Soil FastDNA™ Spin Kit, and DNA extraction was performed according to the kit instructions.

[0118] The results are as follows Figure 12 As shown in Figure B, PC represents the positive control; NC represents the negative control; 1 represents soil sample group 1 (using the kit method); 2 represents soil sample group 2 (using the kit method); 3 represents soil sample group 1 (using the simplified extraction method); and 4 represents soil sample group 2 (using the simplified extraction method). The results indicate that DNA extracted by both methods can amplify positive results, demonstrating that the simplified extraction method described in this invention is also suitable for LAMP detection of soil samples.

[0119] Based on the experimental results from the plant and soil samples above, the LAMP detection system established in this invention exhibits extremely high sensitivity and excellent specificity, with low requirements for the quality of the DNA template. Whether using the cumbersome but high-purity kit method, the isopropanol precipitation method, or the simplified extraction method requiring only NaOH solution and omitting centrifugation, the obtained crude DNA template consistently shows a positive color reaction. Therefore, this invention is perfectly suitable for the rapid detection of Rhizoctonia solani in tomato under field conditions, requiring no complex instruments or cumbersome DNA purification steps, and possesses excellent practical application value and promising prospects for widespread application.

[0120] The above results demonstrate that the loop-mediated isothermal amplification detection method using the primers LAMP-RS of this invention allows for direct visual observation to determine the presence of Rhizoctonia solani in the sample. The primers of this invention exhibit a sensitivity of up to 100 ag / μL and high specificity. When used for tomato disease detection, they can detect the disease in tomato leaves before a clear phenotype is observed. This invention is suitable for early, rapid, sensitive, and visual detection of tomato damping-off.

[0121] It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any equivalent substitutions should be included within the protection scope of the present invention.

Claims

1. A primer for detecting Rhizoctonia solani using LAMP, comprising an outer primer and an inner primer, characterized in that, The outer primers are the forward outer primer F3-RS and the reverse outer primer B3-RS; the inner primers are the forward inner primer FIP-RS and the reverse inner primer BIP-RS. The nucleotide sequences of each primer are as follows: F3-RS: 5'-CGAGTTCTTGTTCTGGACGT-3'; B3-RS: 5'-CTTTGCCCCTTTGACCGC-3'; FIP-RS: 5'-CCTCACCGTAGCTGCCTTCTTCGCATCTGCTCCTCGACTT-3'; BIP-RS: 5'-AGCAGCCATCATGTTCTTGGCATGGCTCTGTTCAATACCGC-3'.

2. A LAMP detection kit for tomato damping-off disease prepared using the primers described in claim 1, characterized in that, Its components include: the primers, LAMP reaction buffer, thermophilic DNA polymerase for LAMP detection, and nucleic acid fluorescent dye; The primers contain equal amounts of the two outer primers and equal amounts of the two inner primers. The molar ratio of inner primer to outer primer is 3~1:1~3.

3. The tomato damping-off LAMP detection kit according to claim 2, characterized in that, The LAMP reaction buffer contains 8 mM Mg 2+ 10×Bst II reaction buffer; The thermophilic DNA polymerase used for LAMP detection is Bst II DNA polymerase.

4. The tomato damping-off LAMP detection kit according to claim 3, characterized in that, The nucleic acid fluorescent dye is SYBR Green I.

5. A method for detecting tomato damping-off using the tomato damping-off LAMP detection kit according to any one of claims 2 to 4, characterized in that, DNA from the sample to be tested is extracted as a template, mixed into a LAMP reaction system, and subjected to a LAMP reaction. If a positive color change occurs after adding a nucleic acid fluorescent dye to the reaction product, the sample to be tested is a positive sample for tomato damping-off.

6. The method for detecting damping-off disease in tomatoes according to claim 5, characterized in that, The LAMP reaction system consisted of the following components: 1×Bst II Buffer, 8 mM Mg 2+ 0.2~1.0U / μL Bst II DNA polymerase, 0.5~1.5mM dNTPs, 0.5 μM FIP-RS, 0.5 μM BIP-RS, 0.25 μM F3-RS, 0.25 μM B3-RS, 1 μL template, and sterile ultrapure water to bring the volume to 20 μL.

7. The method for detecting damping-off disease in tomatoes according to claim 6, characterized in that, The LAMP reaction conditions are 52-66℃ for 20-70 min, followed by inactivation at 85℃ for 5 min.

8. The method for detecting damping-off disease in tomatoes according to claim 7, characterized in that, The LAMP reaction conditions were 56°C for 30 min, followed by inactivation at 85°C for 5 min.

9. The application of the LAMP detection primers for Rhizoctonia solani as described in claim 1 in the early identification of damping-off disease in tomatoes and the detection of Rhizoctonia solani.

10. The application of the LAMP detection kit for tomato damping-off according to any one of claims 2 to 4 in the early identification of tomato damping-off and the detection of Rhizoctonia solani in soil and plant leaves.

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