Fluorescent quantitative PCR (Polymerase Chain Reaction) primer group, kit and method for detecting tomato neck and root rot germs and application

Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) primer group, a kit and a method for detecting tomato neck and root rot germs and application. Belongs to the technical field of gene detection. Sequence homology comparison is carried out on the tomato neck and root rot pathogen and other fusarium oxysporum genomes, a specific site for detecting the tomato neck and root rot pathogen is determined, and the sequence is as shown in SEQ ID NO.19. The site is further utilized to design an amplification primer group, a kit and a detection method, a real-time fluorescent quantitative PCR detection system of the FORL is established, the system is good in specificity, high in sensitivity, strong in repeatability and high in accuracy, can detect genome DNA of the tomato neck and root rot bacteria, the sensitivity is 16.4 pg / mu L, the quantity of the bacteria in infected plants can be rapidly and accurately identified, and the method is suitable for popularization and application. And a scientific basis is provided for early prevention and treatment of tomato neck and root rot.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, and more particularly to a fluorescent quantitative PCR primer set, kit, method and application for detecting tomato collar and root rot pathogen. BACKGROUND

[0002] Tomato collar and root rot is a worldwide soil-borne fungal disease caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL). The mortality rate of tomato plants is more than 80%, causing serious economic losses. Due to the lack of disease-resistant varieties and the limited effect of chemical control, the disease is difficult to control.

[0003] Traditional methods for detecting tomato collar and root rot pathogen mainly include symptom observation and field diagnosis, tissue isolation and purification culture, systematic identification and pathogenicity determination. However, these methods are time-consuming, tedious to operate, cannot achieve quantitative detection, have low identification accuracy, and are prone to misdiagnosis, which affects the prevention and treatment effect. With the continuous development of molecular biology technology, molecular systematic methods have been introduced into the identification of Fusarium. Using fluorescent quantitative PCR technology, the pathogen in the plant can be qualitatively and quantitatively detected before the plant shows symptoms, which is of great significance for preventing and controlling the disease before the spores of the pathogen spread and proliferate.

[0004] Currently, the existing technologies for detecting tomato collar and root rot pathogen are as follows: a molecular marker for identifying Fusarium oxysporum f. sp. radicis-lycopersici and its application, published as CN112176090A. This patent is a SNP molecular marker detection, which is a qualitative detection, used to determine whether the Fusarium oxysporum f. sp. radicis-lycopersici is the tomato collar and root rot pathogen. It is more suitable for pathogen identification and cannot achieve qualitative and quantitative detection of tomato collar and root rot pathogen.

[0005] Therefore, how to provide a fluorescent quantitative PCR primer set, kit, method and application for detecting tomato collar and root rot pathogen, which can improve the sensitivity, specificity and repeatability of detection, is a problem that needs to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the application provides a fluorescent quantitative PCR primer group, kit, method and application for detecting tomato verticillium wilt pathogen.

[0007] In order to achieve the above object, the application adopts the following technical scheme:

[0008] The first object of the application is to provide an application of a DNA molecule in detection of tomato verticillium wilt pathogen, characterized in that the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 19; the DNA molecule is located in the GCA_000260155.3 genome, between lcl|JH651389.1_cds_EXL54089.1_1273 [locus_tag=FOCG_07178]

[0009] and lcl|JH651389.1_cds_EXL54090.1_1274 [locus_tag=FOCG_07179], [location=join(2245510..2245652, 2245703..2245724)].

[0010] The first object of the application is to provide an application of a DNA molecule in detection of tomato verticillium wilt pathogen, characterized in that the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 19; the DNA molecule is located in the GCA_000260155.3 genome, between lcl|JH651389.1_cds_EXL54089.1_1273 [locus_tag=FOCG_07178]

[0011] The first object of the application is to provide an application of a DNA molecule in detection of tomato verticillium wilt pathogen, characterized in that the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 19; the DNA molecule is located in the GCA_000260155.3 genome, between lcl|JH651389.1_cds_EXL54089.1_1273 [locus_tag=FOCG_07178]

[0012] SY-36F: 5'-CCGGTTGTCTCTTTCGCG-3', SEQ ID NO: 13;

[0013] SY-36R: 5'-GGGGCTCCACGTCTTGTC-3', SEQ ID NO: 14.

[0014] As a preferred technical scheme, the tomato verticillium wilt pathogen is Fusarium oxysporum.

[0015] The first object of the application is to provide an application of a DNA molecule in detection of tomato verticillium wilt pathogen, characterized in that the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 19; the DNA molecule is located in the GCA_000260155.3 genome, between lcl|JH651389.1_cds_EXL54089.1_1273 [locus_tag=FOCG_07178]

[0016] Still another object of the present application is to provide a fluorescent quantitative PCR detection method for detecting tomato collar and root rot pathogen for non-disease diagnosis and treatment, comprising the following steps:

[0017] (1) Extraction of the genomic DNA of the sample to be tested;

[0018] (2) Using the fluorescent quantitative PCR primer set described above to perform fluorescent quantitative PCR amplification on the DNA template obtained in step (1) to obtain an amplification curve;

[0019] (3) Detection result judgment standard: when the CT value is ≤32, it is determined to be positive, and when the CT value is >32, it is determined to be negative.

[0020] As a preferred technical solution, the reaction system of PCR amplification in step (2) is: genomic DNA 2 μL, SY-36F 0.4 μL, SY-36R 0.4 μL, 2xTransStartTM Top Green qPCR SuperMix 10 μL, RNase-free ddH2O 7.2 μL, total system 20 μL.

[0021] As a preferred technical solution, the reaction program of PCR amplification in step (2) is: 95℃ pre-denaturation for 30s; 95℃ for 5s, 58℃ annealing for 15s, 72℃ extension for 10s, a total of 45 cycles.

[0022] Still another object of the present application is to provide the application of the fluorescent quantitative PCR primer set or the kit or the detection method in the qualitative and quantitative detection of tomato collar and root rot pathogen for non-disease diagnosis and treatment.

[0023] Through the above technical solution, compared with the prior art, the present application has the following beneficial effects:

[0024] The present application establishes a real-time fluorescent quantitative PCR detection system for FORL, which has good specificity, high sensitivity, strong repeatability and high accuracy, can detect tomato collar and root rot pathogen genomic DNA, has a sensitivity of 16.4 pg / μL, and can quickly and accurately identify the amount of pathogen in the diseased plant, thereby providing a scientific basis for the early prevention and treatment of tomato collar and root rot. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creating any creative labor.

[0026] Figure 1 M: 2000bp Marker; 1, 2, 4, 5: Fusarium oxysporum f. sp. radices-lycopersici (FORL); 3: Fusarium oxysporum f. sp. lycopersici (FOL); 6-8: Fusarium oxysporum f. sp. niveum race 0, 1, 2; 9: Fusarium oxysporum f. sp. cucumerinum (FOCU); 10: Fusarium oxysporum f. sp. melonis (FOM); 11: Fusarium oxysporum f. sp. conglutinans (FOC); 12: Fusarium oxysporum f. sp. fragariae (FOF); 13: Phytophthora cactorum; 14: Colletotrichum fragariae; N: negative control.

[0027] Figure 2 M: 2000bp Marker; 1-4: Fusarium oxysporum f. sp. radices-lycopersici (FORL); 5: Fusarium oxysporum f. sp. lycopersici (FOL); 6-8: Fusarium oxysporum f. sp. niveum race 0, 1, 2; 9: Fusarium oxysporum f. sp. cucumerinum (FOCU); 10: Fusarium oxysporum f. sp. melonis (FOM); 11: Fusarium oxysporum f. sp. conglutinans (FOC); 12: Fusarium oxysporum f. sp. fragariae (FOF); 13: Phytophthora cactorum; 14: Colletotrichum fragariae; N: negative control.

[0028] Figure 3 M: 2000bp Marker; 1-4: Fusarium oxysporum f. sp. radices-lycopersici (FORL); 5: Fusarium oxysporum f. sp. lycopersici (FOL); 6-8: Fusarium oxysporum f. sp. niveum race 0, 1, 2; 9: Fusarium oxysporum f. sp. cucumerinum (FOCU); 10: Fusarium oxysporum f. sp. melonis (FOM); 11: Fusarium oxysporum f. sp. conglutinans (FOC); 12: Fusarium oxysporum f. sp. fragariae (FOF); 13: Phytophthora cactorum; 14: Colletotrichum fragariae; N: negative control.

[0029] Figure 4 M: 2000bp Marker; 1-4: Fusarium oxysporum f. sp. radices-lycopersici (FORL); 5: Fusarium oxysporum f. sp. lycopersici (FOL); 6-8: Fusarium oxysporum f. sp. niveum race 0, 1, 2; 9: Fusarium oxysporum f. sp. cucumerinum (FOCU); 10: Fusarium oxysporum f. sp. melonis (FOM); 11: Fusarium oxysporum f. sp. conglutinans (FOC); 12: Fusarium oxysporum f. sp. fragariae (FOF); 13: Phytophthora cactorum; 14: Colletotrichum fragariae; N: negative control.

[0030] Figure 5 M: 2000bp Marker; 1-4: Fusarium oxysporum f. sp. radices-lycopersici (FORL); 5: Fusarium oxysporum f. sp. lycopersici (FOL); 6-8: Fusarium oxysporum f. sp. niveum race 0, 1, 2; 9: Fusarium oxysporum f. sp. cucumerinum (FOCU); 10: Fusarium oxysporum f. sp. melonis (FOM); 11: Fusarium oxysporum f. sp. conglutinans (FOC); 12: Fusarium oxysporum f. sp. fragariae (FOF); 13: Phytophthora cactorum; 14: Colletotrichum fragariae; N: negative control.

[0031] Figure 6 M: 2000bp Marker; 1-4: Fusarium oxysporum f. sp. radices-lycopersici (FORL); 5: Fusarium oxysporum f. sp. lycopersici (FOL); 6-8: Fusarium oxysporum f. sp. niveum race 0, 1, 2; 9: Fusarium oxysporum f. sp. cucumerinum (FOCU); 10: Fusarium oxysporum f. sp. melonis (FOM); 11: Fusarium oxysporum f. sp. conglutinans (FOC); 12: Fusarium oxysporum f. sp. fragariae (FOF); 13: Phytophthora cactorum; 14: Colletotrichum fragariae; N: negative control.

[0032] Figure 7Fig. 2: Stem section of tomato plants inoculated with F. oxysporum f. sp. radices-lycopersici. A: susceptible variety 'MoneyMaker'; B: resistant variety 'TMS150'.

[0033] Figure 8 Fig. 4: Colony-forming units of F. oxysporum f. sp. radices-lycopersici on tomato plants at different days after inoculation. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0035] Example 1

[0036] Design and synthesis of Real-time PCR primers

[0037] The genomic sequence of F. oxysporum f. sp. radices-lycopersici was searched in the Fusarium oxysporum genomic database (https: / / www.ncbi.nlm.nih.gov / nuccore / JH651389.1 / (Fusarium oxysporum f. sp. radices-lycopersici, GCA_000260155.3), and sequence homology was compared with the genomic data of all other species of Fusarium oxysporum in NCBI to find specific sites in the sequence of the tomato-specific form of Fusarium oxysporum. The specific process is as follows:

[0038] The genome sequences of 11 strains of 7 species of Fusarium oxysporum f. sp. radices-lycopersici (GCA_000260155.3), Fusarium oxysporum f. sp. lycopersici (GCA_015345895.1, GCA_001703175.2, GCA_003977725.1), Fusarium oxysporum f. sp. fragariae (GCA_016166325.2), Fusarium oxysporum f. sp. niveum (GCA_014602815.1, GCA_019593445.1, GCA_014602775.1), Fusarium oxysporum f. sp. conglutinans (GCA_014154955.1), Fusarium oxysporum f. sp. cucumerinum (GCA_001702495.1), and Fusarium oxysporum f. sp. melonis (GCA_001703205.2) were pairwise compared using the Genome VarScan plug-in of TBtools to find the difference sites. Then, using a Venn diagram, the non-intersecting part was found as a special difference site. According to the difference sites, primers were designed using the Batch Target Region Primer Design plug-in. The rationality and effectiveness of the primers were detected using Primer Check, and 9 pairs of primers were designed according to the difference fragments of the tomato Fusarium oxysporum f. sp. radices-lycopersici genome data (Table 1)

[0039] Table 1 Partial primer sequence information

[0040]

[0041] Example 2

[0042] Screening of primer specificity

[0043] (1) Preliminary screening

[0044] Using 9 pairs of primers in Table 1 designed by different fragments of tomato vascular wilt and root rot genome data, the genomic DNA of tomato vascular wilt and root rot fungus, tomato wilt fungus, watermelon wilt fungus, cucumber wilt fungus, melon wilt fungus, cabbage wilt fungus and strawberry anthracnose fungus was used as a template, RNase-free ddH2O was used as a negative control, and ordinary PCR was used for preliminary screening of tomato vascular wilt and root rot fungus specific primers Figure 1 ), and SY-36 was selected as a tomato vascular wilt and root rot fungus detection primer, and the primer sequence was as follows:

[0045] SY-36F: 5'-CCGGTTGTCTCTTTCGCG-3', SEQ ID NO. 13;

[0046] SY-36R: 5'-GGGGCTCCACGTCTTGTC-3', SEQ ID NO. 14.

[0047] The design of SY-36 detection primer is based on the alignment of https: / / www.ncbi.nlm.nih.gov / nuccore / JH651389.1 / (Fusarium oxysporum f. sp. radices-lycopersici, GCA_000260155.3) genome and other Fusarium oxysporum genomes mentioned above, and the difference fragment is designed, which is located in: >lcl|JH651389.1_cds_EXL54089.1_1273 [locus_tag=FOCG_07178]

[0048] [protein=hypothetical protein][protein_id=EXL54089.1]

[0049] [location=2241897..2243324][gbkey=CDS] this gene fragment and

[0050] >lcl|JH651389.1_cds_EXL54090.1_1274 [locus_tag=FOCG_07179]

[0051] [protein=hypothetical protein][protein_id=EXL54090.1]

[0052] [location=join(2245510..2245652,2245703..2245724)][gbkey=CDS] This difference between the sequences of the gene fragments is 2185 p, and the specific nucleotide sequence is as follows:

[0053]

[0054] (2) Secondary screening:

[0055] Further, the SY-36 detection primer preliminarily screened was used for PCR amplification of Fusarium oxysporum f. sp. radices-lycopersici (FORL), Fusarium oxysporum f. sp. lycopersici (FOL), different physiological races of Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. cucumeris (FOCU), Fusarium oxysporum f. sp. melonis (FOM), Fusarium oxysporum f. sp. conglutinans (FOC), Fusarium oxysporum f. sp. fragariae (FOF), Lily Phytophthora blight, strawberry anthracnose Colletotrichum fragariae and the negative control group, and the experimental results are shown in Table 2. Figure 2

[0056] Result analysis: As shown in Table 1, the primer set SY-36 can amplify a specific band of about 170 bp from the genomic DNA of Fusarium oxysporum f. sp. radices-lycopersici, and no amplification is observed for other control strains, indicating that the primer SY-36 has good specificity and can distinguish Fusarium oxysporum f. sp. radices-lycopersici from other related pathogens. The primer is selected as the specific primer for real-time PCR quantitative detection of Fusarium oxysporum f. sp. radices-lycopersici for subsequent experimental study. Figure 2 Example 3

[0057] Primer amplification efficiency and sensitivity

[0058] (1) Nucleic acid gradient concentration preparation: The genomic DNA of Fusarium oxysporum f. sp. radices-lycopersici was used as a standard, and was diluted by 10 times in gradient for 7 times, and the final concentrations were 1640 ng / μL, 164 ng / μL, 16.4 ng / μL, 1.64 ng / μL, 164 pg / μL, 16.4 pg / μL, 1.64 pg / μL, and 0.164 pg / μL, respectively.

[0059] (2) Ordinary PCR amplification: The working solution of different concentration gradient diluted above was taken as a template for real-time fluorescent quantitative PCR reaction.

[0060] Real-time PCR reaction system: DNA 2 μL, upstream primer SY-36-F 0.4 μL (primer concentration 5 μM), downstream primer SY-36-R 0.4 μL (primer concentration 5 μM), 2×TransStartTM Top Green qPCR SuperMix 10 μL, RNase-free ddH2O 7.2 μL, total system 20 μL.

[0061]

[0062] ​​Real-time PCR amplification procedure: 95℃ pre-denaturation 30s; 95℃ 5s, 58℃ annealing 15s, 72℃ extension 10s, 45 cycles in total;

[0063] After Real-time PCR reaction, the melting curve (Fig. 1), amplification curve (Fig. 2) and standard curve (Fig. 3) are automatically generated by the system. Figure 3 Figure 4 Figure 5

[0064] Result analysis: the melting curve has obvious single peak without miscellaneous peak, which indicates that the tomato collar and root rot disease primer used in the application has strong specificity without non-specific amplification.

[0065] The amplification curve shows that the sensitivity lower limit of the detection system is 16.4pg / μL, which is about 100 times higher than that of the conventional PCR method; and the optimal quantitative detection range is 164ng / μL to 16.4pg / μL.

[0066] The standard curve linear equation is y=-3.3934log(x)+26.714, the Ct value and the logarithm of the standard product DNA copy number concentration have good linear relationship, the correlation coefficient (R 2 ) is 0.9996, the slope is-3.3934, and the corresponding amplification efficiency is 97.10%, which meets the evaluation index of the fluorescence quantitative PCR standard curve.

[0067] Example 4

[0068] Actual sample detection

[0069] Tomato collar and root rot disease fungus (Fusarium oxysporum f.sp.radices-lycopersici) is used to infect tomato 'Money Maker' (sensitive variety, Calgene, USA) and 'TMS 150' (resistant variety, Shanghai Huihe Seed Industry Company), and the disease index of 28 samples after 0d, 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d or 25d after inoculation is observed (Table 2), and the real-time fluorescence quantitative PCR detection system established above is used to quantitatively detect the disease fungus (Table 3).

[0070] Table 2 Disease index of resistant and sensitive tomato inoculated with collar and root rot disease fungus

[0071]

[0072] Table 3 Fluorescence quantitative PCR detection of disease fungus content of tomato plants​​​

[0073]

[0074]

[0075] Note: The FORL content is the amount of DNA of Fusarium solani f. sp. radicis-lycopersici (μg) per gram of tomato plant.

[0076] Result analysis: According to the results of Real-time PCR detection (Table 3), the amount of pathogen in ‘Money Maker’ was always significantly higher than that in ‘TMS150’. The amount of pathogen in ‘Money Maker’ decreased slowly from 0 to 3 dpi, which might be due to the fact that part of the pathogen on the root surface entered the soil and part of it invaded the roots. The amount of pathogen increased gradually from 5 to 17 dpi, reaching the maximum value of 10.93 μg / g at 17 dpi. The amount of pathogen decreased slowly from 19 to 25 dpi, and the final amount of pathogen was 5.62 μg / g. The decrease in the amount of pathogen might be related to the wilting and death of the plants at the time of final sampling. Figure 6 The susceptible variety ‘Money Maker’ showed slight wilting symptoms at 9 dpi, with slight browning of the vascular bundle and a disease index of 12.50. The disease index increased gradually from 13 to 19 dpi, and the browning of the vascular bundle also gradually deepened. The disease index reached the maximum value of 95.00 at 25 dpi, and the browning of the vascular bundle was the most serious. Figure 7 This might be related to the wilting and death of the plants after 21 dpi.

[0077] The amount of pathogen in ‘TMS150’ decreased from 0 to 3 dpi, increased gradually from 5 to 13 dpi, reached the maximum value of 3.98 μg / g at 13 dpi, and then decreased slowly after 15 dpi, indicating that the resistant variety could resist the reproduction of the pathogen after being stressed by the pathogen. The amount of pathogen was 0.31 μg / g at 25 dpi. The resistant variety ‘TMS150’ showed slight symptoms at 13 dpi, with a disease index of 3.06. The disease index reached the maximum value of 14.44 at 25 dpi, and the vascular bundle showed slight browning. Figure 7

[0078] Example 5

[0079] Detection of Fusarium solani f. sp. radicis-lycopersici in tomato plants by spread plate method

[0080] ​Using the spread plate method, 28 samples of Money Maker' (susceptible variety), 'TMS150' (resistant variety) were detected for pathogen amount at 0d, 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d, 25d after inoculation with pathogen.

[0081] The specific operation is as follows:

[0082] Take 1g of fresh sample, rinse with sterile water for 3-4 times, put the rinsed sample into a sterilized mortar, grind thoroughly, add 1mL of sterile water, stir evenly, and then dilute continuously for two times with 10-fold gradient. Perform the operation in a clean bench, use a sterile pipette to take a certain volume (50-100μL) of the original liquid and the two times diluted bacterial liquid, drop it on the central area of the plate culture medium, take out a glass coating rod immersed in 75% alcohol, quickly sweep over the outer flame of the alcohol lamp and ignite, let the alcohol burn out, hold the coating rod in the air and cool for 10-15s. The back of the hand is not scalding, use the flat part of the front end of the sterile coating rod to contact the bacterial liquid, gently spread it outwards from the center of the plate to the edge in a clockwise (or counterclockwise) circle by circle, process and coat 5 plates each time, after coating, immediately immerse the coating rod in alcohol again for sterilization to prevent cross contamination. Place the coated plate on the table for 5-10min to allow the bacterial liquid to fully penetrate the medium, finally cover the dish cover and place it in a constant temperature incubator at 28℃ for 4-5d, and record the number of colonies on the medium, the experimental results are shown in Figure 8 .

[0083] Result analysis: The number of colonies on the resistant and susceptible hosts after infection differs greatly, the number of colonies of 'MoneyMaker' is always significantly higher than that of 'TMS150'. At 0d after inoculation, the number of colonies of 'MoneyMaker' is slightly higher than that of 'TMS150', the number of colonies of 'Money Maker' increases continuously from 0-17dpi, reaches the maximum value of 2.07×105cfu / g at 17dpi, and decreases slightly from 19-25dpi; the number of colonies of 'TMS150' increases slowly from 0-13dpi, reaches the maximum value of 1.07×105cfu / g at 13dpi, and decreases as a whole from 15-25dpi. This is basically consistent with the fluorescence quantitative PCR detection Figure 8 ). The results are basically consistent with the detection results of tomato collar and root rot pathogen in tomato plants by PCR quantitative detection system in Example 4, verifying the accuracy of the PCR quantitative detection system.

[0084] Each embodiment in the specification is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same and similar parts between each embodiment can be referred to each other.

[0085] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of a DNA molecule in the detection of tomato neck rot and root rot pathogens, characterized in that, The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.

19.

2. A set of fluorescent quantitative PCR primers for detecting the pathogen causing tomato neck rot and root rot, characterized in that, The sequence of the primer set is as follows: SY-36F: 5'-CCGGTTGTTCTCTTCGCG-3', SEQ ID NO: 13; SY-36R: 5'-GGGGCTCCACGTCTTGTC-3', SEQ ID NO:

14.

3. The fluorescent quantitative PCR primer set for detecting tomato neck rot and root rot pathogens according to claim 2, characterized in that, The pathogen causing tomato neck and root rot is *Fusarium oxysporum*.

4. A real-time PCR detection kit for detecting the pathogen causing tomato neck and root rot, characterized in that, Includes the fluorescent quantitative PCR primer set as described in claim 2 or 3.

5. A real-time quantitative PCR method for detecting the pathogen causing tomato neck rot and root rot, used for non-disease diagnosis and treatment, characterized in that, Includes the following steps: (1) Extraction of genomic DNA from the sample to be tested; (2) Use the fluorescent quantitative PCR primer set described in claim 2 or 3 to perform fluorescent quantitative PCR amplification on the DNA template obtained in step (1) to obtain an amplification curve; (3) Criteria for judging test results: When the CT value is ≤32, it is judged as positive; when the CT value is >32, it is judged as negative.

6. The method for detecting the pathogen of tomato neck rot and root rot using real-time quantitative PCR for non-disease diagnosis and treatment according to claim 5, characterized in that, The PCR amplification reaction system described in step (2) is as follows: 2 μL genomic DNA, 0.4 μL SY-36F, 0.4 μL SY-36R, 10 μL 2×TransStart™ Top Green qPCR SuperMix, 7.2 μL RNase-free ddH2O, and a total system volume of 20 μL.

7. The method for detecting the pathogen of tomato neck rot and root rot using real-time quantitative PCR for non-disease diagnosis and treatment according to claim 5, characterized in that, The PCR amplification reaction program described in step (2) is as follows: 95℃ pre-denaturation for 30s; 95℃ for 5s, 58℃ annealing for 15s, 72℃ extension for 10s, for a total of 45 cycles.

8. The application of the fluorescent quantitative PCR primer set according to claim 2 or 3, the kit according to claim 4, or the detection method according to any one of claims 5-7 in the qualitative and quantitative detection of the pathogen of tomato neck rot and root rot in non-disease diagnosis and treatment.

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