Fluorescent quantitative PCR (Polymerase Chain Reaction) primer group for detecting tomato root rot pathogenic bacteria and application of fluorescent quantitative PCR primer group

Abstract

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) primer group for detecting pathogenic bacteria of tomato root rot and application, and relates to the technical field of molecular biology and plant disease detection, the primer group comprises a primer pair BT-F / BT-R for specifically detecting the pathogenic bacteria of phytophthora and a primer pair EF-F / EF-R for specifically detecting fusarium oxysporum, the nucleotide sequences of the primers are respectively shown as SEQ ID NO: 1-4. The invention also provides a kit containing the primer group and a method for detecting tomato root rot pathogenic bacteria, and the method comprises the following steps: extracting genome DNA of a sample to be detected, performing SYBR Green I real-time fluorescent quantitative PCR amplification by taking the obtained genome DNA as a template and adopting the primer group, and detecting the tomato root rot pathogenic bacteria according to the fluorescent quantitative PCR amplification result. And judging whether the to-be-detected sample contains phytophthora pathogenic bacteria and fusarium oxysporum or not. According to the invention, tomato root rot pathogens phytophthora and fusarium oxysporum can be specifically detected, and accurate identification and quantitative detection of the phytophthora and fusarium oxysporum can be realized.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and plant disease detection technology, and in particular to a fluorescent quantitative PCR primer set and its application for detecting the pathogen of tomato root rot. Background Technology

[0002] Tomato root rot is a common and serious soil-borne disease in tomato cultivation, severely affecting the health of the plant's root system and stem base. This disease primarily infects the root system and stem base, especially the root and stem areas. In the early stages, water-soaked lesions appear at the base of the stem; as the disease progresses, the lesions gradually turn brown and rot, causing the entire plant to wilt, and in severe cases, die. If the root collar is affected, it is often accompanied by temporary wilting of young leaves. After the lesions expand into ring-shaped rot, the above-ground leaves wilt irreversibly, and older leaves turn yellow starting from the leaf tips. In severe cases, this leads to overall yellowing and wilting, root rot, and ultimately, plant death. This disease is prevalent in major tomato-producing areas of North and Northeast my country, often causing severe yield losses and quality degradation, resulting in significant economic losses for agricultural production.

[0003] The pathogens causing tomato root rot mainly include fungi of the genus *Phytophthora* and *Fusarium oxysporum*. Both types of pathogens are characterized by strong soil-borne transmission, a wide host range, and a long survival period, making them difficult to control. Currently, the diagnosis of this disease still mainly relies on traditional methods such as field symptom observation, pathogen isolation and culture, and morphological identification. These methods are not only cumbersome and time-consuming, but their accuracy is also easily affected by subjective experience and environmental factors, making it difficult to achieve early, rapid, and highly sensitive detection of pathogens, often delaying the optimal control period.

[0004] Therefore, developing a molecular detection technology capable of accurately and rapidly identifying the main pathogens of tomato root rot is of significant practical importance for early diagnosis of this disease, guiding scientific pesticide application, improving control efficiency, and reducing pesticide overuse. Quantitative real-time PCR (qPCR) technology, due to its high sensitivity, strong specificity, and accurate quantification, has become the mainstream tool for molecular detection of pathogenic microorganisms. Developing specific primer sets for the main pathogens of tomato root rot and establishing a reliable qPCR detection system will help improve the monitoring and integrated control capabilities of this disease. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a set of fluorescent quantitative PCR primers for detecting the pathogens of tomato root rot and their application, which can specifically detect the pathogens of tomato root rot, Phytophthora and Fusarium oxysporum, and achieve accurate identification and quantitative detection of the two.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a set of fluorescent quantitative PCR primers for detecting the pathogen of tomato root rot, comprising a first primer pair and a second primer pair; The first primer pair is used for the specific detection of pathogens of the genus Phytophthora, including primer BT-F with the sequence shown in SEQ ID NO:1 and primer BT-R with the sequence shown in SEQ ID NO:2; The second primer pair is used for the specific detection of Fusarium oxysporum and includes primer EF-F with the sequence shown in SEQ ID NO:3 and primer EF-R with the sequence shown in SEQ ID NO:4.

[0007] Secondly, the present invention provides a kit for detecting pathogens causing tomato root rot, comprising the aforementioned primer set.

[0008] As a preferred embodiment, the kit also includes reagents required for the SYBR Green I real-time quantitative PCR reaction.

[0009] Thirdly, the present invention provides a method for detecting pathogens causing tomato root rot, comprising the following steps: S1, Extract genomic DNA from the sample to be tested; S2, using the genomic DNA obtained in step S1 as a template, perform SYBR Green I real-time quantitative PCR amplification using the primer set described above; S3. Based on the results of quantitative real-time PCR amplification, determine whether the sample to be tested contains pathogens of the genus Phytophthora and Fusarium oxysporum.

[0010] As a preferred option, the sample to be tested is tomato plant tissue or soil from which tomatoes are grown.

[0011] As a preferred embodiment, in step S2, the reaction conditions for the quantitative PCR amplification of *Phytophthora* pathogens include: 95℃, 10 min; 95℃, 30 s; 56℃, 55 s; 75℃, 30 s, 34 cycles; and the reaction conditions for the quantitative PCR amplification of *Fusarium oxysporum* include: 95℃, 10 min; 95℃, 30 s; 57℃, 55 s; 75℃, 30 s, 34 cycles; with fluorescence signal collected during the extension at 75℃.

[0012] As a preferred embodiment, in step S3, the PCR amplification products are detected by agarose gel electrophoresis. If a specific DNA band of 700 bp appears, the sample is determined to contain Phytophthora pathogens; if a specific DNA band of 260 bp appears, the sample is determined to contain Fusarium oxysporum.

[0013] As a preferred method, the Ct value obtained by real-time quantitative PCR is substituted into the standard curve to calculate the content of Phytophthora and Fusarium oxysporum in the sample to be tested.

[0014] Fourthly, the present invention provides the application of the above-mentioned primer set or the above-mentioned kit in the detection of pathogens causing tomato root rot.

[0015] According to the above technical solution, the beneficial effects of the present invention are: 1. Based on a specifically designed primer set, this invention successfully established a real-time quantitative PCR method capable of simultaneously detecting and identifying two major pathogens of tomato root rot (Phytophthora infestans and Fusarium oxysporum). This method exhibits extremely high specificity, showing no cross-reactivity with other common tomato pathogens, and overcomes the drawbacks of traditional methods that rely on morphological identification and are prone to misinterpretation.

[0016] 2. The detection system of this invention possesses high sensitivity and quantifiability, with minimum detection concentrations of 6 × 10⁻⁶ for Phytophthora and Fusarium oxysporum. 2 CFU / ml and 3.95×10 2 The method, using CFU / ml, enables trace detection and precise quantification of pathogens, providing a reliable tool for early disease warning and dynamic monitoring. Furthermore, it is simple to operate, has a short detection cycle, and produces reliable results, making it suitable for rapid screening and precise control of field samples. This is of great significance for reducing pesticide overuse and ensuring safe tomato production. Attached Figure Description

[0017] Figure 1 This is an electrophoresis diagram verifying the specificity of primer pair BT-F / BT-R in Example 2 of the present invention. Lanes 1-3: Phytophthora spp. pathogens; Lane 4: Botrytis cinerea; Lane 5: Fusarium oxysporum; Lane 6: Bacillus natto; Lane 7: Lactic acid bacteria; Lane 8: Bacillus subtilis.

[0018] Figure 2 This is an electrophoresis diagram verifying the specificity of primer pair EF-F / EF-R in Example 2 of the present invention. Lane 1: Botrytis cinerea; Lane 2: Phytophthora; Lane 3: Fusarium oxysporum; Lane 4: Bacillus natto; Lane 5: Lactic acid bacteria; Lane 6: Bacillus subtilis.

[0019] Figure 3 This is a graph showing the sensitivity detection results of real-time quantitative PCR against pathogens of the genus *Phytophthora* in Example 3 of this invention. Curves 1-6 in the graph correspond to template concentrations of 10⁻⁶ and 10⁻⁶, respectively. 7 CFU / ml, 2 is 10 6 CFU / ml, 3 is 10 5 CFU / ml, 4 is 10 4 CFU / ml, 5 is 10 3CFU / ml, 6 is 10 2 The samples were serially diluted to CFU / ml, and curve 7 served as the negative control.

[0020] Figure 4 This is a graph showing the sensitivity detection results of real-time quantitative PCR against Fusarium oxysporum in Example 3 of the present invention. Curves 1-6 in the graph correspond to template concentrations of 10⁻⁶ and 10⁻⁶, respectively. 7 CFU / ml, 2 is 10 6 CFU / ml, 3 is 10 5 CFU / ml, 4 is 10 4 CFU / ml, 5 is 10 3 CFU / ml, 6 is 10 2 The samples were serially diluted to CFU / ml, and curve 7 served as the negative control.

[0021] Figure 5 The fluorescence quantitative PCR amplification curve was used to verify the effectiveness of the detection system of this invention.

[0022] Figure 6 This is a standard curve established based on the gradient dilution of positive standards for pathogenic fungi of the genus Phytophthora.

[0023] Figure 7 This is a standard curve established based on the gradient dilution of Fusarium oxysporum positive standards. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough understanding of the disclosure of the invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0026] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.

[0027] This embodiment provides a method for detecting pathogens causing tomato root rot, including the following steps: S1, Extract genomic DNA from the sample to be tested; S2, using the genomic DNA obtained in step S1 as a template, perform SYBR Green I real-time quantitative PCR amplification using the primer set described above; S3. Based on the results of quantitative real-time PCR amplification, determine whether the sample to be tested contains pathogens of the genus Phytophthora and Fusarium oxysporum.

[0028] Example 1 Establishment of a real-time quantitative PCR method for detecting tomato root rot.

[0029] 1. DNA Extraction: Genomic DNA was extracted from tomato plant tissues using the CTAB method. This included the following steps: (1) Take an appropriate amount of tissue into a sterile mortar, add liquid nitrogen and grind it into a fine powder; (2) Place the ground tissue sample into a 2mL centrifuge tube. Add 600-1000μL of CTAB extraction buffer preheated to 65℃ and mix thoroughly by aspiration.

[0030] (3) Place the centrifuge tube in a 65°C water bath for 1 hour, and gently invert and mix it every 10 minutes during the water bath.

[0031] (4) After the water bath, remove the centrifuge tube and add an equal volume of extraction solution I (phenol: chloroform: isoamyl alcohol = 25:24:1) into the tube. Vortex to mix and centrifuge at 4℃ and 12000r / min for 8min.

[0032] (5) Take 500 μL of supernatant into a new 1.5 mL centrifuge tube, add an equal volume of extraction buffer II (chloroform: isoamyl alcohol = 24:1), vortex to mix, and centrifuge at 4 °C and 12000 r / min for 8 min.

[0033] (6) Pipette the supernatant into a new 1.5mL centrifuge tube, add 3 times the volume of anhydrous ethanol (pre-cooled to -20℃), mix gently, and let stand in a -20℃ refrigerator for 30min.

[0034] (7) After standing, remove the centrifuge tube and centrifuge at 4℃ and 10000r / min for 8min.

[0035] (8) Discard the supernatant, add 700 μL of 75% ethanol to wash the precipitate, centrifuge at 4°C and 10000 r / min for 8 min, discard the supernatant and collect the precipitate.

[0036] (9) Repeat step 8.

[0037] (10) Place the centrifuge tube in a clean bench and blow air until the precipitate is completely dry. Then add 50-100 μL of ddH2O to dissolve the precipitate. Use an ultra-micro UV spectrophotometer to determine the purity and concentration of the DNA for later use.

[0038] 2. Real-time PCR reaction system and conditions: The 20 μL reaction system shown in Tables 1 and 2 was used for detection. The reaction was performed on a real-time quantitative PCR instrument under the following conditions: 95℃ (pre-denaturation) for 2 min; 95℃ (denaturation) for 15 s, 57℃ (annealing) for 15 s, 40 cycles (PCR amplification); fluorescence signal was collected during extension at 72℃, using the FAM (SYBR signal channel).

[0039] 3. Quantitative fluorescence detection Mix all components of the reaction systems shown in Tables 1 and 2 thoroughly, place them in a real-time quantitative PCR instrument, and perform 40 cycles of amplification reaction according to the corresponding program. Collect fluorescence signals during the 72°C extension phase of each cycle.

[0040] The amplification activity of the detection system was determined by real-time monitoring of fluorescence signal accumulation. The results showed that a significant enhancement of specific fluorescence signal was observed when using the above system and procedure to amplify the genomic DNA of the tomato root rot pathogen. Figure 5 As shown, the amplification curve of the target pathogen exhibits typical exponential growth and plateau phases, with significant fluorescence signal intensity, proving that the established detection method can effectively and specifically detect the pathogen causing tomato root rot.

[0041] Based on the results obtained above, the strong fluorescence signal intensity and good amplification efficiency exhibited by this detection system demonstrate its reliable performance. Therefore, it has been determined as the standard method for all subsequent detection experiments. The preferred 20 μL system in this embodiment is shown in Tables 1 and 2, but it is not limited to this.

[0042] Example 2 Specific detection of pathogens causing tomato root rot.

[0043] The first pair of primers is used for the specific detection of pathogens in the genus *Phytophthora*. BT-F: 5'-GTATCATGTGCACGTACTCGG-3' BT-R: 5'-CAAGAAAGCCTTACGACGGA-3' The second pair of primers is used for the specific detection of Fusarium oxysporum: EF-F: 5'-CATCGAGAAGTTCGAGAAGG-3' EF-R: 5'-TACTTGAAGGAACCCTACC-3' To verify the specificity of the two primer pairs provided in this invention, genomic DNA was extracted from the following test strains as templates: Target strains: Phytophthora tomatoae, Fusarium oxysporum.

[0044] Non-target strains: Botrytis cinerea, Bacillus natto, lactic acid bacteria, Bacillus subtilis.

[0045] Using the primer set described above, conventional PCR amplification was performed using DNA from each strain as a template.

[0046] The reaction system consisted of: 12.5 μL of 2×PCR Master Mix, 1 μL each of upstream and downstream primers (10 μM), 1 μL of template DNA, and 9.5 μL of ddH2O.

[0047] The reaction program for *Phytophthora* was: pre-denaturation at 94 °C for 10 min; 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min; and a final extension at 72 °C for 10 min. The reaction program for *Fusarium oxysporum* was: pre-denaturation at 94 °C for 10 min; 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min; and a final extension at 72 °C for 10 min.

[0048] The PCR products were detected by 1.5% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, the first primer pair (BT-F and BT-R) amplified a specific band of approximately 700 bp only in the *Phytophthora* pathogen template (lanes 1-3), and no amplified bands were found in any non-target bacteria or the ddH2O control (lanes 4-8). Figure 2 As shown, the second primer pair (EF-F and EF-R) amplified a specific band of approximately 260 bp only in the *Fusarium oxysporum* template (lane 3), and no amplification was observed in any non-target bacteria or the ddH2O control (lanes 1-2, 4-6). These results clearly demonstrate that both primer pairs provided in this invention possess good interspecies specificity and can be used for the specific identification and detection of pathogens causing tomato root rot.

[0049] Example 3 Sensitivity for detecting pathogens causing tomato root rot.

[0050] DNA fragments from *Phytophthora* and *Fusarium oxysporum* were extracted using the CTAB method described in Example 1. After amplification with BT and EF primers, the fragments were purified and transformed into *Trans 2-Blue* *E. coli*, from which plasmid DNA was extracted. Recombinant plasmids containing *Phytophthora* and *Fusarium oxysporum*-specific gene fragments were serially diluted 10-fold (from 10...). 7 CFU / ml up to 10 2 The concentration of CFU / ml was detected using the real-time quantitative PCR method established in Example 1.

[0051] The results show that the method of the present invention has high sensitivity against both *Phytophthora* and *Fusarium oxysporum*. Figure 3 and Figure 4 As shown in the amplification curves, the detection limit of this method for both pathogens can reach 10. 2 The melting curves for each concentration gradient showed a single peak, indicating good amplification specificity. This method is highly sensitive and suitable for early detection of trace pathogens in field samples. The limits of detection (LOD) for *Phytophthora* and *Fusarium oxysporum* using this method were determined to be 6 × 10⁻⁶ CFU / ml. 2 CFU / ml and 3.95×10 2 CFU / ml.

[0052] Example 4 Actual sample testing.

[0053] To verify the practical application value of the present invention, samples of tomato plants suspected of being infected with root rot were collected from the field and tested using the primer set and detection method provided by the present invention, while the traditional pathogen isolation, culture and identification method was used as a control.

[0054] The results showed that the detection rate of the fluorescence quantitative PCR method established in this invention was significantly higher than that of the traditional isolation and culture method, and it could accurately identify whether the infection was caused by a single infection of Phytophthora or Fusarium oxysporum or a combination of both, proving the accuracy and reliability of this invention in practical applications.

[0055] Example 5 Quantitative detection of the sample to be tested.

[0056] Based on the Ct values ​​obtained from the serially diluted standards in Example 3, standard curves for Phytophthora and Fusarium oxysporum were plotted with the logarithm of the initial template copy number as the x-axis and the Ct value as the y-axis.

[0057] like Figure 6 As shown, the standard curve regression equation for *Phytophthora infestans* is y = -3.3343x + 31.7548, with a coefficient of determination (R²). 2 The value is 0.9977. For example... Figure 7 As shown, the standard curve regression equation for Fusarium oxysporum is y = -3.0766x + 25.6916, and the coefficient of determination (R²) is... 2 The value was 0.9934. The two standard curves showed good fitting, proving that the method established in this invention can accurately quantify the content of the two pathogens in the sample.

[0058] It should be noted that the above embodiments are only used to illustrate the present invention, but the present invention is not limited to the above embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A set of fluorescent quantitative PCR primers for detecting the pathogen of tomato root rot, characterized in that: Including the first primer pair and the second primer pair; The first primer pair is used for the specific detection of pathogens of the genus Phytophthora, including primer BT-F with the sequence shown in SEQ ID NO:1 and primer BT-R with the sequence shown in SEQ ID NO:2; The second primer pair is used for the specific detection of Fusarium oxysporum and includes primer EF-F with the sequence shown in SEQ ID NO:3 and primer EF-R with the sequence shown in SEQ ID NO:

4.

2. A kit for detecting the pathogen of tomato root rot, characterized in that: Includes the primer set as described in claim 1.

3. The reagent kit according to claim 2, characterized in that: The kit also includes reagents required for the SYBR Green I real-time quantitative PCR reaction.

4. A method for detecting the pathogen of tomato root rot, characterized in that: Includes the following steps: S1, Extract genomic DNA from the sample to be tested; S2, using the genomic DNA obtained in step S1 as a template, perform SYBR GreenI real-time quantitative PCR amplification using the primer set described in claim 1; S3. Based on the results of quantitative real-time PCR amplification, determine whether the sample to be tested contains pathogens of the genus Phytophthora and Fusarium oxysporum.

5. The method according to claim 4, characterized in that: The sample to be tested is tomato plant tissue or soil from which tomatoes are grown.

6. The method according to claim 4, characterized in that: In step S2, the reaction conditions for the SYBR Green I real-time quantitative PCR amplification are as follows: pre-denaturation at 95℃ for 2 min; followed by 40 cycles of amplification, each cycle including denaturation at 95℃ for 15 s, annealing at 57℃ for 15 s, and collection of fluorescence signal during extension at 72℃.

7. The method according to claim 4, characterized in that: In step S3, the PCR amplification products are detected by agarose gel electrophoresis. If a specific DNA band of 700 bp appears, the sample is determined to contain Phytophthora spp. pathogens; if a specific DNA band of 260 bp appears, the sample is determined to contain Fusarium oxysporum.

8. The method according to claim 7, characterized in that: Substitute the Ct values ​​obtained from real-time quantitative PCR into the standard curve to calculate the content of Phytophthora and Fusarium oxysporum pathogens in the sample to be tested.

9. The application of the primer set of claim 1 or the kit of claim 2 in the detection of pathogens causing tomato root rot.

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